Publications

Single-stranded DNA (ssDNA) intermediates which emerge during DNA metabolic processes are shielded by replication protein A (RPA). RPA binds to ssDNA and acts as a gatekeeper to direct the ssDNA towards downstream DNA metabolic pathways with exceptional specificity. Understanding the mechanistic basis for such RPA-dependent functional specificity requires knowledge of the structural conformation of ssDNA when RPA-bound. Previous studies suggested a stretching of ssDNA by RPA. However, structural investigations uncovered a partial wrapping of ssDNA around RPA. Therefore, to reconcile the models, in this study, we measured the end-to-end distances of free ssDNA and RPAssDNA complexes using single-molecule FRET and double electronelectron resonance (DEER) spectroscopy and found only a small systematic increase in the end-to-end distance of ssDNA upon RPA binding. This change does not align with a linear stretching model but rather supports partial wrapping of ssDNA around the contour of DNA binding domains of RPA. Furthermore, we reveal how phosphorylation at the key Ser-384 site in the RPA70 subunit provides access to the wrapped ssDNA by remodeling the DNA-binding domains. These findings establish a precise structural model for RPA-bound ssDNA, providing valuable insights into how RPA facilitates the remodeling of ssDNA for subsequent downstream processes.

Histidine (His) presents a unique challenge for modeling disordered protein conformations, as it is versatile and occurs in both the neutral (His⁰) and positively charged (His⁺) states. These His charge states, which are enabled by its imidazole side chain, influence the electrostatic and short-range interactions of His residues, which potentially engage in cation−π, π-π, and charge-charge interactions. Existing coarse-grained (CG) models often simplify His representation by assigning it an average charge, thereby neglecting these potential short-range interactions. To address this gap, we developed a model for intrinsically disordered proteins (IDPs) that accounts for the properties of histidine (H). The resulting IDPH model is a 21-amino acid CG model incorporating both His charge states. We show that interactions involving previously neglected His⁰ are critical for accurate modeling at high pH, where they significantly influence the compaction of His-rich IDPs such as Histatin-5 and CPEB4. These interactions contribute to structural stabilizations primarily via His⁰-His⁰ and His⁰-Arg interactions, which are overlooked in models focusing solely on the charged His⁺ state.

Aromatic residues can participate in various biomolecular interactions, such as π-π, cation−π, and CH−π interactions, which are essential for protein structure and function. Here, we re-evaluate the geometry and energetics of these interactions using quantum mechanical (QM) calculations, focusing on pairwise interactions involving the aromatic amino acids Phe, Tyr, and Trp and the cationic amino acids Arg and Lys. Our findings reveal that π-π interactions, while energetically favorable, are less abundant in structured proteins than commonly assumed and are often overshadowed by previously underappreciated, yet prevalent, CH−π interactions. Cation−π interactions, particularly those involving Arg, show strong binding energies and a specific geometric preference toward stacked conformations, despite the global QM minimum, suggesting that a rather perpendicular T-shape conformation should be more favorable. Our results support a more nuanced understanding of protein stabilization via interactions involving aromatic residues. On the one hand, our results challenge the traditional emphasis on π-π interactions in structured proteins by showing that CH−π and cation−π interactions contribute significantly to their structure. On the other hand, π-π interactions appear to be key stabilizers in solvated regions and thus may be particularly important to the stabilization of intrinsically disordered proteins.

Lujo virus (LUJV) is a human pathogen that was the cause of a deadly hemorrhagic fever outbreak in Africa. LUJV is a divergent member of the Arenaviridae with some similarities to both the \u201cOld World\u201d and \u201cNew World\u201d serogroups, but it uses a cell-entry receptor, neuropilin-2 (NRP2), that is distinct from the receptors of OW and NW viruses. Though the receptor binding domain of LUJV has been characterized structurally, the overall organization of the trimeric spike complex and how NRP2 is recognized in this context were unknown. Here, we present the structure of the membrane-embedded LUJV spike complex determined by cryo-electron microscopy. Analysis of the structure suggested that a single NRP2 molecule is bound at the apex of the trimeric spike and that multiple subunits of the trimer contact the receptor. The binding of NRP2 involves an intriguing arginine-methionine interaction, which we analyzed using quantum mechanical modeling methods. We compare the LUJV spike structure with the only other available structure of a complete arenaviral spike, which is the Lassa virus. The similarities and differences between them shed light on Arenavirus evolution, inform vaccine design, and provide information that will be useful in combating future Arenavirus outbreaks.

Mechanical energy, specifically in the form of ultrasound, can induce pressure variations and temperature fluctuations when applied to an aqueous media. These conditions can both positively and negatively affect protein complexes, consequently altering their stability, folding patterns, and self-assembling behavior. Despite much scientific progress, our current understanding of the effects of ultrasound on the self-assembly of amyloidogenic proteins remains limited. In the present study, we demonstrate that when the amplitude of the delivered ultrasonic energy is sufficiently low, it can induce refolding of specific motifs in protein monomers, which is sufficient for primary nucleation; this has been revealed by MD. These ultrasound-induced structural changes are initiated by pressure perturbations and are accelerated by a temperature factor. Furthermore, the prolonged action of low-amplitude ultrasound enables the elongation of amyloid protein nanofibrils directly from natively folded monomeric lysozyme protein, in a controlled manner, until it reaches a critical length. Using solution X-ray scattering, we determined that nanofibrillar assemblies, formed either under the action of sound or from natively fibrillated lysozyme, share identical structural characteristics. Thus, these results provide insights into the effects of ultrasound on fibrillar protein self-assembly and lay the foundation for the potential use of sound energy in protein chemistry.

The kinetics of proteinDNA recognition, along with its thermodynamic properties, including affinity and specificity, play a central role in shaping biological function. ProteinDNA recognition kinetics are characterized by two key elements: the time taken to locate the target site amid various nonspecific alternatives; and the kinetics involved in the recognition process, which may necessitate overcoming an energetic barrier. In this study, we developed a coarse-grained (CG) model to investigate interactions between a transcription factor called the sex-determining region Y (SRY) protein and DNA, in order to probe how DNA conformational changes affect SRYDNA recognition and binding kinetics. We find that, not only does a requirement for such a conformational DNA transition correspond to a higher energetic barrier for binding and therefore slower kinetics, it may further impede the recognition kinetics by increasing unsuccessful binding events (skipping events) where the protein partially binds its DNA target site but fails to form the specific proteinDNA complex. Such skipping events impose the need for additional cycles protein search of nonspecific DNA sites, thus significantly extending the overall recognition time. Our results highlight a trade-off between the speed with which the protein scans nonspecific DNA and the rate at which the protein recognizes its specific target site. Finally, we examine molecular approaches potentially adopted by natural systems to enhance proteinDNA recognition despite its intrinsically slow kinetics.

Histidine (His) stands out as the most versatile natural amino acid due to its side chains facile propensity to protonate at physiological pH, leading to a transition from aromatic to cationic characteristics and thereby enabling diverse biomolecular interactions. In this study, our objective was to quantify the energetics and geometries of pairwise interactions involving His at varying pH levels. Through quantum chemical calculations, we discovered that His exhibits robust participation in both π-π and cation-π interactions, underscoring its ability to adopt a π or cationic nature, akin to other common residues. Of particular note, we found that the affinity of protonated His for aromatic residues (via cation-π interactions) is greater than the affinity of neutral His for either cationic residues (also via cation-π interactions) or aromatic residues (via π-π interactions). Furthermore, His frequently engages in CH-π interactions, and notably, depending on its protonation state, we found that some instances of hydrogen bonding by His exhibit greater stability than is typical for interamino acid hydrogen bonds. The strength of the pH-dependent pairwise energies of His with aromatic residues is supported by the abundance of pairwise interactions with His of low and high predicted pK_a values. Overall, our findings illustrate the contribution of His interactions to protein stability and its potential involvement in conformational changes despite its relatively low abundance in proteins.

The Origin Recognition Complex (ORC) seeds replication-fork formation by binding to DNA replication origins, which in budding yeast contain a 17bp DNA motif. High resolution structure of the ORC-DNA complex revealed two base-interacting elements: a disordered basic patch (Orc1-BP4) and an insertion helix (Orc4-IH). To define the ORC elements guiding its DNA binding in vivo, we mapped genomic locations of 38 designed ORC mutants, revealing that different ORC elements guide binding at different sites. At silencing-associated sites lacking the motif, ORC binding and activity were fully explained by a BAH domain. Within replication origins, we reveal two dominating motif variants showing differential binding modes and symmetry: a non-repetitive motif whose binding requires Orc1-BP4 and Orc4-IH, and a repetitive one where another basic patch, Orc1-BP3, can replace Orc4-IH. Disordered basic patches are therefore key for ORC-motif binding in vivo, and we discuss how these conserved, minor-groove interacting elements can guide specific ORC-DNA recognition.

Replication Protein A (RPA) is a single stranded DNA (ssDNA) binding protein that coordinates diverse DNA metabolic processes including DNA replication, repair, and recombination. RPA is a heterotrimeric protein with six functional oligosaccharide/oligonucleotide (OB) domains and flexible linkers. Flexibility enables RPA to adopt multiple configurations and is thought to modulate its function. Here, using single molecule confocal fluorescence microscopy combined with optical tweezers and coarse-grained molecular dynamics simulations, we investigated the diffusional migration of single RPA molecules on ssDNA under tension. The diffusion coefficient D is the highest (20,000 nucleotides²/s) at 3 pN tension and in 100 mM KCl and markedly decreases when tension or salt concentration increases. We attribute the tension effect to intersegmental transfer which is hindered by DNA stretching and the salt effect to an increase in binding site size and interaction energy of RPA-ssDNA. Our integrative study allowed us to estimate the size and frequency of intersegmental transfer events that occur through transient bridging of distant sites on DNA by multiple binding sites on RPA. Interestingly, deletion of RPA trimeric core still allowed significant ssDNA binding although the reduced contact area made RPA 15-fold more mobile. Finally, we characterized the effect of RPA crowding on RPA migration. These findings reveal how the high affinity RPA-ssDNA interactions are remodeled to yield access, a key step in several DNA metabolic processes.

Biomolecular condensates are essential for cellular functionality, yet the complex interplay among the diverse molecular interactions that mediate their formation remains poorly understood. Here, using coarse-grained molecular dynamics simulations, we address the contribution of cation-π interactions to the stability of condensates formed via liquid-liquid phase separation. We found greater stabilization of up to 80% via cation-π interactions in condensates formed from peptides with higher aromatic residue content or less charge clustering. The contribution of cation-π interactions to droplet stability increases with increasing ionic strength, suggesting a trade-off between cation-π and electrostatic interactions. Cation-π interactions, therefore, can compensate for reduced electrostatic interactions, such as occurs at higher salt concentrations and in sequences with less charged residue content or clustering. Designing condensates with desired biophysical characteristics therefore requires quantification not only of the individual interactions but also cross-talks involving charge-charge, π-π, and cation-π interactions.

Intrinsically disordered proteins (IDPs) adopt a wide array of different conformations that can be constrained by the presence of proline residues, which are frequently found in IDPs. To assess the effects of proline, we designed a series of peptides that differ with respect to the number of prolines in the sequence and their organization. Using high-resolution atomistic molecular dynamics simulations, we found that accounting for whether the proline residues are clustered or isolated contributed significantly to explaining deviations in the experimentally-determined gyration radii of IDPs from the values expected based on the Flory scaling-law. By contrast, total proline content makes smaller contribution to explaining the effect of prolines on IDP conformation. Proline residues exhibit opposing effects depending on their organizational pattern in the IDP sequence. Clustered prolines (i.e., prolines with ≤2 intervening non-proline residues) result in expanded peptide conformations whereas isolated prolines (i.e., prolines with >2 intervening non-proline residues) impose compacted conformations. Clustered prolines were estimated to induce an expansion of ∼20% in IDP dimension (via formation of PPII structural elements) whereas isolated prolines were estimated to induce a compaction of ∼10% in IDP dimension (via the formation of backbone turns). This dual role of prolines provides a mechanism for conformational switching that does not rely on the kinetically much slower isomerization of cis proline to the trans form. Bioinformatic analysis demonstrates high populations of both isolated and clustered prolines and implementing them in coarse-grained molecular dynamics models illustrates that they improve the characterization of the conformational ensembles of IDPs.

We describe for the first time the co-existence of the parallel and antiparallel conformation of the heterodimeric E3/K3 and E3/R3 coiled-coil systems in solution. The introduction of a furanylated amino acid in the (EIAALEK)3 sequence allowed, upon photo-induced covalent crosslinking, freezing of the respective coiled-coil complexes present in solution. The occurrence of both parallel and antiparallel conformations in solution was supported by computational simulations and further confirmed by fluorescence experiments based on pyrene-pyrene stacking.

In eukaryotes, many DNA/RNA-binding proteins possess intrinsically disordered regions (IDRs) with large negative charge, some of which involve a consecutive sequence of aspartate (D) or glutamate (E) residues. We refer to them as D/E repeats. The functional role of D/E repeats is not well understood, though some of them are known to cause autoinhibition through intramolecular electrostatic interaction with functional domains. In this work, we investigated the impacts of D/E repeats on the target DNA search kinetics for the high-mobility group box 1 (HMGB1) protein and the artificial protein constructs of the Antp homeodomain fused with D/E repeats of varied lengths. Our experimental data showed that D/E repeats of particular lengths can accelerate the target association in the overwhelming presence of non-functional high-Affinity ligands ('decoys'). Our coarse-grained molecular dynamics (CGMD) simulations showed that the autoinhibited proteins can bind to DNA and transition into the uninhibited complex with DNA through an electrostatically driven induced-fit process. In conjunction with the CGMD simulations, our kinetic model can explain how D/E repeats can accelerate the target association process in the presence of decoys. This study illuminates an unprecedented role of the negatively charged IDRs in the target search process.

Diffusion is a pervasive process present in a broad spectrum of cellular reactions. Its mathematical description has existed for nearly two centuries and permits the construction of simple rules for evaluating the characteristic timescales of diffusive processes and some of their determinants. Although the term diffusion originally referred to random motions in three-dimensional (3D) media, several biological diffusion processes in lower dimensions have been reported. One-dimensional (1D) diffusions have been reported, for example, for translocations of various proteins along DNA or protein (e.g., microtubule) lattices and translation of helical peptides along the coil-coiled interface. Two-dimensional (2D) diffusion has been shown for dynamics of proteins along membranes. The microscopic mechanisms of these 1-3D diffusions may vary significantly depending on the nature of the diffusing molecules, the substrate, and the interactions between them. In this review, we highlight some key examples of 1-3D biomolecular diffusion processes and illustrate the roles that electrostatic interactions and intrinsic disorder may play in modulating these processes.

Many proteins have intrinsically disordered regions (IDRs), which are often characterized by a high fraction of charged residues with polyampholytic (i.e., mixed charge) or polyelectrolytic (i.e., uniform charge) characteristics. Polyelectrolytic IDRs include consecutive positively charged Lys or Arg residues (K/R repeats) or consecutive negatively charged Asp or Glu residues (D/E repeats). In previous research, D/E repeats were found to be about five times longer than K/R repeats and to be much more common in eukaryotes. Within these repeats, a preference is often observed for E over D and for K over R. To understand the greater prevalence of D/E over K/R repeats and the higher abundance of E and K, we simulated the conformational ensemble of charged homo-polypeptides (polyK, polyR, polyD, and polyE) using molecular dynamics simulations. The conformational preferences and dynamics of these polyelectrolytic polypeptides change with changes in salt concentration. In particular, polyD and polyE are more sensitive to salt than polyK and polyR, as polyD and polyE tend to adsorb more divalent cations, which leads to their having more compact conformations. We conclude with a discussion of biophysical explanations for the relative abundance of charged amino acids and particularly for the greater abundance of D/E repeats over K/R repeats.

In a process known as facilitated diffusion, DNA-binding proteins find their target sites by combining three-dimensional diffusion and one-dimensional scanning of the DNA. Following the trade-off between speed and stability, agile exploration of DNA requires loose binding, whereas, at the DNA target site, the searching protein needs to establish tight interactions with the DNA. To enable both efficient search and stable binding, DNA-binding proteins and DNA often switch conformations upon recognition. Here, we study the one-dimensional diffusion and DNA binding of the dimeric lac repressor (LacI), which was reported to adopt two different conformations when binding different conformations of DNA. Using coarse-grained molecular dynamic simulations, we studied the diffusion and the sequence-specific binding of these conformations of LacI, as well as their truncated or monomeric variants, with two DNA conformations: straight and bent. The simulations were compared to experimental observables. This study supports that linear diffusion along DNA combines tight rotation-coupled groove tracking and rotation-decoupled hopping, where the protein briefly dissociates and reassociates just a few base pairs away. Tight groove tracking is crucial for target-site recognition, while hopping speeds up the overall search process. We investigated the diffusion of different LacI conformations on DNA and show how the flexibility of LacIs hinge regions ensures agility on DNA as well as faithful groove tracking. If the hinge regions instead form α-helices at the proteinDNA interface, tight groove tracking is not possible. On the contrary, the helical hinge region is essential for tight binding to bent, specific DNA, for the formation of the specific complex. Based on our study of different encounter complexes, we argue that the conformational change in LacI and DNA bending are somewhat coupled. Our findings underline the importance of two distinct protein conformations for facilitated diffusion and specific binding, respectively.

A way of modulating the solid-state electron transport (ETp) properties of oligopeptide junctions is presented by charges and internal hydrogen bonding, which affect this process markedly. The ETp properties of a series of tyrosine (Tyr)-containing hexa-alanine peptides, self-assembled in monolayers and sandwiched between gold electrodes, are investigated in response to their protonation state. Inserting a Tyr residue into these peptides enhances the ETp carried their junctions. Deprotonation of the Tyr-containing peptides causes a further increase of ETp efficiency that depends on this residue's position. Combined results of molecular dynamics simulations and spectroscopic experiments suggest that the increased conductance upon deprotonation is mainly a result of enhanced coupling between the charged C-terminus carboxylate group and the adjacent Au electrode. Moreover, intra-peptide hydrogen bonding of the Tyr hydroxyl to the C-terminus carboxylate reduces this coupling. Hence, the extent of such a conductance change depends on the Tyr-carboxylate distance in the peptide's sequence.

Silk is a unique, remarkably strong biomaterial made of simple protein building blocks. To date, no synthetic method has come close to reproducing the properties of natural silk, due to the complexity and insufficient understanding of the mechanism of the silk fiber formation. Here, we use a combination of bulk analytical techniques and nanoscale analytical methods, including nano-infrared spectroscopy coupled with atomic force microscopy, to probe the structural characteristics directly, transitions, and evolution of the associated mechanical properties of silk protein species corresponding to the supramolecular phase states inside the silkworm's silk gland. We found that the key step in silk-fiber production is the formation of nanoscale compartments that guide the structural transition of proteins from their native fold into crystalline β-sheets. Remarkably, this process is reversible. Such reversibility enables the remodeling of the final mechanical characteristics of silk materials. These results open a new route for tailoring silk processing for a wide range of new material formats by controlling the structural transitions and self-assembly of the silk protein's supramolecular phases.

Peptide-RNA coacervates can result in the concentration and compartmentalization of simple biopolymers. Given their primordial relevance, peptide-RNA coacervates may have also been a key site of early protein evolution. However, the extent to which such coacervates might promote or suppress the exploration of novel peptide conformations is fundamentally unknown. To this end, we used electron paramagnetic resonance spectroscopy (EPR) to characterize the structure and dynamics of an ancient and ubiquitous nucleic acid binding element, the helix-hairpin-helix (HhH) motif, alone and in the presence of RNA, with which it forms coacervates. Double electron-electron resonance (DEER) spectroscopy applied to singly labeled peptides containing one HhH motif revealed the presence of dimers, even in the absence of RNA. Moreover, dimer formation is promoted upon RNA binding and was detectable within peptide-RNA coacervates. DEER measurements of spin-diluted, doubly labeled peptides in solution indicated transient α-helical character. The distance distributions between spin labels in the dimer and the signatures of α-helical folding are consistent with the symmetric (HhH)2-Fold, which is generated upon duplication and fusion of a single HhH motif and traditionally associated with dsDNA binding. These results support the hypothesis that coacervates are a unique testing ground for peptide oligomerization and that phase-separating peptides could have been a resource for the construction of complex protein structures via common evolutionary processes, such as duplication and fusion.

Intrinsically disordered regions (IDRs) of proteins are often characterized by a high fraction of charged residues, but differ in their overall net charge and in the organization of the charged residues. The function-encoding information stored via IDR charge composition and organization remains elusive. Here, we aim to decipher the sequencefunction relationship in IDRs by presenting a comprehensive bioinformatic analysis of the charge properties of IDRs in the human, mouse, and yeast proteomes. About 50% of the proteins comprise at least a single IDR, which is either positively or negatively charged. Highly negatively charged IDRs are longer and possess greater net charge per residue compared with highly positively charged IDRs. A striking difference between positively and negatively charged IDRs is the characteristics of the repeated units, specifically, of consecutive Lys or Arg residues (K/R repeats) and Asp or Glu (D/E repeats) residues. D/E repeats are found to be about five times longer than K/R repeats, with the longest found containing 49 residues. Long stretches of consecutive D and E are found to be more prevalent in nucleic acid-related proteins. They are less common in prokaryotes, and in eukaryotes their abundance increases with genome size. The functional role of D/E repeats and the profound differences between them and K/R repeats are discussed.

The association between two intrinsically disordered proteins (IDPs) may produce a fuzzy complex characterized by a high binding affinity, similar to that found in the ultrastable complexes formed between two well-structured proteins. Here, using coarse-grained simulations, we quantified the biophysical forces driving the formation of such fuzzy complexes. We found that the high-affinity complex formed between the highly and oppositely charged H1 and ProTα proteins is sensitive to electrostatic interactions. We investigated 52 variants of the complex by swapping charges between the two oppositely charged proteins to produce sequences whose negatively or positively charged residue content was more homogeneous or heterogenous (i.e., polyelectrolytic or polyampholytic, having higher or lower absolute net charges, respectively) than the wild type. We also changed the distributions of oppositely charged residues within each participating sequence to produce variants in which the charges were segregated or well mixed. Both types of changes significantly affect binding affinity in fuzzy complexes, which is governed by both enthalpy and entropy. The formation of H1ProTa is supported by an increase in configurational entropy and by entropy due to counterion release. The latter can be twice as large as the former, illustrating the dominance of counterion entropy in modulating the binding thermodynamics. Complexes formed between proteins with greater absolute net charges are more stable, both enthalpically and entropically, indicating that enthalpy and entropy have a mutually reinforcing effect. The sensitivity of the thermodynamics of the complex to net charge and the charge pattern within each of the binding constituents may provide a means to achieve binding specificity between IDPs.

Highly negatively charged segments containing only aspartate or glutamate residues (\u201cD/E repeats\u201d) are found in many eukaryotic proteins. For example, the C-terminal 30 residues of the HMGB1 protein are entirely D/E repeats. Using nuclear magnetic resonance (NMR), fluorescence, and computational approaches, we investigated how the D/E repeats causes the autoinhibition of HMGB1 against its specific binding to cisplatin-modified DNA. By varying ionic strength in a wide range (40900 mM), we were able to shift the conformational equilibrium between the autoinhibited and uninhibited states toward either of them to the full extent. This allowed us to determine the macroscopic and microscopic equilibrium constants for the HMGB1 autoinhibition at various ionic strengths. At a macroscopic level, a model involving the autoinhibited and uninhibited states can explain the salt concentration-dependent binding affinity data. Our data at a microscopic level show that the D/E repeats and other parts of HMGB1 undergo electrostatic fuzzy interactions, each of which is weaker than expected from the macroscopic autoinhibitory effect. This discrepancy suggests that the multivalent nature of the fuzzy interactions enables strong autoinhibition at a macroscopic level despite the relatively weak intramolecular interaction at each site. Both experimental and computational data suggest that the D/E repeats interact preferentially with other intrinsically disordered regions (IDRs) of HMGB1. We also found that mutations mimicking post-translational modifications relevant to nuclear export of HMGB1 can moderately modulate DNA-binding affinity, possibly by impacting the autoinhibition. This study illuminates a functional role of the fuzzy interactions of D/E repeats.

Despite the continuous discovery of host and guest proteins in membraneless organelles, complex hostguest interactions hinder the understanding of the molecular grammar governing liquidliquid phase separation. In this study, we characterized the localization and dynamic properties of guest proteins in liquid droplets using single-molecule fluorescence microscopy. Eighteen guest proteins of different sizes, structures, and oligomeric states were examined in host p53 liquid droplets. Recruitment did not significantly depend on the structural properties of the guest proteins, but was moderately correlated with their length, total charge, and number of R and Y residues. In contrast, the diffusion of disordered guest proteins was comparable to that of host p53, whereas that of folded proteins varied widely. Molecular dynamics simulations suggest that folded proteins diffuse within the voids of the liquid droplet while interacting weakly with neighboring host proteins, whereas disordered proteins adapt their structures to form tight interactions with the host proteins. Our study provides insights into the key molecular principles of the localization and dynamics of guest proteins in liquid droplets.

Microtubules (MTs), an essential component of the eukaryotic cytoskeleton, are a lattice of polymerized tubulin dimers and are crucial for various cellular processes. The genetic and chemical diversity of tubulin and their disordered tails gives rise to a "tubulin code". The functional role of tubulin post-translational modifications (PTMs), which contribute to the chemical diversity of the tubulin code, is gradually being unraveled. However, variation in the length and spatial organization of tubulin poly-modifications leads to an enormous combinatorial PTM space, which is difficult to study experimentally. Hence, the impact of the combinatorial tubulin PTM space on the biophysical properties of tubulin tails and their interactions with other proteins remains elusive. Here, we combine all-atom and coarse-grained molecular dynamics simulations to elucidate the biophysical implications of the large combinatorial tubulin PTM space in the context of an MT lattice. We find that tail-body interactions are more dominant in the tubulin dimer than in an MT lattice, and are more significant for the tails of α compared with β tubulin. In addition, poly-glutamylation, but not poly-glycylation, expands the dimensions of the tubulin tails. Poly-glutamylation also leads to a decrease in the diffusion rate of MT-associated protein EB1 on MTs, while poly-glycylation often increases diffusion rate. These observations are generally not sensitive to the organization of the poly-modifications. The effect of PTMs on MT charge density and tail dynamics are also discussed. Overall, this study presents a molecular quantification of the biophysical properties of tubulin tails and their poly-modifications, and provides predictions on the functional importance of tubulin PTMs.

DNA-binding proteins rely on linear diffusion along the longitudinal DNA axis, supported by their nonspecific electrostatic affinity for DNA, to search for their target recognition sites. One may therefore expect that the ability to engage in linear diffusion along DNA is universal to all DNA-binding proteins, with the detailed biophysical characteristics of that diffusion differing between proteins depending on their structures and functions. One key question is whether the linear diffusion mechanism is defined by translation coupled with rotation, a mechanism that is often termed sliding. We conduct coarse-grained and atomistic molecular dynamics simulations to investigate the minimal requirements for protein sliding along DNA. We show that coupling, while widespread, is not universal. DNA-binding proteins that slide along DNA transition to uncoupled translationrotation (i.e., hopping) at higher salt concentrations. Furthermore, and consistently with experimental reports, we find that the sliding mechanism is the less dominant mechanism for some DNA-binding proteins, even at low salt concentrations. In particular, the toroidal PCNA protein is shown to follow the hopping rather than the sliding mechanism.

Intrinsically disordered proteins play a crucial role in cellular phase separation, yet the diverse molecular forces driving phase separation are not fully understood. It is of utmost importance to understand how peptide sequence, and particularly the balance between the peptides short- and long-range interactions with other peptides, may affect the stability, structure, and dynamics of liquidliquid phase separation in protein condensates. Here, using coarse-grained molecular dynamics simulations, we studied the liquid properties of the condensate in a series of polymers in which the ratio of short-range dispersion interactions to long-range electrostatic interactions varied. As the fraction of mutations that participate in short-range interactions increases at the expense of long-range electrostatic interactions, a significant decrease in the critical temperature of phase separation is observed. Nevertheless, sequences with a high fraction of short-range interactions exhibit stabilization, which suggests compensation for the loss of long-range electrostatic interactions. Decreased condensate stability is coupled with decreased translational diffusion of the polymers in the condensate, which may result in the loss of liquid characteristics in the presence of a high fraction of uncharged residues. The effect of exchanging long-range electrostatic interactions for short-range interactions can be explained by the kinetics of breaking intermolecular contacts with neighboring polymers and the kinetics of intramolecular fluctuations. While both time scales are coupled and increase as electrostatic interactions are lost, for sequences that are dominated by short-range interactions, the kinetics of intermolecular contact breakage significantly slows down. Our study supports the contention that different types of interactions can maintain protein condensates, however, long-range electrostatic interactions enhance its liquid-like behavior.

DNA mismatch repair (MMR) is an important postreplication process that eliminates mispaired or unpaired nucleotides to ensure genomic replication fidelity. In humans, Msh2-Msh6 and Msh2-Msh3 are the two mismatch repair initiation factors that recognize DNA lesions. While X-ray crystal structures exist for these proteins in complex with DNA lesions, little is known about their structures during the initial search along nonspecific double-stranded DNA, because they are short-lived and difficult to determine experimentally. In this study, various computational approaches were used to sidestep these difficulties. All-atom and coarse-grained simulations based on the crystal structures of Msh2-Msh3 and Msh2-Msh6 showed no translation along the DNA, suggesting that the initial search conformation differs from the lesion-bound crystal structure. We modeled probable search-mode structures of MSH proteins and showed, using coarse-grained molecular dynamics simulations, that they can perform rotation-coupled diffusion on DNA, which is a suitable and efficient search mechanism for their function and one predicted earlier by fluorescence resonance energy transfer and fluorescence microscopy studies. This search mechanism is implemented by electrostatic interactions among the mismatch-binding domain (MBD), the clamp domains, and the DNA backbone. During simulations, their diffusion rate did not change significantly with an increasing salt concentration, which is consistent with observations from experimental studies. When the gap between their DNA-binding clamps was increased, Msh2-Msh3 diffused mostly via the clamp domains while Msh2-Msh6 still diffused using the MBD, reproducing the experimentally measured lower diffusion coefficient of Msh2-Msh6. Interestingly, Msh2-Msh3 was capable of dissociating from the DNA, whereas Msh2-Msh6 always diffused on the DNA duplex. This is consistent with the experimental observation that Msh2-Msh3, unlike Msh2-Msh6, can overcome obstacles such as nucleosomes. Our models provide a molecular picture of the different mismatch search mechanisms undertaken by Msh2-Msh6 and Msh2-Msh3, despite the similarity of their structures.

The respiratory and intestinal tracts are exposed to physical and biological hazards accompanying the intake of air and food. Likewise, the vasculature is threatened by inflammation and trauma. Mucin glycoproteins and the related von Willebrand factor guard the vulnerable cell layers in these diverse systems. Colon mucins additionally house and feed the gut microbiome. Here, we present an integrated structural analysis of the intestinal mucin MUC2. Our findings reveal the shared mechanism by which complex macromolecules responsible for blood clotting, mucociliary clearance, and the intestinal mucosal barrier form protective polymers and hydrogels. Specifically, cryo-electron microscopy and crystal structures show how disulfide-rich bridges and pH-tunable interfaces control successive assembly steps in the endoplasmic reticulum and Golgi apparatus. Remarkably, a densely O-glycosylated mucin domain performs an organizational role in MUC2. The mucin assembly mechanism and its adaptation for hemostasis provide the foundation for rational manipulation of barrier function and coagulation.

Proteins with intrinsically disordered regions have a tendency to condensate via liquid-liquid phase separation both in vitro and in vivo. Such biomolecular coacervates play various significant roles in biologically important regulatory processes. The present work explores the structural and dynamic features of coacervates formed by model polyampholytes, being intrinsically disordered proteins, that differ in terms of their charged amino acid patterns. Differences in the distribution of charged amino acids along the polyampholyte sequence lead to distinctly different structural features in the dense phase and hence to different liquid properties. Increased charge clustering raises the critical temperature for phase separation and results in each polyampholyte experiencing a larger number of inter-chain contacts with neighboring proteins in the condensate. Consequently, polyampholytes with greater charge clustering adopt a much more extended conformation, having a radius of gyration up to twice that observed in the dilute bulk phase. Translational diffusion within the droplet is pronounced, being just 4-20 times slower than in the bulk, consistently with the high conformational entropy in the dense phase and high exchange rate of the network of intermolecular interactions in the condensate. Coupled to the faster diffusion, the condensate also adopts a more elongated shape and exhibits imperfect packing, which results in cavities. This study quantifies the fundamental microscopic properties of condensates including the effect of long-range electrostatic forces and particularly how they can be modulated by the charge pattern.

Protein diffusion in lower-dimensional spaces is used for various cellular functions. For example, sliding on DNA is essential for proteins searching for their target sites, and protein diffusion on microtubules is important for proper cell division and neuronal development. On the one hand, these linear diffusion processes are mediated by long-range electrostatic interactions between positively charged proteins and negatively charged biopolymers and have similar characteristic diffusion coefficients. On the other hand, DNA and microtubules have different structural properties. Here, using computational approaches, we studied the mechanism of protein diffusion along DNA and microtubules by exploring the diffusion of both protein types on both biopolymers. We found that DNA-binding and microtubule-binding proteins can diffuse on each other's substrates; however, the adopted diffusion mechanism depends on the molecular properties of the diffusing proteins and the biopolymers. On the protein side, only DNA-binding proteins can perform rotation-coupled diffusion along DNA, with this being due to their higher net charge and its spatial organization at the DNA recognition helix. By contrast, the lower net charge on microtubule-binding proteins enables them to diffuse more quickly than DNA-binding proteins on both biopolymers. On the biopolymer side, microtubules possess intrinsically disordered, negatively charged C-terminal tails that interact with microtubule-binding proteins, thus supporting their diffusion. Thus, although both DNA-binding and microtubule-binding proteins can diffuse on the negatively charged biopolymers, the unique molecular features of the biopolymers and of their natural substrates are essential for function.

Thermodynamic stability is an important property of proteins that is linked to many of the trade-offs that characterize a protein molecule and therefore its function. Designing a protein with a desired stability is a complicated task given the intrinsic trade-off between enthalpy and entropy which applies for both the folded and unfolded states. Traditionally, protein stability is manipulated by point mutations which regulate the folded state enthalpy. In some cases, the entropy of the unfolded state has also been manipulated by means that drastically restrict its conformational dynamics such as engineering disulfide bonds. In this mini-review, we survey various approaches to modify protein stability by manipulating the entropy of either the unfolded or the folded states. We show that point mutations that involve elimination of long-range contacts may have a greater destabilization effect than mutations that eliminate shorter-range contacts. Protein conjugation can also affect the entropy of the unfolded state and thus the overall stability. In addition, we show that entropy can contribute to shape the folded state and yield greater protein stabilization. Hence, we argue that the entropy component can be practically manipulated both in the folded and unfolded state to modify protein stability.

DNA sequences are often recognized by multi-domain proteins that may have higher affinity and specificity than single-domain proteins. However, the higher affinity to DNA might be coupled with slower recognition kinetics. In this study, we address this balance between stability and kinetics for multi-domain Cys(2)His(2)- (C2H2-) type zinc-finger (ZF) proteins. These proteins are the most prevalent DNA-binding domain in eukaryotes and C2H2 type zinc-finger proteins (C2H2-ZFPs) constitute nearly one-half of all known and predicted transcription factors in human. Extensive contact with DNA via tandem ZF domains confers high stability on the sequence-specific complexes. However, this can limit target search efficiency, especially for low abundance ZFPs. Earlier, we found that asymmetrical distribution of electrostatic charge among the three ZF domains of the low abundance transcription factor Egr-1 facilitates its DNA search process. Here, on a diverse set of 273 human C2H2-ZFP comprised of 3-15 tandem ZF domains, we find that, in many cases, electrostatic charge and binding specificity are asymmetrically distributed among the ZF domains so that neighbouring domains have different DNA-binding properties. For proteins containing 3-6 ZF domains, we show that the low abundance proteins possess a higher degree of non-specific asymmetry and vice versa. Our findings suggest that where the electrostatics of tandem ZF domains are similar (i.e., symmetrical), the ZFPs are more abundant to optimize their DNA search efficiency. This study reveals new insights into the fundamental determinants of recognition by C2H2-ZFPs of their DNA binding sites in the cellular landscape. The importance of electrostatic asymmetry with respect to binding site recognition by C2H2-ZFPs suggests the possibility that it may also be important in other ZFP systems and reveals a new design feature for zinc finger engineering.Author summaryOptimal recognition of proteins to DNA is governed by various factors among them the thermodynamics, kinetics and specificity of the protein-DNA complex. Multi-domain DNA-binding proteins are expected to have higher affinity and specificity due to the extensive interface they form with DNA. However, larger interface may result with higher friction when these proteins scan the DNA for the target site via the sliding mechanism. A way to overcome this drawback is to have asymmetry in the protein so that the interface with DNA is smaller. Alternatively, higher abundance can also increase the search speed. Here, using computational analysis of large data set of multi-domain zinc finger DNA-binding proteins, we report a trade-off between asymmetry and abundance.

Microtubules (MTs) are essential components of the eukaryotic cytoskeleton that serve as "highways" for intracellular trafficking. In addition to the well-known active transport of cargo by motor proteins, many MT-binding proteins seem to adopt diffusional motility as a transportation mechanism. However, because of the limited spatial resolution of current experimental techniques, the detailed mechanism of protein diffusion has not been elucidated. In particular, the precise role of tubulin tails and tail modifications in the diffusion process is unclear. Here, using coarse-grained molecular dynamics simulations validated against atomistic simulations, we explore the molecular mechanism of protein diffusion along MTs. We found that electrostatic interactions play a central role in protein diffusion; the disordered tubulin tails enhance affinity but slow down diffusion, and diffusion occurs in discrete steps. While diffusion along wild-type MT is performed in steps of dimeric tubulin, the removal of the tails results in a step of monomeric tubulin. We found that the energy barrier for diffusion is larger when diffusion on MTs is mediated primarily by the MT tails rather than the MT body. In addition, globular proteins (EB1 and PRC1) diffuse more slowly than an intrinsically disordered protein (Tau) on MTs. Finally, we found that polyglutamylation and polyglycylation of tubulin tails lead to slower protein diffusion along MTs, although polyglycylation leads to faster diffusion across MT protofilaments. Taken together, our results explain experimentally observed data and shed light on the roles played by disordered tubulin tails and tail modifications in the molecular mechanism of protein diffusion along MTs.

Replication protein A (RPA) plays a critical role in all eukaryotic DNA processing involving singlestranded DNA (ssDNA). Contrary to the notion that RPA provides solely inert protection to transiently formed ssDNA, the RPA-ssDNA complex acts as a dynamic DNA processing unit. Here, we studied the diffusion of RPA along 60 nt ssDNA using a coarse-grained model in which the ssDNA-RPA interface was modeled by both aromatic and electrostatic interactions. Our study provides direct evidence of bulge formation during the diffusion of ssDNA along RPA. Bulges can form at a few sites along the interface and store 1-7 nt of ssDNA whose release, upon bulge dissolution, leads to propagation of ssDNA diffusion. These findings thus support the reptation mechanism, which involves bulge formation linked to the aromatic interactions, whose short range nature reduces cooperativity in ssDNA diffusion. Greater cooperativity and a larger diffusion coefficient for ssDNA diffusion along RPA are observed for RPA variants with weaker aromatic interactions and for interfaces homogenously stabilized by electrostatic interactions. ssDNA propagation in the latter instance is characterized by lower probabilities of bulge formation; thus, it may fit the sliding-without-bulge model better than the reptation model. Thus, the reptation mechanism allows ssDNA mobility despite the extensive and high affinity interface of RPA with ssDNA. The short-range aromatic interactions support bulge formation while the long-range electrostatic interactions support the release of the stored excess ssDNA in the bulge and thus the overall diffusion.

Proteins are subject to various conflicting forces that trade-off against each other. For example, during folding, the protein achieves lower enthalpy at the cost of lower entropy. Similarly, the trade-off for increased stability may be decreased flexibility, which may abolish allosteric pathways. Accordingly, stability trades-off against function, which may also trade-off against folding kinetics and mechanism. Furthermore, attaining increased stability may reduce a protein's ability to adopt novel functions. Understanding the biophysics and function of proteins requires quantification of the driving forces involved in each of the trade-offs. Indeed, quantification of the linkages in the network of trade-offs is essential to obtaining a more complete understanding of protein structure and function.

The opening of a Watson-Crick double helix is required for crucial cellular processes, including replication, repair, and transcription. It has long been assumed that RNA or DNA base pairs are broken by the concerted symmetric movement of complementary nucleobases. By analyzing thousands of base-pair opening and closing events from molecular simulations, here, we uncover a systematic stepwise process driven by the asymmetric flipping-out probability of paired nucleobases. We demonstrate experimentally that such asymmetry strongly biases the unwinding efficiency of DNA helicases toward substrates that bear highly dynamic nucleobases, such as pyrimidines, on the displaced strand. Duplex substrates with identical thermodynamic stability are thus shown to be more easily unwound from one side than the other, in a quantifiable and predictable manner. Our results indicate a possible layer of gene regulation coded in the direction-dependent unwindability of the double helix.

The current report extends the facilitated diffusion model to account for conflict between the search and recognition binding modes adopted by DNA-binding proteins (DBPs) as they search DNA and subsequently recognize and bind to their specific binding site. The speed of the search dynamics is governed by the energetic ruggedness of the protein-DNA landscape, whereas the rate for the recognition process is mostly dictated by the free energy barrier for the transition between the DBP's search and recognition binding modes. We show that these two modes are negatively coupled, such that fast 1D sliding and rapid target site recognition probabilities are unlikely to coexist. Thus, a tradeoff occurs between optimizing the timescales for finding and binding the target site. We find that these two kinetic properties can be balanced to produce a fast timescale for the total target search and recognition process by optimizing frustration. Quantification of the facilitated diffusion model by including a frustration term enables it to explain several experimental observations concerning search and recognition speeds. The extended model captures experimental estimate of the energetic ruggedness of the protein-DNA landscape and predicts how various molecular properties of protein-DNA binding affect recognition kinetics. Particularly, point mutations may change the frustration and so affect protein association with DNA, thus providing a means to modulate protein-DNA affinity by manipulating the protein's association or dissociation reactions.

Lassa virus (LASV)is a notorious human pathogen in West Africa. Its class I trimeric spike complex displays a distinct architecture, and its cell entry mechanism involves unique attributes not shared by other related viruses. We determined the crystal structure of the GP2 fusion glycoprotein from the spike complex of LASV (GP2 _LASV )in its post-fusion conformation. GP2 _LASV adopts a canonical helical bundle configuration similarly to other viruses in its family. The core packing of GP2 _LASV , however, is more organized compared to GP2 from other viruses reducing the formation of internal hydrophobic cavities. We demonstrate a link between the formation of such unfavorable hydrophobic cavities and the efficiencies of membrane fusion and cell entry. Our study suggests that LASV has evolved a more efficient membrane fusogen compared to other viruses from its family by optimizing the post-fusion configuration of its GP2 module.

The alpha-helical coiled coil (CC) is a common protein motif that because of the simplicity of its sequence/structure relationship, it has been studied extensively to address fundamental questions in protein science as well as to develop strategies for designing protein with novel architectures. Nevertheless, a complete understanding of CC structures and their dynamics is still far from achieved. Particularly, spontaneous sliding at interfaces of CC proteins was observed for some systems, but its mechanism and usage as an intrinsic conformational change at CCs in protein-protein interfaces is unclear. Using coarse-grained and atomistic simulations, we study various sequences of homodimeric CC, in both parallel and antiparallel configurations. Both the strength of the hydrophobic core and the existence of salt bridges at the periphery of the interface affect sliding dynamics at the CC interface. Although the energy landscape for sliding along a CC interface is different for parallel and antiparallel configurations, both are characterized by a free energy of 1-1.5 kcal/mol, depending on the residues that constitute the CC interface. These barrier heights suggest that sliding kinetics is relatively slow in CC systems and are not expected to be of long length scale, yet they can be involved in functional motions. Our study explains the sliding that has been experimentally observed for the antiparallel CC of the dynein stalk region and the nuclear pore complex and suggests that this one-dimensional motion is an intrinsic feature in CC systems that can be involved in other CC systems.

Recognition of single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA) is important for many fundamental cellular functions. A variety of single-stranded DNA-binding proteins (ssDBPs) and single-stranded RNA-binding proteins (ssRBPs) have evolved that bind ssDNA and ssRNA, respectively, with varying degree of affinities and specificities to form complexes. Structural studies of these complexes provide key insights into their recognition mechanism. However, computational modeling of the specific recognition process and to predict the structure of the complex is challenging, primarily due to the heterogeneity of their binding energy landscape and the greater flexibility of ssDNA or ssRNA compared with double-stranded nucleic acids. Consequently, considerably fewer computational studies have explored interactions between proteins and single-stranded nucleic acids compared with protein interactions with double-stranded nucleic acids. Here, we report a newly developed energy-based coarse-grained model to predict the structure of ssDNA-ssDBP and ssRNA-ssRBP complexes and to assess their sequence-specific interactions and stabilities. We tuned two factors that can modulate specific recognition: base-aromatic stacking strength and the flexibility of the single-stranded nucleic acid. The model was successfully applied to predict the binding conformations of 12 distinct ssDBP and ssRBP structures with their cognate ssDNA and ssRNA partners having various sequences. Estimated binding energies agreed well with the corresponding experimental binding affinities. Bound conformations from the simulation showed a funnel-shaped binding energy distribution where the native-like conformations corresponded to the energy minima. The various ssDNA-protein and ssRNA-protein complexes differed in the balance of electrostatic and aromatic energies. The lower affinity of the ssRNA-ssRBP complexes compared with the ssDNA-ssDBP complexes stems from lower flexibility of ssRNA compared to ssDNA, which results in higher rate constants for the dissociation of the complex (k(off)) for complexes involving the former.Author summary Quantifying bimolecular self-assembly is pivotal to understanding cellular function. In recent years, a large progress has been made in understanding the structure and biophysics of protein-protein interactions. Particularly, various computational tools are available for predicting these structures and to estimate their stability and the driving forces of their formation. The understating of the interactions between proteins and nucleic acids, however, is still limited, presumably due to the involvement of non-specific interactions as well as the high conformational plasticity that may demand an induced-fit mechanism. In particular, the interactions between proteins and single-stranded nucleic acids (i.e., single-stranded DNA and RNA) is very challenging due to their high flexibility. Furthermore, the interface between proteins and single-stranded nucleic acids is often chemically more heterogeneous than the interface between proteins and double-stranded DNA. In this study, we developed a coarse-grained computational model to predict the structure of complexes between proteins and single-stranded nucleic acids. The model was applied to estimate binding affinities and the estimated binding energies agreed well with the corresponding experimental binding affinities. The kinetics of association as well as the specificity of the complexes between proteins and ssDNA are different than those with ssRNA, mostly due to differences in their conformational flexibility.

Protein backbone alternation due to insertion/deletion or mutation operation often results in a change of fundamental biophysical properties of proteins. The proposed work intends to encode the protein stability changes associated with single point deletions (SPDs) of amino acids in proteins. The encoding will help in the primary screening of detrimental backbone modifications before opting for expensive in vitro experimentations. In the absence of any benchmark database documenting SPDs, we curate a data set containing SPDs that lead to both folded conformations and unfolded state. We differentiate these SPD instances with the help of simple structural and physicochemical features and eventually classify the foldability resulting out of SPDs using a Random Forest classifier and an Elliptic Envelope based outlier detector. Adhering to leave one out cross validation, the accuracy of the Random Forest classifier and the Elliptic Envelope is of 99.4% and 98.1%, respectively. The newly defined database and the delineation of SPD instances based on its resulting foldability provide a head start toward finding a solution to the given problem.

Huntington's disease is a neurodegenerative disorder resulting from an expanded polyglutamine (polyQ) repeat of the Huntingtin (Htt) protein. Affected tissues often contain aggregates of the N-terminal Htt exon 1 (Htt-Ex1) fragment. The N-terminal N17 domain proximal to the polyQ tract is key to enhance aggregation and modulate Htt toxicity. Htt-Ex1 is intrinsically disordered, yet it has been postulated that under physiological conditions membranes induce the N17 to adopt an alpha-helical structure, which then plays a key role in regulating Htt protein aggregation. The present study leverages the recently available assignment of NMR peaks in an N17Q17 construct, in order to provide a look into the changes occurring in vitro upon exposing this fragment to various brain extract fragments as well as to synthetic bilayers. Residue-specific changes were observed by 3D HNCO NMR, whose nature was further clarified with ancillary CD and aggregation studies, as well as with molecular dynamic calculations. From this combination of measurements and computations, a unified picture emerges, whereby transient structures consisting of alpha-helices spanning a fraction of the N17 residues form during N17Q17-membrane interactions. These interactions are fairly dynamic, but they qualitatively mimic more rigid variants that have been discussed in the literature. The nature of these interactions and their potential influence on the aggregation process of these kinds of constructs under physiological conditions are briefly assessed.

Previous studies conducted on flexible loop regions in proteins revealed that the energetic consequences of changing loop length predominantly arise from the entropic cost of ordering a loop during folding. However, in an earlier study of human acylphosphatase (hmAcP) using experimental and computational approaches, we showed that thermodynamic stabilization upon loop truncation can be attributed mainly to the increased entropy of the folded state. Here, using ¹⁵N NMR spectroscopy, we studied the effect of loop truncation on hmAcP backbone dynamics on the picosecond-nanosecond timescale with the aim of confirming the effect of folded state entropy on protein stability. NMR-relaxation-derived N-H squared generalized order parameters reveal that loop truncation results in a significant increase in protein conformational flexibility. Comparison of these results with previously acquired all-atom molecular dynamics simulation, analyzed here in terms of squared generalized NMR order parameters, demonstrates general agreement between the two methods. The NMR study not only provides direct evidence for the enhanced conformational entropy of the folded state of hmAcP upon loop truncation but also gives a quantitative measure of the observed effects.

Predicting the effect of a single point mutation on protein thermodynamic stability (Delta Delta G) is an ongoing challenge with high relevance for both fundamental and applicable aspects of protein science. Drawbacks that limit the predictive power of stability prediction tools include the lack of representations for the explicit energetic terms of the unfolded state. Using coarse-grained simulations and analytical modeling analysis, we found that a mutation that involves the breaking of long-range contacts may lead to an increase in the unfolded state entropy, which can lead to an overall destabilization of the protein. A bioinformatics analysis indicates that the effect of mutation on the unfolded state is greater for hydrophobic or charged (compared with polar) residues that participate in long-range contacts through a loop length longer than 18 amino acids and whose formation probabilities are relatively high.

Entropy-enthalpy compensation is observed in many reactions, particularly for polymeric biomolecules that often involve large changes in entropy and enthalpy. The imperfect cancelation of entropy and enthalpy dictates many biophysical characteristics, such as protein thermodynamic stability and the free energy barrier for protein folding. In this study, we examine how tethering a conjugate to a protein may affect the thermodynamic stability of the protein. We found that a conjugate mostly affects the unfolded state by eliminating formation of some residual interactions. Consequently, both the enthalpy and the entropy of the unfolded state are affected. We suggest that, because this effect is not localized, the gain in conformational flexibility (i.e., increased entropy) is larger than the loss of some residual interaction (i.e., increased enthalpy). Therefore, the unfolded state of the conjugated protein has a lower free energy than that of the free protein, resulting in thermodynamic destabilization. (C) 2018 Elsevier B.V. All rights reserved.

Many mutations that cause familial hypercholesterolemia localize to ligand-binding domain 5 (LA5) of the low-density lipoprotein receptor, motivating investigation of the folding and misfolding of this small, disulfide-rich, calcium-binding domain. LA5 folding is known to involve non-native disulfide isomers, yet these folding intermediates have not been structurally characterized. To provide insight into these intermediates, we used nuclear magnetic resonance (NMR) to follow LA5 folding in real time. We demonstrate that misfolded or partially folded disulfide intermediates are indistinguishable from the unfolded state when focusing on the backbone NMR signals, which provide information on the formation of only the final, native state. However, ¹³C labeling of cysteine side chains differentiated transient intermediates from the unfolded and native states and reported on disulfide bond formation in real time. The cysteine pairings in a dominant intermediate were identified using ¹³C-edited three-dimensional NMR, and coarse-grained molecular dynamics simulations were used to investigate the preference of this disulfide set over other non-native arrangements. The transient population of LA5 species with particular non-native cysteine connectitivies during folding supports the conclusion that cysteine pairing is not random and that there is a bias toward certain structural ensembles during the folding process, even prior to the binding of calcium.

Several DNA-binding proteins, such as topoisomerases, helicases and sliding clamps, have a toroidal (i.e. ring) shape that topologically traps DNA, with this quality being essential to their function. Many DNA-binding proteins that function, for example, as transcription factors or enzymes were shown to be able to diffuse linearly (i.e. slide) along DNA during the search for their target binding sites. The protein's sliding properties and ability to search DNA, which often also involves hopping and dissociation, are expected to be different when it encircles the DNA. In this study, we explored the linear diffusion of four ring-shaped proteins of very similar structure: three sliding clamps (PCNA, beta-clamp, and the gp45) and the 9-1-1 protein, with a particular focus on PCNA. Coarse-grained molecular dynamics simulations were performed to decipher the sliding mechanism adopted by these ring-shaped proteins and to determine how the molecular properties of the inner and outer ring govern its search speed. We designed in silico variants to dissect the contributions of ring geometry and electrostatics to the sliding speed of ring-shaped proteins along DNA. We found that the toroidal proteins diffuse when they are tilted relative to the DNA axis and able to rotate during translocation, but that coupling between rotation and translocation is quite weak. Their diffusion speed is affected by the shape of the inner ring and, to a lesser extent, by its electrostatic properties. However, breaking the symmetry of the electrostatic potential can result in deviation of the DNA from the center of the ring and cause slower linear diffusion. The findings are discussed in light of earlier computational and experimental studies on the sliding of clamps.

Intersegmental transfer that involves direct relocation of a DNA-binding protein from one nonspecific DNA site to another was previously shown to contribute to speeding up the identification of the DNA target site. This mechanism is promoted when the protein is composed of at least two domains that have different DNA binding affinities and thus show a degree of mobility. In this study, we investigate the effect of particle crowding on the ability of a multi-domain protein to perform intersegmental transfer. We show that although crowding conditions often favor 1D diffusion of proteins along DNA over 3D diffusion, relocation of one of the tethered domains to initiate intersegmental transfer is possible even under crowding conditions. The tendency to perform intersegmental transfer by a multi-domain protein under crowding conditions is much higher for larger crowding particles than smaller ones and can be even greater than under no-crowding conditions. We report that the asymmetry of the two domains is even magnified by the crowders. The observations that crowding supports intersegmental transfer serve as another example that in vivo complexity does not necessarily slow down DNA search kinetics by proteins.

In prokaryotes, the RecA protein catalyzes the repair and strand exchange of double-stranded DNA. RecA binds to single-stranded DNA (ssDNA) and forms a presynaptic complex in which the protein polymerizes around the ssDNA to form a right-handed helical nucleoprotein filament structure. In the present work, the mechanism for the formation of the RecA-ssDNA filament structure is modeled using coarse-grained molecular dynamics simulations. Information from the X-ray structure was used to model the protein itself but not its interactions; the interactions between the protein and the ssDNA were modeled solely by electrostatic, aromatic, and repulsive energies. For the present study, the monomeric, dimeric, and trimeric units of RecA and 4, 8, and 11 NT-long ssDNA, respectively, were studied. Our results indicate that monomeric RecA is not sufficient for nucleoprotein filament formation; rather, dimeric RecA is the elementary binding unit, with higher multimeric units of RecA facilitating filament formation. Our results reveal that loop region flexibility at the primary binding site of RecA is essential for it to bind the incoming ssDNA, that the aromatic residues present in the loop region play an important role in ssDNA binding, and that ATP may play a role in guiding the ssDNA by changing the electrostatic potential of the RecA protein.

Proteins, like other biomolecules, have a modular and hierarchical structure. Various building blocks are used to construct proteins of high structural complexity and diverse functionality. In multidomain proteins, for example, domains are fused to each other in different combinations to achieve different functions. Although the LEGO brick metaphor is justified as a means of simplifying the complexity of three-dimensional protein structures, several fundamental properties (such as allostery or the induced-fit mechanism) make deviation from it necessary to respect the plasticity, softness, and cross-talk that are essential to protein function. In this work, we illustrate recently reported protein behavior in multidomain proteins that deviates from the LEGO brick analogy. While earlier studies showed that a protein domain is often unaffected by being fused to another domain or becomes more stable following the formation of a new interface between the tethered domains, destabilization due to tethering has been reported for several systems. We illustrate that tethering may sometimes result in a multidomain protein behaving as "less than the sum of its parts". We survey these cases for which structure additivity does not guarantee thermodynamic additivity. Protein destabilization due to fusion to other domains may be linked in some cases to biological function and should be taken into account when designing large assemblies.

Differential signaling of the type I interferon receptor (IFNAR) has been correlated with the ability of its subunit, IFNAR1, to differentially recognize a large spectrum of different ligands, which involves intricate conformational re-arrangements of multiple interacting domains. To shed light onto the structural determinants governing ligand recognition, we compared the force-induced unfolding of the IFNAR1 ectodomain when bound to interferon and when free, using the atomic force microscope and steered molecular dynamics simulations. Unexpectedly, we find that IFNAR1 is easier to mechanically unfold when bound to interferon than when free. Analysis of the structures indicated that the origin of the reduction in unfolding forces is a conformational change in IFNAR1 induced by ligand binding.

Interactions between water and biomolecules can significantly change the former's structural, dynamic, and thermodynamic properties relative to the bulk. Experimental, theoretical, and computational studies show that changes in water properties can be observed at distances of more than 10 angstrom from a biomolecule. The effects of biopolymers on hydration water molecules can be attributed to several factors: the chemical nature of the amino acid residues involved, the spatial arrangement of the biomolecule, and its conformational flexibility. In the current study, we concentrate on the effect of protein chain flexibility on the properties of hydration water, using short peptides as a model. We constructed 18 linear peptides with the sequence (XXGG) x 5, where X represents one of the common amino acids, other than glycine and proline. Using molecular dynamics (MD) simulations, we studied how restricting the chain flexibility can affect the structural, dynamic, and thermodynamic properties of hydration water. We found that restricting the peptide dynamics can slow down the translational motions of water molecules to a distance of at least 12-13 angstrom. Analysis of the 'slow' water molecules (residence time >= 100 ps) together with a thermodynamic analysis of water within 4.5 angstrom of the peptide revealed significant differences between the hydration properties of the peptides. The balance between the entropic and enthalpic solvation effects defines the final contribution to the hydration free energy of the restricted system. Our study implies that different regions of the proteins that have different configurational entropies may also have different solvation entropies and therefore different contributions to the overall thermodynamic stability. Therefore, mutations of a solvent exposed residue may modify the thermodynamic stability depending solely on the flexibility of the mutated sites due to their different solvation characteristics.

Rapid recognition by a protein of its DNA target site is achieved through a combination of one- and three-dimensional (1D and 3D) diffusion, which allows efficient scanning of the many alternative sites. This facilitated diffusion mechanism is expected to be affected by cellular conditions, particularly crowding, given that up to 40% of the total cellular volume may by occupied by macromolecules. Both experimental and theoretical studies showed that crowding particles can enhance facilitated diffusion and accelerate search kinetics. This effect may originate from crowding forcing a trade-off between 3D and 1D diffusion. In this study, using coarse-grained molecular dynamic simulations, we investigate how the molecular properties of the crowders may modulate the effect exerted by crowding on a searcher protein. We show that crowders with an affinity to the DNA are less effective search facilitators than particles whose contribution is solely entropic. Crowders that have affinity to DNA may occupy DNA sites and thereby function as obstacles or roadblocks that slow down the searcher protein, and they may also produce a smaller excluded volume effect and so reduce usage of the hopping searching mode in favor of less-effective 3D diffusion in the bulk. We discuss how strong repulsive interactions between the crowding particles themselves may affect the overall dynamics of the crowders and their excluded volume effect. Our study shows that search kinetics and its mechanism are modulated not only by salt concentration and crowding occupancy, but also by the properties of the crowding particles.

The key feature explaining the rapid recognition of a DNA target site by its protein lies in the combination of one- and three-dimensional (1D and 3D) diffusion, which allows efficient scanning of the many alternative sites. This facilitated diffusion mechanism is expected to be affected by cellular conditions, particularly crowding, given that up to 40% of the total cellular volume may by occupied by Macromolecules. Using coarse-grained molecular dynamics and Monte Carlo simulations, we show that the crowding particles can enhance facilitated diffusion and accelerate search kinetics. This effect originates from a trade-off between 3D and 1D diffusion. The 3D diffusion coefficient is lower under crowded conditions, but it has little influence because the excluded volume effect of molecular crowding restricts its use. Largely prevented from using 3D diffusion, the searching protein dramatically increases its use of the hopping search mode, which results in a higher linear diffusion coefficient. The coefficient of linear diffusion also increases under crowded conditions as a result of increased collisions between the crowding particles and the searching protein. Overall, less 3D diffusion coupled with an increase in the use of the hopping and speed of 1D diffusion :results in faster search kinetics Under crowded conditions. Our study shows, that the search kinetics and mechanism are modulated not only by the crowding occupancy but also by the properties of the crowding particles and the salt concentration.

Laminin, an ∼800-kDa heterotrimeric protein, is a major functional component of the extracellular matrix, contributing to tissue development and maintenance. The unique architecture of laminin is not currently amenable to determination at high resolution, as its flexible and narrowsegments complicate both crystallization and single-particle reconstruction by electronmicroscopy. Therefore, we used cross-linking and MS, evaluated using computational methods, to address key questions regarding laminin quaternary structure. This approach was particularly well suited to the ∼750-Å coiled coil that mediates trimer assembly, and our results support revision of the subunit order typically presented in laminin schematics. Furthermore, information on the subunit register in the coiled coil and cross-links to downstream domains provide insights into the self-assembly required for interaction with other extracellular matrix and cell surface proteins.

Understanding the molecular mechanism and the fast kinetics of DNA target site recognition by a protein is essential to decipher genetic activity in the cell. The speed of searching DNA may depend on the structural complexity of the proteins and the DNA molecules as well as the cellular environment. Coarse-grained (CG) molecular dynamics simulations are powerful means to investigate the molecular details of the search performed by protein to locate the target sites. Recent studies showed how different proteins scan DNA and how the search efficiency can be enhanced and regulated by the protein properties. In this review, we discuss computational approaches to study the physical chemistry of DNA search processes using CG molecular dynamics simulations and their advantage in covering long time-scale biomolecular processes. WIREs Comput Mol Sci 2016, 6:515531. doi: 10.1002/wcms.1262. For further resources related to this article, please visit the WIREs website.

Chirality-induced spin selectivity is a recently-discovered effect, which results in spin selectivity for electrons transmitted through chiral peptide monolayers. Here, we use this spin selectivity to probe the organization of self-assembled α-helix peptide monolayers and examine the relation between structural and spin transfer phenomena. We show that the α-helix structure of oligopeptides based on alanine and aminoisobutyric acid is transformed to a more linear one upon cooling. This process is similar to the known cold denaturation in peptides, but here the self-assembled monolayer plays the role of the solvent. The structural change results in a flip in the direction of the electrical dipole moment of the adsorbed molecules. The dipole flip is accompanied by a concomitant change in the spin that is preferred in electron transfer through the molecules, observed via a new solid-state hybrid organic-inorganic device that is based on the Hall effect, but operates with no external magnetic field or magnetic material.

Protein loops are essential structural elements that influence not only function but also protein stability and folding rates. It was recently reported that shortening a loop in the AcP protein may increase its native state conformational entropy. This effect on the entropy of the folded state can be much larger than the lower entropic penalty of ordering a shorter loop upon folding, and can therefore result in a more pronounced stabilization than predicted by polymer model for loop closure entropy. In this study, which aims at generalizing the effect of loop length shortening on native state dynamics, we use all-atom molecular dynamics simulations to study how gradual shortening a very long or solvent-exposed loop region in four different proteins can affect their stability. For two proteins, AcP and Ubc7, we show an increase in native state entropy in addition to the known effect of the loop length on the unfolded state entropy. However, for two permutants of SH3 domain, shortening a loop results only with the expected change in the entropy of the unfolded state, which nicely reproduces the observed experimental stabilization. Here, we show that an increase in the native state entropy following loop shortening is not unique to the AcP protein, yet nor is it a general rule that applies to all proteins following the truncation of any loop. This modification of the loop length on the folded state and on the unfolded state may result with a greater effect on protein stability.

Proteins form large macromolecular assemblies with RNA that govern essential molecular processes. RNA-binding proteins have often been associated with conformational flexibility, yet the extent and functional implications of their intrinsic disorder have never been fully assessed. Here, through large-scale analysis of comprehensive protein sequence and structure datasets we demonstrate the prevalence of intrinsic structural disorder in RNA-binding proteins and domains. We addressed their functionality through a quantitative description of the evolutionary conservation of disordered segments involved in binding, and investigated the structural implications of flexibility in terms of conformational stability and interface formation. We conclude that the functional role of intrinsically disordered protein segments in RNA-binding is two-fold: first, these regions establish extended, conserved electrostatic interfaces with RNAs via induced fit. Second, conformational flexibility enables them to target different RNA partners, providing multi-functionality, while also ensuring specificity. These findings emphasize the functional importance of intrinsically disordered regions in RNA-binding proteins.

Ubiquitination is one of the most common post-translational modifications of proteins, and mediates regulated protein degradation among other cellular processes. A fundamental question regarding the mechanism of protein ubiquitination is whether and how ubiquitin affects the biophysical nature of the modified protein. For some systems, it was shown that the position of ubiquitin within the attachment site is quite flexible and ubiquitin does not specifically interact with its substrate. Nevertheless, it was revealed that polyubiquitination can decrease the thermal stability of the modified protein in a site-specific manner because of alterations of the thermodynamic properties of the folded and unfolded states. In this study, we used detailed atomistic simulations to focus on the molecular effects of ubiquitination on the native structure of the modified protein. As a model, we used Ubc7, which is an E2 enzyme whose in vivo ubiquitination process is well characterized and known to lead to degradation. We found that, despite the lack of specific direct interactions between the ubiquitin moiety and Ubc7, ubiquitination decreases the conformational flexibility of certain regions of the substrate Ubc7 protein, which reduces its entropy and thus destabilizes it. The strongest destabilizing effect was observed for systems in which Lys48-linked tetra-ubiquitin was attached to sites used for in vivo degradation. These results reveal how changes in the configurational entropy of the folded state may modulate the stability of the protein's native state. Overall, our results imply that ubiquitination can modify the biophysical properties of the attached protein in the folded state and that, in some proteins, different ubiquitination sites will lead to different biophysical outcomes. We propose that this destabilizing effect of polyubiquitin on the substrate is linked to the functions carried out by the modification, and in particular, regulatory control of protein half-life through proteasomal degradation.

The Engrailed Homeodomain (EnHD) transcription factor of Drosophila melanogaster was fused to the enhanced green fluorescent protein (eGFP) either at its C- or N-terminus via three- or ten-residue flexible linkers. Here, we show that EnHD undergoes destabilization upon fusing it to eGFP regardless of the linker length used and whether the tethering is to its N- or C-terminus. The destabilization is reflected in melting points that are lower by up to 9°C. Thermodynamic analysis and coarse-grained molecular dynamic simulations indicate that this destabilization is due to eGFP-promoted entropic stabilization of the denatured state ensemble of EnHD. Our results provide, therefore, an example for destabilizing interdomain allostery. They are also important given the widespread use of eGFP tagging in cell biology, as they indicate that such tagging can cause unintended protein destabilization and concomitant effects.

Glycosylation plays not only a functional role but can also modify the biophysical properties of the modified protein. Usually, natural glycosylation results in protein stabilization; however, in vitro and in silico studies showed that sometimes glycosylation results in thermodynamic destabilization. Here, we applied coarse-grained and all-atom molecular dynamics simulations to understand the mechanism underlying the loss of stability of the MM1 protein by glycosylation. We show that the origin of the destabilization is a conformational distortion of the protein caused by the interaction of the monosaccharide with the protein surface. Though glycosylation creates new short-range glycan-protein interactions that stabilize the conjugated protein, it breaks long-range protein-protein interactions. This has a destabilizing effect because the probability of long- and short-range interactions forming differs between the folded and unfolded states. The destabilization originates not from simple loss of interactions but due to a trade-off between the short- and long-range interactions.

Although engineering of transcription factors and DNA-modifying enzymes has drawn substantial attention for artificial gene regulation and genome editing, most efforts focus on affinity and specificity of the DNA-binding proteins, typically overlooking the kinetic properties of these proteins. However, a simplistic pursuit of high affinity can lead to kinetically deficient proteins that spend too much time at nonspecific sites before reaching their targets on DNA. We demonstrate that structural dynamic knowledge of the DNA-scanning process allows for kinetically and thermodynamically balanced engineering of DNA-binding proteins. Our current study of the zinc-finger protein Egr-1 (also known as Zif268) and its nuclease derivatives reveals kinetic and thermodynamic roles of the dynamic conformational equilibrium between two modes during the DNAscanning process: one mode suitable for search and the other for recognition. By mutagenesis, we were able to shift this equilibrium, as confirmed by NMR spectroscopy. Using fluorescence and biochemical assays as well as computational simulations, we analyzed how the shifts of the conformational equilibrium influence binding affinity, target search kinetics, and efficiency in displacing other proteins from the target sites. A shift toward the recognition mode caused an increase in affinity for DNA and a decrease in search efficiency. In contrast, a shift toward the search mode caused a decrease in affinity and an increase in search efficiency. This accelerated site-specific DNA cleavage by the zinc-finger nuclease, without enhancing off-target cleavage. Our study shows that appropriate modulation of the dynamic conformational ensemble can greatly improve zinc-finger technology, which has used Egr-1 (Zif268) as a major scaffold for engineering.

Many novel applications in bioelectronics rely on the interaction between biomolecules and electronically conducting substrates. However, crucial knowledge about the relation between electronic transport via peptides and their amino-acid composition is still absent. Here, we report results of electronic transport measurements via several homopeptides as a function of their structural properties and temperature. We demonstrate that the conduction through the peptide depends on its length and secondary structure as well as on the nature of the constituent amino acid and charge of its residue. We support our experimental observations with high-level electronic structure calculations and suggest off-resonance tunneling as the dominant conduction mechanism via extended peptides. Our findings indicate that both peptide composition and structure can affect the efficiency of electronic transport across peptides.

ssDNA binding proteins (SSBs) protect ssDNA from chemical and enzymatic assault that can derail DNA processing machinery. Complexes between SSBs and ssDNA are often highly stable, but predicting their structures is challenging, mostly because of the inherent flexibility of ssDNA and the geometric and energetic complexity of the interfaces that it forms. Here, we report a newly developed coarse-grained model to predict the structure of SSB-ssDNA complexes. The model is successfully applied to predict the binding modes of six SSBs with ssDNA strands of lengths of 6-65 nt. In addition to charge-charge interactions (which are often central to governing protein interactions with nucleic acids by means of electrostatic complementarity), an essential energetic term to predict SSB-ssDNA complexes is the interactions between aromatic residues and DNA bases. For some systems, flexibility is required from not only the ssDNA but also, the SSB to allow it to undergo conformational changes and the penetration of the ssDNA into its binding pocket. The association mechanisms can be quite varied, and in several cases, they involve the ssDNA sliding along the protein surface. The binding mechanism suggests that coarse-grained models are appropriate to study the motion of SSBs along ssDNA, which is expected to be central to the function carried out by the SSBs.

Water molecules are abundant in protein-DNA interfaces, especially in their nonspecific complexes. In this study, we investigated the organization and energetics of the interfacial water by simplifying the geometries of the proteins and the DNA to represent them as two equally and oppositely charged planar surfaces immersed in water. We found that the potential of mean force for bringing the two parallel surfaces into close proximity comprises energetic barriers whose properties strongly depend on the charge density of the surfaces. We demonstrated how the organization of the water molecules into discretized layers and the corresponding energetic barriers to dehydration can be modulated by the charge density on the surfaces, salt, and the structure of the surfaces. The 1-2 layers of ordered water are tightly bound to the charged surfaces representing the nonspecific protein-DNA complex. This suggests that water might mediate one-dimensional diffusion of proteins along DNA (sliding) by screening attractive electrostatic interactions between the positively charged molecular surface on the protein and the negatively charged DNA backbone and, in doing so, reduce intermolecular friction in a manner that smoothens the energetic landscape for sliding, and facilitates the 1D diffusion of the protein.

PEGylation of protein side chains has been used for more than 30 years to enhance the pharmacokinetic properties of protein drugs. However, there are no structure- or sequence-based guidelines for selecting sites that provide optimal PEG-based pharmacokinetic enhancement with minimal losses to biological activity. We hypothesize that globally optimal PEGylation sites are characterized by the ability of the PEG oligomer to increase protein conformational stability; however, the current understanding of how PEG influences the conformational stability of proteins is incomplete. Here we use the WW domain of the human protein Pin 1 (WW) as a model system to probe the impact of PEG on protein conformational stability. Using a combination of experimental and theoretical approaches, we develop a structure-based method for predicting which sites within WW are most likely to experience PEG-based stabilization, and we show that this method correctly predicts the location of a stabilizing PEGylation site within the chicken Src SH3 domain. PEG-based stabilization in WW is associated with enhanced resistance to proteolysis, is entropic in origin, and likely involves disruption by PEG of the network of hydrogen-bound solvent molecules that surround the protein. Our results highlight the possibility of using modern site-specific PEGylation techniques to install PEG oligomers at predetermined locations where PEG will provide optimal increases in conformational and proteolytic stability.

The recognition of DNA-binding proteins (DBPs) to their specific site often precedes by a search technique in which proteins slide, hop along the DNA contour or perform inter-segment transfer and 3D diffusion to dissociate and re-associate to distant DNA sites. In this study, we demonstrated that the strength and nature of the non-specific electrostatic interactions, which govern the search dynamics of DBPs, are strongly correlated with the conformation of the DNA. We tuned two structural parameters, namely curvature and the extent of helical twisting in circular DNA. These two factors are mutually independent of each other and can modulate the electrostatic potential through changing the geometry of the circular DNA conformation. The search dynamics for DBPs on circular DNA is therefore markedly different compared with linear B-DNA. Our results suggest that, for a given DBP, the rotation-coupled sliding dynamics is precluded in highly curved DNA (as well as for over-twisted DNA) because of the large electrostatic energy barrier between the inside and outside of the DNA molecule. Under such circumstances, proteins prefer to hop in order to explore interior DNA sites. The change in the balance between sliding and hopping propensities as a function of DNA curvature or twisting may result in different search efficiency and speed.

Multidomain transcription factors, which are especially abundant in eukaryotic genomes, are advantageous to accelerate the search kinetics for target site because they can follow the intersegment transfer via the monkey-bar mechanism in which the protein forms a bridged intermediate between two distant DNA regions. Monkey-bar dynamics highly depends on the properties of the multidomain protein (the affinity of each of the constituent domains to the DNA and the length of the linker) and the DNA molecules (their inter-distance and inter-angle). In this study, we investigate using coarse-grained molecular dynamics simulations how the local conformation of the DNA may affect the DNA search performed by a multidomain protein Pax6 in comparison to that of the isolated domains. Our results suggest that in addition to the common rotation-coupled translation along the DNA major groove, for curved DNA the tethered domains may slide in a rotation-decoupled sliding mode. Furthermore, themultidomain proteins move by longer jumps on curved DNA compared with those performed by the single domain protein. The long jumps originate from the DNA curvature bringing two sequentially distant DNA sites into close proximity with each other and they suggest that multidomain proteins may move on highly curved DNA faster than linear DNA.

DNA recognition by DNA-binding proteins, which is a pivotal event in most gene regulatory processes, is often preceded by an extensive search for the correct site. A facilitated diffusion process, in which a DBP combines 3D diffusion in solution with 1D sliding along DNA, has been suggested to explain how proteins can locate their target sites on DNA much faster than predicted by 3D diffusion alone. One of the key mechanisms in the localization of the target by a DNA-binding protein is intersegment transfer in which the protein forms a bridged intermediate between two distant DNA regions. This jumping mechanism is more enhanced when the DNA-binding protein is asymmetric in its structure or its dynamics. We suggest that asymmetry supports the \u201cmonkey bar\u201d mechanism, in which different domains of the protein interact with different DNA regions. In this minireview, we discuss how the molecular architectures of the proteins and DNA may modulate the efficiency of monkey bar dynamics.

A fundamental step in gene-regulatory activities, such as repression, transcription, and recombination, is the binding of regulatory DNA-binding proteins (DBPs) to specific targets in the genome. To rapidly localize their regulatory genomic sites, DBPs reduce the dimensionality of the search space by combining three-dimensional (3D) diffusion in solution with one-dimensional (1D) sliding along DNA. However, the requirement to form a thermodynamically stable protein-DNA complex at the cognate genomic target sequence imposes a challenge on the protein because, as it navigates one-dimensionally along the genome, it may come in close contact with sites that share partial or even complete sequence similarity with the functional DNA sequence. This puzzling issue creates a conflict between two basic requirements: finding the cognate site quickly and stably binding it. Here, we structurally assessed the interface adopted by a variety of DBPs to bind DNA specifically and nonspecifically, and found that many DBPs utilize one interface to specifically recognize a DNA sequence and another to assist in propagating along the DNA through nonspecific associations. While these two interfaces overlap each other in some proteins, they present partial overlap in others and frustrate the protein-DNA interface. Using coarse-grained molecular dynamics simulations, we demonstrate that the existence of frustration in DBPs is a compromise between rapid 1D diffusion along other regions in the genome (high frustration smoothens the landscape for sliding) and rapid formation of a stable and essentially active protein-DNA complex (low frustration reduces the free energy barrier for switching between the two binding modes).

We focus on dimeric DNA-binding proteins from two well-studied families: orthodox type II restriction endonucleases (REs) and transcription factors (TFs). Interactions of the protein's recognition sites with the DNA and, particularly, the contribution of each of the monomers to one-dimensional (1D) sliding along nonspecific DNA were studied using computational tools. Coarse-grained molecular dynamics simulations of DNA scanning by various TFs and REs provide insights into how the symmetry of a homodimer can be broken while they nonspecifically interact with DNA. The characteristics of protein sliding along DNA, such as the average sliding length, partitioning between 1D and 3D search, and the one-dimensional diffusion coefficient D₁, strongly depend on the salt concentration, which in turn affects the probability of the two monomers adopting a cooperative symmetric sliding mechanism. Indeed, we demonstrate that maximal DNA search efficiency is achieved when the protein adopts an asymmetric search mode in which one monomer slides while its partner hops. We find that proteins classified as TFs have a higher affinity for the DNA, longer sliding lengths, and an increased probability of symmetric sliding in comparison with REs. Moreover, TFs can perform their biological function over a much wider range of salt concentrations than REs. Our results demonstrate that the different biological functions of DNA-binding proteins are related to the different nonspecific DNA search mechanisms they adopt.

Entropic stabilization of native protein structures typically relies on strategies that serve to decrease the entropy of the unfolded state. Here we report, using a combination of experimental and computational approaches, on enhanced thermodynamic stability conferred by an increase in the configurational entropy of the folded state. The enhanced stability is observed upon modifications of a loop region in the enzyme acylphosphatase and is achieved despite significant enthalpy losses. The modifications that lead to increased stability, as well as those that result in destabilization, however, strongly compromise enzymatic activity, rationalizing the preservation of the native loop structure even though it does not provide the protein with maximal stability or kinetic foldability.

DNA recognition by DNA-binding proteins (DBPs), which is a pivotal event in most gene regulatory processes, is often preceded by an extensive search for the correct site. A facilitated diffusion process in which a DBP combines three-dimensional diffusion in solution with one-dimensional sliding along DNA has been suggested to explain how proteins can locate their target sites on DNA much faster than predicted by three-dimensional diffusion alone. Although experimental and theoretical studies have recently advanced understanding of the biophysical principles underlying the search mechanism, the process under in vivo cellular conditions is poorly understood. In this study, we used various computational approaches to explore how the presence of obstacle proteins on the DNA influences search efficiency. At a low obstacle occupancy (i.e., when few obstacles occupy sites on the DNA), sliding by the searching DBP may be confined, which may impair search efficiency. The obstacles, however, can be bypassed during hopping events, and the number of bypasses is larger for higher obstacle occupancies. Dynamism on the part of the obstacles may even further facilitate search kinetics. Our study shows that the nature and efficiency of the search process may be governed not only by the intrinsic properties of the DBP and the salt concentration of the medium, but also by the in vivo association of DNA with other macromolecular obstacles, their location, and occupancy.

Extensive contact with DNA via multiple zinc fingers allows highly specific DNA-binding of zinc-finger-class transcription factors, but can also slow the target search process. Here we introduce recent insights into how zinc-finger proteins can rapidly scan DNA. Potential application of the new knowledge to the zinc-finger-based technology is also discussed.

Most eukaryotic and a substantial fraction of prokaryotic proteins are composed of more than one domain. The tethering of these evolutionary, structural, and functional units raises, among others, questions regarding the folding process of conjugated domains. Studying the folding of multidomain proteins in silico enables one to identify and isolate the tethering-induced biophysical determinants that govern crosstalks generated between neighboring domains. For this purpose, we carried out coarse-grained and atomistic molecular dynamics simulations of two two-domain constructs from the immunoglobulin-like β-sandwich fold. Each of these was experimentally shown to behave as the "sum of its parts," that is, the thermodynamic and kinetic folding behavior of the constituent domains of these constructs seems to occur independently, with the folding of each domain uncoupled from the folding of its partner in the two-domain construct. We show that the properties of the individual domains can be significantly affected by conjugation to another domain. The tethering may be accompanied by stabilizing as well as destabilizing factors whose magnitude depends on the size of the interface, the length, and the flexibility of the linker, and the relative stability of the domains. Accordingly, the folding of a multidomain protein should not be viewed as the sum of the folding patterns of each of its parts, but rather, it involves abrogating several effects that lead to this outcome. An imbalance between these effects may result in either stabilization or destabilization owing to the tethering. Proteins 2012;

Poorly structured domains in proteins enhance their susceptibility to proteasomal degradation. To learn whether the presence of such a domain near either end of a protein determines its direction of entry into the proteasome, directional translocation was enforced on several proteasome substrates. Using archaeal PAN-20S complexes, mammalian 26S proteasomes, and cultured cells, we identified proteins that are degraded exclusively from either the C or N terminus and some showing no directional preference. This property results from interactions of the substrate's termini with the regulatory ATPase and could be predicted based on the calculated relative stabilities of the N and C termini. Surprisingly, the direction of entry into the proteasome affected markedly the spectrum of peptides released and consequently influenced the efficiency of MHC class I presentation. Thus, easily unfolded termini are translocated first, and the direction of translocation influences the peptides generated and presented to the immune system.

We present an integrated experimental and computational study of the molecular mechanisms by which myristoylation affects protein folding and function, which has been little characterized to date. Myristoylation, the covalent linkage of a hydrophobic C14 fatty acyl chain to the N-terminal glycine in a protein, is a common modification that plays a critical role in vital regulated cellular processes by undergoing reversible energetic and conformational switching. Coarse-grained folding simulations for the model pH-dependent actin- and membrane-binding protein hisactophilin reveal that nonnative hydrophobic interactions of the myristoyl with the protein as well as nonnative electrostatic interactions have a pronounced effect on folding rates and thermodynamic stability. Folding measurements for hydrophobic residue mutations of hisactophilin and atomistic simulations indicate that the nonnative interactions of the myristoyl group in the folding transition state are nonspecific and robust, and so smooth the energy landscape for folding. In contrast, myristoyl interactions in the native state are highly specific and tuned for sensitive control of switching functionality. Simulations and amide hydrogen exchange measurements provide evidence for increases as well as decreases in stability localized on one side of the myristoyl binding pocket in the protein, implicating strain and altered dynamics in switching. The effects of folding and function arising from myristoylation are profoundly different from the effects of other post-translational modifications.

Repeat proteins have unique elongated structures that, unlike globular proteins, are quite modular. Despite their simple one-dimensional structure, repeat proteins exhibit intricate folding behavior with a complexity similar to that of globular proteins. Therefore, repeat proteins allow one to quantify fundamental aspects of the biophysics of protein folding. One important feature of repeat proteins is the interfaces between the repeating units. In particular, the distribution of stabilities within and between the repeats was previously suggested to affect their folding characteristics. In this study, we explore how the interface affects folding kinetics and cooperativity by investigating two families of repeat proteins, namely, the Ankyrin and tetratricopeptide repeat proteins, which differ in the number of interfacial contacts that are formed between their units as well as in their folding behavior. By using simple topology-based models, we show that modulating the energetic strength of the interface relative to that of the repeat itself can drastically change the protein stability, folding rate, and cooperativity. By further dissecting the interfacial contacts into several subsets, we isolated the effects of each of these groups on folding kinetics. Our study highlights the importance of interface connectivity in determining the folding behavior.

Protein ubiquitination is central to the regulation of various pathways in eukaryotes. The process of ubiquitination and its cellular outcome were investigated in hundreds of proteins to date. Despite this, the evolution of this regulatory mechanism has not yet been addressed comprehensively. Here, we quantify the rates of evolutionary changes of ubiquitination and SUMOylation (Small Ubiquitin-like MOdifier) sites. We estimate the time at which they first appeared, and compare them to acetylation and phosphorylation sites and to unmodified residues. We observe that the various modification sites studied exhibit similar rates. Mammalian ubiquitination sites are weakly more conserved than unmodified lysine residues, and a higher degree of relative conservation is observed when analyzing bona fide ubiquitination sites. Various reasons can be proposed for the limited level of excess conservation of ubiquitination, including shifts in locations of the sites, the presence of alternative sites, and changes in the regulatory pathways. We observe that disappearance of sites may be compensated by the presence of a lysine residue in close proximity, which is significant when compared to evolutionary patterns of unmodified lysine residues, especially in disordered regions. This emphasizes the importance of analyzing a window in the vicinity of functional residues, as well as the capability of the ubiquitination machinery to ubiquitinate residues in a certain region. Using prokaryotic orthologs of ubiquitinated proteins, we study how ubiquitination sites were formed, and observe that while sometimes sequence additions and rearrangements are involved, in many cases the ubiquitination machinery utilizes an already existing sequence without significantly changing it. Finally, we examine the evolution of ubiquitination, which is linked with other modifications, to infer how these complex regulatory modules have evolved. Our study gives initial insights into the formation of ubiquitination sites, their degree of conservation in various species, and their co-evolution with other posttranslational modifications.

Egr-1 is an inducible transcription factor that recognizes 9-bp target DNA sites via three zinc finger domains and activates genes in response to cellular stimuli such as synaptic signals and vascular stresses. Using spectroscopic and computational approaches, we have studied structural, dynamic, and kinetic aspects of the DNAscanning process in which Egr-1 is nonspecifically bound to DNA and perpetually changes its location on DNA. Our NMR data indicate that Egr-1 undergoes highly dynamic domain motions when scanning DNA. In particular, the zinc finger 1 (ZF1) of Egr-1 in the nonspecific complex is mainly dissociated fromDNA and undergoes collective motions on a nanosecond timescale, whereas zinc fingers 2 and 3 (ZF2 and ZF3, respectively) are bound to DNA. This was totally unexpected because the previous crystallographic studies of the specific complex indicated that all of Egr-1's three zinc fingers are equally involved in binding to a target DNA site. Mutations that are expected to enhance ZF1's interactions with DNA and with ZF2 were found to reduce ZF1's domain motions in the nonspecific complex suggesting that these interactions dictate the dynamic behavior of ZF1. By experiment and computation, we have also investigated kinetics of Egr-1's translocation between two nonspecific DNA duplexes. Our data on the wild type and mutant proteins suggest that the domain dynamics facilitate Egr-1's intersegment transfer that involves transient bridging of two DNA sites. These results shed light on asymmetrical roles of the zinc finger domains for Egr-1 to scan DNA efficiently in the nucleus.

Intrinsically disordered regions, terminal tails, and flexible linkers are abundant in DNA-binding proteins and play a crucial role by increasing the affinity and specificity of DNA binding. Disordered tails often undergo a disorder-to-order transition during interactions with DNA and improve both the kinetics and thermodynamics of specific DNA binding. The DNA search by proteins that interact nonspecifically with DNA can be supported by disordered tails as well. The disordered tail may increase the overall protein-DNA interface and thus increase the affinity of the protein to the DNA and its sliding propensity while slowing linear diffusion. The exact effect of the disordered tails on the sliding rate depends on the degree of positive charge clustering, as has been shown for homeodomains and p53 transcription factors. The disordered tails, which may be viewed as DNA recognizing subdomains, can facilitate intersegment transfer events that occur via a "monkey bar" mechanism in which the domains bridge two different DNA fragments simultaneously. The "monkey bar" mechanism can be facilitated by internal disordered linkers in multidomain proteins that mediate the cross-talks between the constituent domains and especially their brachiation dynamics and thus their overall capability to search DNA efficiently. The residue sequence of the disordered tails has unique characteristics that were evolutionarily selected to achieve the optimized function that is unique to each protein. Perturbation of the electrostatic characteristics of the disordered tails by post-translational modifications, such as acetylation and phosphorylation, may affect protein affinity to DNA and therefore can serve to regulate DNA recognition. Modifying the disordered protein tails or the flexibility of the inter-domain linkers of multidomain proteins may affect the cross-talk between the constituent domains so as to facilitate the search kinetics of non-specific DNA sequences and increase affinity to the specific sequences.

DNA-binding proteins execute many different genetic tasks, such as transcription, repression, replication, and the repair of damaged DNA. Given the size of the genome, the large number of molecular species in vivo, and the crowded environment in which these processes take place, the remarkable efficiency and specificity of proteinDNA recognition present a major theoretical puzzle. In particular, two fundamental issues of great interest in protein DNA recognition are: how proteins achieve a high degree of selectivity among a huge background of competing nonspecific DNA sequences and the aspect of the rapid recognition rate of the DNA target sequence by the protein.

Intrinsically disordered regions, particularly disordered tails, are very common in DNA-binding proteins (DBPs). The ability of disordered tails to modulate specific and nonspecific interactions with DNA is tightly linked to their being rich in positively charged residues that are often non-randomly distributed along the tail. Perturbing the composition and distribution of charged residues in the disordered regions by post-translational modifications, such as phosphorylation and acetylation, may impair the ability of the tail to interact nonspecifically with DNA by reducing its DNA affinity. In this study, we analyzed datasets of 3398 and 8943 human proteins that undergo acetylation or phosphorylation, respectively. Both modifications are common on the disordered tails of DBPs (3.1 ± 0.2 (0.07 ± 0.007) and 2.0 ± 0.2 (0.02 ± 0.003) acetylation and phosphorylation sites per tail (per tail residue), respectively). Phosphorylation sites are abundant in disordered regions and particularly in flexible tails for both DBPs and non-DBPs. While acetylation sites are also frequently occurred in the disordered tails of DBPs, in non-DBPs they are often found in ordered regions. This difference may indicate that acetylation has different function in DBPs and non-DBPs. Post-translational modifications, which often take place at disordered sites of DBPs, can modulate the interactions of proteins with DNA by changing the local and global properties of the tails. The effect of the modulation can be tuned by adjusting the number of modifications and the cross-talks between them.

Abstract The following sections are included: Introduction Methods Statistical analysis of acetylation and phosphorylation in DNA-binding proteins Simulation model for searching the DNA of modified proteins Results The occurrence of acetylation and phosphorylation at disordered regions The occurrence of acetylation and phosphorylation at disordered tails The interplay between post-translational modifications of disordered Conclusions Acknowledgments

Rapid recognition of DNA target sites involves facilitated diffusion through which alternative sites are searched on genomic DNA. A key mechanism facilitating the localization of the target by a DNA-binding protein (DBP) is one-dimensional diffusion (sliding) in which electrostatic forces attract the protein to the DNA. As the protein reaches its target DNA site, it switches frompurely electrostatic binding to a specific set of interactions with the DNA bases that also involves hydrogen bonding and van der Waals forces. High overlap between the DBP patches used for nonspecific and specific interactions with DNA may enable an immediate transition between the two binding modes following target site localization. By contrast, an imperfect overlap may result in greater frustration between the two potentially competing binding modes and consequently slower switching between them. A structural analysis of 125 DBPs indicates frustration between the two binding modes that results in a large difference between the orientations of the protein to the DNA when it slides compared to when it specifically interacts with DNA. Coarse-grained molecular dynamics simulations of in silico designed peptides comprising the full range of frustrations between the two interfaces show slower transition from nonspecific to specific DNA binding as the overlap between the patches involved in the two binding modes decreases. The complex search kinetics may regulate the search by eliminating trapping of the protein in semispecific sites while sliding.

Conjugating flexible polymers (such as oligosaccharides) to proteins or confining a protein in a restricted volume often increases protein thermal stability. In this communication, we investigate the interplay between conjugation and confinement which is not trivial as the magnitude and the mechanism of stabilization are different in each instance. Using coarse-grained computational approach the folding biophysics is studied when the protein is placed in a sphere of variable radius and is conjugated to 0-6 mono- or penta-saccharides. We observe a synergistic effect on thermal stability when short oligosaccharides are attached and the modified protein is confined in a small cage. However, when large oligosaccharides are added, a conflict between confinement and glycosylation arises as the stabilizing effect of the cage is dramatically reduced and it is almost impossible to further stabilize the protein beyond the mild stabilization induced by the sugars.

The ubiquitin-proteasome system is responsible for the degradation of numerous proteins in eukaryotes. Degradation is an essential process in many cellular pathways and involves the proteasome degrading a wide variety of unrelated substrates while retaining specificity in terms of its targets for destruction and avoiding unneeded proteolysis. How the proteasome achieves this task is the subject of intensive research. Many proteins are targeted for degradation by being covalently attached to a poly-ubiquitin chain. Several studies have indicated the importance of a disordered region for efficient degradation. Here, we analyze a data set of 482 in vivo ubiquitinated substrates and a subset in which ubiquitination is known to mediate degradation. We show that, in contrast to phosphorylation sites and other regulatory regions, ubiquitination sites do not tend to be located in disordered regions and that a large number of substrates are modified at structured regions. In degradation-mediated ubiquitination, there is a significant bias of ubiquitination sites to be in disordered regions; however, a significant number is still found in ordered regions. Moreover, in many cases, disordered regions are absent from ubiquitinated substrates or are located far away from the modified region. These surprising findings raise the question of how these proteins are successfully unfolded and ultimately degraded by the proteasome. They indicate that the folded domain must be perturbed by some additional factor, such as the p97 complex, or that ubiquitination may induce unfolding.

The p53 protein is a homotetrameric transcription factor whose monomers comprise several domains. Although its organization with and without DNA was elucidated recently, characterizing the p53-DNA complex at the atomic level remains challenging because of its many disordered regions. Here we use computational models to predict the wiring of the four chains composing p53 and study its sliding dynamics along DNA in different oligomeric states. We find that helical sliding along the major groove is the most feasible DNA search mechanism for a large range of salt concentrations. Tighter packing of the tetrameric core domain is associated with a greater nonspecific affinity for DNA and the slowest linear diffusion dynamics along DNA. C-tails facilitate linear diffusion but restrict the association of two primary dimers into a tetramer. This restriction can disappear at higher salt concentrations, which decrease the affinity of C-tails for DNA, or upon interaction of the C-tail with other DNA segments. Our results support evidence for the positive regulation of p53 function by the C-tails and suggest that posttranslational charge modifications may alter the affinity of the tails for DNA. Conversely, the N-termini have little effect on sliding characteristics. Changes in the electrostatic potentials of the core domain via missense mutations corresponding to cancer development can also affect sliding by p53. Our study provides molecular insight into the role of various p53 domains during DNA search and indicates that the complex interdomain and protein-DNA cross-talks in which p53 engages may be related to its repertoire of cellular functions.

Cystine disulfide bond is a common feature in numerous biologically active peptides and proteins and accordingly its replacement by various surrogates presents a potential route to obtain analogs with improved pharmacokinetic characteristics. The purpose of the present study was to assess whether an azo-bridge can serve as such a surrogate. In view of the marked clinical significance of somatostatin and the brain natriuretic peptide (BNP) we choose these peptides as a model. Three cyclic-azo somatostatin analogs and three cyclic-azo BNP analogs were effectively prepared in solution through azo bond formation between p-amino phenylalanine and His or Tyr residues that were positioned in the peptide sequences in place of the native Cys residues. The peptides binding affinities to the sst₂ and ANP-receptor (NPR-A) expressed on rat acinar pancreating carcinoma AR4-2J cell membranes and HeLa cells, respectively, were examined. The somatostatin analogs displayed good to moderate affinities to the rat sst₂ in the nM range with best results obtained with peptide 2, that is, IC₅₀ = 8.1 nM. Molecular dynamics simulations on these peptides suggests on a correlation between the observed binding potencies and the degree of conformational space overlapping with that of somatostatin. The BNP analogs exhibited binding affinities to the NPR-A in the nM range with best results obtained with BNP-1, that is, IC₅₀ = 60 nM.

Intrinsically disordered tails are common in DNA-binding proteins and can affect their search efficiency on nonspecific DNA by promoting the brachiation dynamics of intersegment transfer. During brachiation, the protein jumps between distant DNA regions via an intermediate state in which the tail and globular moieties are bound to different DNA segments. While the disordered tail must be long and positively charged to facilitate DNA search, the effect of its residue sequence on brachiation is unknown. We explored this issue using the NK-2 and Antp homeodomain transcription factors. We designed 566 NK-2 tail-variants and 55 Antp tailvariants having different net charges and positive charge distributions and studied their dynamics and DNA search efficiencies using coarse-grained molecular dynamics simulations. More intersegment transfers occur when the tail is moderately positively charged and the positive charges are clustered together in the middle of the tail or towards its N terminus. The presence of a negatively charged residue does not significantly affect protein brachiation, although it is likely that the presence of many negatively charged residues will complicate the DNA search mechanism. A bioinformatic analysis of 1,384 wild-type homeodomains illustrates that the charge composition and distribution in their N-tail sequences are consistent with an optimal charge pattern to promote intersegment transfer. Our study thus indicates that the residue sequence of the disordered tails of DNA-binding proteins has unique characteristics that were evolutionarily selected to achieve optimized function and suggests that the sequence-structure-function paradigm known for structured proteins is valid for intrinsically disordered proteins as well.

Asparagine glycosylation is one of the most common and important post-translational modifications of proteins in eukaryotic cells. N-Glycosylation occurs when a triantennary glycan precursor is transferred en bloc to a nascent polypeptide (harboring the N-X-T/S sequon) as the peptide is cotranslationally translocated into the endoplasmic reticulum (ER). In addition to facilitating binding interactions with components of the ER proteostasis network, N-glycans can also have intrinsic effects on protein folding by directly altering the folding energy landscape. Previous work from our laboratories (Hanson et al. Proc. Natl. Acad. Sci. U.S.A. 2009, 109, 3131-3136; Shental-Bechor, D.; Levy, Y. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 8256-8261) suggested that the three sugar residues closest to the protein are sufficient for accelerating protein folding and stabilizing the resulting structure in vitro; even a monosaccharide can have a dramatic effect. The highly conserved nature of these three proximal sugars in N-glycans led us to speculate that introducing an N-glycosylation site into a protein that is not normally glycosylated would stabilize the protein and increase its folding rate in a manner that does not depend on the presence of specific stabilizing protein-saccharide interactions. Here, we test this hypothesis experimentally and computationally by incorporating an N-linked GlcNAc residue at various positions within the Pin WW domain, a small β-sheet-rich protein. The results show that an increased folding rate and enhanced thermodynamic stability are not general, context-independent consequences of N-glycosylation. Comparison between computational predictions and experimental observations suggests that generic glycan-based excluded volume effects are responsible for the destabilizing effect of glycosylation at highly structured positions. However, this reasoning does not adequately explain the observed destabilizing effect of glycosylation within flexible loops. Our data are consistent with the hypothesis that specific, evolved protein-glycan contacts must also play an important role in mediating the beneficial energetic effects on protein folding that glycosylation can confer.

More than 70% of eukaryotic proteins are composed of multiple domains. However, most studies of the search for DNA focus on individual protein domains and do not consider potential cross talk within a multidomain transcription factor. In this study, the molecular features of the DNA search mechanism were explored for two multidomain transcription factors: human Pax6 and Oct-1. Using a simple computational model, we compared a DNA search of multidomain proteins with a search of isolated domains. Furthermore, we studied how manipulating the binding affinity of a single domain to DNA can affect the overall DNA search of the multidomain protein. Tethering the two domains via a flexible linker increases their affinity to the DNA, resulting in a higher propensity for sliding along the DNA, which is more significant for the domain with the weaker DNA-binding affinity. In this case, the domain that binds DNA more tightly anchors the multidomain protein to the DNA and, via the linker, increases the local concentration of the weak DNA-binding domain (DBD). The tethered domains directly exchange between two parallel DNA molecules via a bridged intermediate, where intersegmental transfer is promoted by the weaker DBD. We found that, in general, the relative affinity of the two domains can significantly affect the cross talk between them and thus their overall capability to search DNA efficiently. The results we obtained by examining various multidomain DNA-binding proteins support the necessity of discrepancies between the DNA-binding affinities of the constituent domains.

Protein flexibility is thought to play key roles in numerous biological processes, including antibody affinity maturation, signal transduction, and enzyme catalysis, yet only limited information is available regarding the molecular details linking protein dynamics with function. A single point mutation at the distal site of the endogenous tissue inhibitor of metalloproteinase 1 (TIMP-1) enables this clinical target protein to tightly bind and inhibit membrane type 1 matrix metalloproteinase (MT1-MMP) by increasing only the association constant. The high-resolution X-ray structure of this complex determined at 2 could not explain the mechanism of enhanced binding and pointed to a role for protein conformational dynamics. Molecular dynamics (MD) simulations reveal that the high-affinity TIMP-1 mutants exhibit significantly reduced binding interface flexibility and more stable hydrogen bond networks. This was accompanied by a redistribution of the ensemble of substrates to favorable binding conformations that fit the enzyme catalytic site. Apparently, the decrease in backbone flexibility led to a lower entropy cost upon formation of the complex. This work quantifies the effect of a single point mutation on the protein conformational dynamics and function of TIMP-1. Here we argue that controlling the intrinsic protein dynamics of MMP endogenous inhibitors may be utilized for rationalizing the design of selective novel protein inhibitors for this class of enzymes.

Protein ubiquitination controls the cellular fate of numerous eukaryotic proteins. Despite its importance, many fundamental questions remain regarding its mechanism. One such question is how ubiquitination alters the biophysical properties of the modified protein and whether these alterations are significant in the cellular context. In this study, we investigate the effects of ubiquitination on the folding thermodynamics and mechanism of various substrates using computational tools and find that ubiquitination changes the thermal stability of modified proteins in a manner relevant to cellular processes. These changes depend on the substrate modification site and on the type of ubiquitination. Ubiquitination of the substrate Ubc7 at the residues that are modified in vivo prior to proteasomal degradation uniquely results in significant thermal destabilization and a local unwinding near the modification site, which indicates that ubiquitination possibly facilitates the unfolding process and improves substrate degradation efficiency. With respect to the substrate p19^4inkd, our results support a synergetic effect of ubiquitination and phosphorylation on the degradation process via enhanced thermal destabilization. Our study implies that, in addition to its known role as a recognition signal, the ubiquitin attachment may be directly involved in the cellular process it regulates by changing the biophysical properties of the substrate.

Biomolecular folding and function are often coupled. During molecular recognition events, one of the binding partners may transiently or partially unfold, allowing more rapid access to a binding site. We describe a simple model for this fly-casting mechanism based on the capillarity approximation and polymer chain statistics. The model shows that fly casting is most effective when the protein unfolding barrier is small and the part of the chain which extends toward the target is relatively rigid. These features are often seen in known examples of fly casting in protein - DNA binding. Simulations of protein - DNA binding based on wellfunneled native-topology models with electrostatic forces confirm the trends of the analytical theory.

The search through nonspecific DNA for a specific site by proteins is known to be facilitated by sliding, hopping, and intersegment transfer between separate DNA strands, yet the driving forces of these protein dynamics from the molecular perspective are unclear. In this study, molecular features of the DNA search mechanism were explored for three homologous proteins (the HoxD9, Antp, and NK-2 homeodomains) using a simple computational model in which protein-DNA interactions are represented solely by electrostatic forces. In particular, we studied the impact that disordered N-terminal tails (N-tails), which are more common in DNA-binding proteins than in other proteins, have on the efficiency of DNA search. While the three homeodomain proteins were found to use similar binding interfaces in specific and nonspecific interactions with DNAs, their different electrostatic potentials affect the nature of their sliding dynamics. The different lengths and net charges of the N-tails of the homeodomains affect their motion along the DNA. The presence of an N-tail increases sliding propensity but slows linear diffusion along the DNA. When the search is performed in the presence of two parallel DNA molecules, a direct transfer, which is facilitated by the protein tail, from one nonspecific DNA to another occurs. The tailed proteins jump between two DNA molecules through an intermediate in which the recognition helix of the protein is adsorbed to one DNA fragment and the N-tail is adsorbed to the second, suggesting a "monkey bar" mechanism. Our study illustrates how the molecular architecture of proteins controls the efficiency of DNA scanning.

This review aims at discussing the molecular details of the folding mechanisms of conjugated proteins using computational tools. Almost all studies of protein folding focus on individual proteins and do not consider how interactions with posttranslational modifications and between domains might affect folding. However, different chemical conjugations may introduce a variety of effects on the protein biophysics. These effects depend both on the chemical characteristics of the protein substrate as well as on the chemical and physical properties of the attachment. We review the folding of various types of conjugated proteins, glycoproteins, proteins with tails, ubiquitinated proteins, and multidomain proteins, to explore the underlying biophysical principles of these complex folding processes and in particular to quantify the cross-talk between the protein and its conjugated polymer.

Summary: Protein structures can be viewed as networks of contacts (edges) between amino-acid residues (nodes). Here we dissect proteins into sub-graphs consisting of six nodes and their corresponding edges, with an edge being either a backbone hydrogen bond (H-bond) or a covalent interaction. Six thousand three hundred and twenty-two such sub-graphs were found in a large non-redundant dataset of high-resolution structures, from which 35 occur much more frequently than in a random model. Many of these significant sub-graphs (also called network motifs) correspond to sub-structures of α helices and β-sheets, as expected. However, others correspond to more exotic sub-structures such as 3₁₀ helix, Schellman motif and motifs that were not defined previously. This topological characterization of patterns is very useful for producing a detailed differences map to compare protein structures. Here we analyzed in details the differences between NMR, molecular dynamics (MD) simulations and X-ray structures for Lysozyme, SH3 and the lambda repressor. In these cases, the same structures solved by NMR and simulated by MD showed small but consistent differences in their motif composition from the crystal structures, despite a very small root mean square deviation (RMSD) between them. This may be due to differences in the pair-wise energy functions used and the dynamic nature of these proteins.

(Graph Presented) DNA binding specificity of homeodomain transcription factors is critically affected by disordered N-terminal tails (N-tails) that undergo a disorder-to-order transition upon interacting with DNA. The mechanism of the binding process and the molecular basis of selectivity are largely unknown. The coupling between folding and DNA binding of Antp and NK-2 homeodomains was investigated by coarse-grained molecular dynamics simulations using the native protein-DNA complex. The disordered N-tails were found to decrease the stability of the free proteins by competing with the native intramolecular interactions and increasing the radius of gyration of the homeodomain cores. In the presence of DNA, however, the N-tails increase the stability of the homeodomains by reducing the coupling between folding and DNA binding. Detailed studies on Antp demonstrate that the N-tail anchors the homeodomain to DNA and accelerates formation of specific interactions all along the protein-DNA interface. The tidal electrostatic forces between the N-tail and DNA induce faster and tighter binding of the homeodomain core to the DNA; this mechanism conforms to a fly-casting mechanism. In agreement with experiments, the N-tail of Antp also improves the binding affinity for DNA, with a major contribution by the released waters. These results imply that varying the degree of folding upon binding and thereby modulating the size of the buried surface-disordered N-tails of homeodomains can fine-tune the binding strength for specific DNA sequences. Overall, both the kinetics and thermodynamics of specific DNA binding by homeodomains can be improved by N-tails using a mechanism that is inherent in their disordered state.

In recent years, a growing number of protein folding studies have focused on the unfolded state, which is now recognized as playing a major role in the folding process. Some of these studies show that interactions occurring in the unfolded state can significantly affect the stability and kinetics of the protein folding reaction. In this study, we modeled the effect of electrostatic interactions, both native and nonnative, on the folding of three protein systems that underwent selective charge neutralization or reversal or complete charge suppression. In the case of the N-terminal L9 protein domain, our results directly attribute the increase in thermodynamic stability to destabilization of the unfolded ensemble, reaffirming the experimental observations. These results provide a deeper structural insight into the ensemble of the unfolded state and predict a new mutation site for increased protein stability. In the second case, charge reversal mutations of RNase Sa affected protein stability, with the destabilizing mutations being less destabilizing at higher salt concentrations, indicating the formation of charge-charge interactions in the unfolded state. In the N-terminal L9 and RNase Sa systems, changes in electrostatic interactions in the unfolded state that cause an increase in free energy had an overall compaction effect that suggests a decrease in entropy. In the third case, in which we compared the p-lactalbumin and hen egg-white lysozyme protein homologues, we successfully eliminated differences between the folding kinetics of the two systems by suppressing electrostatic interactions, supporting previously reported findings. Our coarse-grained molecular dynamics study not only reproduces experimentally reported findings but also provides a detailed molecular understanding of the elusive unfolded-state ensemble and how charge-charge interactions can modulate the biophysical characteristics of folding. (C) 2009 Elsevier Ltd. All rights reserved.

Glycosylation is among the most common post-translational modifications that proteins undergo that may affect many of their activities. It may also modify the underlying energy landscape of glycoproteins in a way that their altered biophysical characteristics are linked to their bioactivity. Yet, the capability of glycosylation to modify thermodynamic and kinetic properties varies greatly between glycoproteins. Deciphering the 'glycosylation code' that dictates the interplay between the nature of the carbohydrates or the proteins and the biophysical properties of the glycosylated proteins is essential. In this article, we discuss how the size, number, and structure of glycans, as well as the attachment sites, may modulate the folding of glycoproteins. Understanding the cross-talks between the protein and the attached glycans at the molecular level may assist in tailoring the biophysical properties of proteins in general.

In the cell, protein complexes form by relying on specific interactions between their monomers. Excluded volume effects due to molecular crowding would lead to correlations between molecules even without specific interactions. What is the interplay of these effects in the crowded cellular environment? We study dimerization of a model homodimer when the mondimers are free and when they are tethered to each other. We consider a structured environment: Two monomers first diffuse into a cavity of size Land then fold and bind within the cavity. The folding and binding are simulated by using molecular dynamics based on a simplified topology based model. The confinement in the cell is described by an effective molecular concentration C - L ^-3. A two-state coupled folding and binding behavior is found. We show the maximal rate of dimerization occurred at an effective molecular concentration C ^op 1 mM, which is a relevant cellular concentration. In contrast, for tethered chains the rate keeps at a plateau when C < C ^op but then decreases sharply when C > C ^op. For both the free and tethered cases, the simulated variation of the rate of dimerization and thermodynamic stability with effective molecular concentration agrees well with experimental observations. In addition, a theoretical argument for the effects of confinement on dimerization is also made.

Efficient search of DNA by proteins is fundamental to the control of cellular regulatory processes. It is currently believed that protein sliding, hopping, and transfer between adjacent DNA segments, during which the protein nonspecifically interacts with DNA, are central to the speed of their specific recognition. In this study, we focused on the structural and dynamic features of proteins when they scan the DNA. Using a simple computational model that represents protein-DNA interactions by electrostatic forces, we identified that the protein makes use of identical binding interfaces for both nonspecific and specific DNA interactions. Accordingly, in its one-dimensional diffusion along the DNA, the protein is bound at the major groove and performs a helical motion, which is stochastic and driven by thermal diffusion. A microscopic structural insight into sliding from our model, which is governed by electrostatic forces, corroborates previous experimental studies suggesting that the active site of some regulatory proteins continually faces the interior of the DNA groove while sliding along sugar-phosphate rails. The diffusion coefficient of spiral motion along the major groove of the DNA is not affected by salt concentration, but the efficiency of the search can be significantly enhanced by increasing salt concentration due to a larger number of hopping events. We found that the most efficient search comprises ∼ 20% sliding along the DNA and ∼ 80% hopping and three-dimensional diffusion. The presented model that captures various experimental features of facilitated diffusion has the potency to address other questions regarding the nature of DNA search, such as the sliding characteristics of oligomeric and multidomain DNA-binding proteins that are ubiquitous in the cell.

Energy landscape theory requires that the protein-folding mechanism is generally globally directed or funneled toward the native state. The collective nature of transition state ensembles further suggests that sufficient averaging of the native interactions can occur so that the knowledge of the native topology may suffice for predicting the mechanism. Nevertheless, while simple homogeneously weighted native topology-based models predict the folding mechanisms for many proteins, for other proteins knowledge of the native topology, by itself, seems not to suffice in determining the folding mechanism. Simulations of proteins with differing topologies reveal that the failure of homogeneously weighted topology-based models can, however, be completely understood within the framework of a funneled energy landscape and can be quantified by comparing the fluctuation of entropy cost for forming contacts to the expected fluctuations in contact energy. To be precise, we find the transition state ensembles of proteins with all-α topologies, which are more uniform in the specific entropy cost of contact formation, have transition state ensembles that are more readily perturbed by differences in energetic weights than are the transition state ensembles of proteins with significant amounts of β-structure, where the specific entropy costs of contact formation are more widely distributed. This behavior is consistent with a random-field Ising model analogy that follows from the free energy functional approach to folding.

Multimeric proteins are ubiquitous in many cellular processes that require high levels of regulation. Eukaryotic gene expression is often regulated by a mechanism of combinatorial control that involves the binding of dimeric transcription factors to DNA together with the coordinated activity of additional proteins. In this study, we investigated the dimerization of the Arc-repressor on DNA with the aim of achieving microscopic insight into the possible advantages of interacting with DNA as a complex rather than as a monomeric single-domain protein. We used a computational coarse-grained model in which the protein dynamics was governed by native interactions and protein-DNA interactions were dictated by electrostatic forces. Inspired by previous experimental work that showed an enhanced refolding rate for the Arc-repressor in the presence of DNA and other polyanions, we focused on the mechanism and kinetics of the assembly of Arc monomers in the presence of single-(ssDNA) and double-stranded DNA (dsDNA) molecules in a low-salt concentration environment. The electrostatic interactions that attract the protein to the dsDNA were shown to be fundamental in colocalizing the unfolded Arc chains and in accelerating refolding. Arc monomers bind the dsDNA efficiently and nonspecifically, and search for each other via one-dimensional diffusion. The fastest folding of Arc is observed for DNA of 30 bp. Longer DNA is significantly less efficient in accelerating the Arc refolding rate, since the two subunits search distinct regions of the one-dimensional DNA and are therefore much less colocalized. The probability that the two unfolded chains will meet on 200 bp DNA is similar to that in the bulk. The colocalization of Arc subunits on ssDNA results in much faster folding compared to that obtained on dsDNA of the same length. Differences in the rate of Arc refolding, cooperativity, and the structure of its transition state ensemble introduced by ssDNA and dsDNA molecules demonstrate the important role of colocalization in biological self-assembly processes.

The biennial CASP experiment is a crucial way to evaluate, in an unbiased way, the progress in predicting novel 3D protein structures. In this article, we assess the quality of prediction of template free models, that is, ab initio prediction of 3D structures of proteins based solely on the amino acid sequences, that is, proteins that did not have significant sequence identity to any protein in the Protein Data Bank. There were 13 targets in this category and 102 groups submitted predictions. Analysis was based on the GDT-TS analysis, which has been used in previous CASP experiments, together with a newly developed method, the OK-Rank, as well as by visual inspection. There is no doubt that in recent years many obstacles have been removed on the long and elusive way to deciphering the protein-folding problem. Out of the 13 targets, six were predicted well by a number of groups. On the other hand, it must be stressed that for four targets, none of the models were judged to be satisfactory. Thus, for template free model prediction, as evaluated in this CASP, successes have been achieved for most targets; however, a great deal of research is still required, both in improving the existing methods and in development of new approaches.

The unfolded state ensemble of proteins has been described as a structurally featureless state. While this approach is supported by the fact that many unfolded proteins follow the scaling law behavior of a random coil, there is evidence that the unfolded states of various proteins are stabilized by native or non-native interactions. Recently, the existence of extensive non-native structure was reported for a repeat protein, which resulted in a scaling law exponent that is significantly smaller than that of a random polymer [Cortajarena et al., J. Mol. Biol. 382(1), 203-212 (2008)]. It was concluded that the high compactness of this protein stems from a significant fraction of interacting PPII helical segments in the unfolded state. In this study, we aim at providing possible molecular understanding of this anomalous compactness of the unfolded state and to investigate its origin. Using a hierarchy of computational models, we ask whether in general the unfolded state of a repeat protein is likely to be intrinsically more compact than the unfolded state of globular proteins, or whether this phenomenon depends mostly on the occurrence of a specific sequence that promotes PP _II conformations. Our results suggest that the formation of the PP _II conformation is indeed essential, yet the recurring sequence of repeat proteins promotes the interactions between these PPII segments and the formation of non-native interactions in the unfolded state.

The complexity of the mechanisms by which proteins fold has been shown by many studies to be governed by their native-state topologies. This was manifested in the ability of the native topology-based model to capture folding mechanisms and the success of folding rate predictions based on various topological measures, such as the contact order. However, while the finer details of topological complexity have been thoroughly examined and related to folding kinetics, simpler characteristics of the protein, such as its overall shape, have been largely disregarded. In this study, we investigated the folding of proteins with an unusual elongated geometry that differs substantially from the common globular structure. To study the effect of the elongation degree on the folding kinetics, we used repeat proteins, which become more elongated as they include more repeating units. Some of these have apparently anomalous experimental folding kinetics, with rates that are often less than expected on the basis of rates for globular proteins possessing similar topological complexity. Using experimental folding rates and a larger set of rates obtained from simulations, we have shown that as the protein becomes increasingly elongated, its folding kinetics becomes slower and deviates more from the rate expected on the basis of topology measures fitted for globular proteins. The observed slow kinetics is a result of a more complex pathway in which stable intermediates composed of several consecutive repeats can appear. We thus propose a novel measure, an elongation-sensitive contact order, that takes into account both the extent of elongation and the topological complexity of the protein. This new measure resolves the apparent discrimination between the folding of globular and elongated repeat proteins. Our study extends the current capabilities of folding-rate predictions by unifying the kinetics of repeat and globular proteins.

Proteins with a long flexible polymeric tail attached at their N- or C-terminus are studied using molecular dynamics (MD) simulations of a coarse-grained model for protein folding where the temperature is regulated by either the Berendsen or the Langevin thermostat. These thermostats show different abilities to regulate the temperature of these systems that include flexible and more rigid regions. In the simulations with the Berendsen thermostat, the flexible tail is significantly hotter than the protein, both in its folded and unfolded states. Upon weakening the strength of the Berendsen thermostat, the temperature gradient between the fast and the slow degrees of freedom is significantly decreased, yet linkage between the temperatures of the flexible tail and the protein remains. The Langevin thermostat is proven to regulate the temperature of these inhomogenous systems reliably, without discriminating between the slow and fast degrees of freedom. The Langevin thermostat is less sensitive than is the Berendsen thermostat to the strength of the coupling between the protein system and the thermal bath. Our study calls for special care in choosing the thermostat for MD simulations of systems with inhomogenous degrees of freedom. Using the Berendsen thermostat with strong coupling would result in mistaken thermodynamic descriptions of such systems. (C) 2008 Wiley Periodicals, Inc.

Glycosylation is one of the most common posttranslational modifications to occur in protein biosynthesis, yet its effect on the thermodynamics and kinetics of proteins is poorly understood. A minimalist model based on the native protein topology, in which each amino acid and sugar ring was represented by a single bead, was used to study the effect of glycosylation on protein folding. We studied in silico the folding of 63 engineered SH3 domain variants that had been glycosylated with different numbers of conjugated polysaccharide chains at different sites on the protein's surface. Thermal stabilization of the protein by the polysaccharide chains was observed in proportion to the number of attached chains. Consistent with recent experimental data, the degree of thermal stabilization depended on the position of the glycosylation sites, but only very weakly on the size of the glycans. A thermodynamic analysis showed that the origin of the enhanced protein stabilization by glycosylation is destabilization of the unfolded state rather than stabilization of the folded state. The higher free energy of the unfolded state is enthalpic in origin because the bulky polysaccharide chains force the unfolded ensemble to adopt more extended conformations by prohibiting formation of a residual structure. The thermodynamic stabilization induced by glycosylation is coupled with kinetic stabilization. The effects introduced by the glycans on the biophysical properties of proteins are likely to be relevant to other protein polymeric conjugate systems that regularly occur in the cell as posttranslational modifications or for biotechnological purposes.

Human immunodeficiency virus type 1 (HIV-1) protease (PR) plays an essential role in the life cycle of the virus. Consequently, its inhibition can control acquired immunodeficiency syndrome (AIDS). Any pharmacological treatment targeting the active site of the protease is known to generate escape mutants. On the other hand, if a drug targets a site crucial for the correct folding of the protease, mutations affecting this region would denaturate the protein and thus will not be expressed. We review the progress in our understanding of the folding of the protease, which has been instrumental in the design of a (non-conventional) folding inhibitor. The transferability of these results to other proteins testify to the universality of the folding-inhibition scenario for the design of leads of drugs which are unlikely to generate resistance.

Conformational transitions play a central role in regulating protein function. Structure-based models with multiple basins have been used to understand the mechanisms governing these transitions. A model able to accommodate multiple folding basins is proposed to explore the mutational effects in the folding of the Rop-dimer (Rop). In experiments, Rop mutants show unusually strong increases in folding rates with marginal effects on stability. We investigate the possibility of two competing conformations representing a parallel (P) and the wild-type antiparallel (AP) arrangement of the monomers as possible native conformations. We observe occupation of both distinct states and characterize the transition pathways. An interesting observation from the simulations is that, for equivalent energetic bias, the transition to the P basin (non-wild-type basin) shows a lower free-energy barrier. Thus, the rapid kinetics observed in experiments appear to be the result of two competing states with different kinetic behavior, triggered upon mutation by the opening of a trapdoor arising from Rop's symmetric structure. The general concept of having competing conformations for the native state goes beyond explaining Rop's mutational behaviors and can be applied to other systems. A switch between competing native structures might be triggered by external factors to allow, for example, allosteric control or signaling.

Conformational heterogeneity in proteins is known to often be the key to their function. We present a coarse grained model to explore the interplay between protein structure, folding and function which is applicable to allosteric or non-allosteric proteins. We employ the model to study the detailed mechanism of the reversible conformational transition of Adenylate Kinase (AKE) between the open to the closed conformation, a reaction that is crucial to the protein's catalytic function. We directly observe high strain energy which appears to be correlated with localized unfolding during the functional transition. This work also demonstrates that competing native interactions from the open and closed form can account for the large conformational transitions in AKE. We further characterize the conformational transitions with a new measure Φ_Func, and demonstrate that local unfolding may be due, in part, to competing intra-protein interactions.

Molecular plasticity, the key to many biomolecular self-assembly processes, and electrostatic steering, which guides proteins to DNA, are shown to be coupled and to facilitate DNA search. While protein flexibility is involved in induced-fit recognition and to a larger extent in intrinsically unstructured DNA binding proteins, we show that through a "tidal force" the electrostatic field of the DNA can induce flexibility and the partial unfolding of a two-state folding protein, thereby reducing its folding barrier and, thus, stimulating fly-casting. The protein binds DNA nonspecifically in a partially folded state and completes its folding when it binds the specific site. The interplay between fly-casting and electrostatics is observed even for weak electrostatic forces and is expected to vary with the electrostatic screening due to salt and the intrinsic folding barrier, both of which can be modulated experimentally.

Many cellular functions rely on interactions among proteins and between proteins and nucleic acids. Our understanding of the principles that govern protein folding has been advanced in the recent years using the energy landscape theory and thanks to tight collaborations between experimentalists and theoreticians. It is likely that our current understanding of protein folding can be applied to understand more complex cellular self-organization processes. The limited success of binding predictions may suggest that the physical and chemical principles of protein binding have to be revisited to correctly capture the essence of protein recognition. In this review, we discuss the power of reduced models to study the physics of protein assembly. Since energetic frustration is sufficiently small, native topology-based models, which correspond to perfectly unfrustrated energy landscapes, have shown that binding mechanisms are robust and governed primarily by the protein's native topology. These models impressively capture many of the binding characteristics found in experiments and highlights the fundamental role of flexibility in binding. The essential role of solvent molecules and electrostatic interactions in binding is also discussed. Despite the success of the minimally frustrated models to describe the dynamics and mechanisms of binding, the actual degree of frustration has to be explored to quantify the capacity of a protein to bind specifically to other proteins. We have found that introducing mutations can significantly reduce specificity by introducing an additional binding mode. Deciphering and quantifying the key ingredients for biological self-assembly is invaluable to reading out genomic sequences and understanding cellular interaction networks.

Human transthyretin (TTR) is an amyloidogenic protein whose aggregation is associated with several types of amyloid diseases. The following mechanism of TTR amyloid formation has been proposed. TTR tetramer at first dissociates into native monomers, which is the rate-limiting step in fibril formation. The monomeric species then partially unfold to form amyloidogenic intermediates that subsequently undergo a downhill self-assembly process. The amyloid deposit can be facilitated by disease-associated point mutations. However, only subtle structural differences were observed between the crystal structures of the wild type and the disease-associated variants. To investigate how single-point mutations influence the effective energy landscapes of TTR monomers, molecular dynamics (MD) simulations were performed on wild-type TTR and two pathogenic variants. Principal coordinate analysis on MD-generated ensembles has revealed multiple unfolding pathways for each protein. Amyloidogenic intermediates with the dislocated C strand-loop-D strand motif were observed only on the unfolding pathways of V30M and L55P variants and not for wild-type TTR. Our study suggests that the sequence-dependent unfolding pathway plays a crucial role in the amyloidogenicity of TTR. Analyses of side chain concerted motions indicate that pathogenic mutations on "edge strands" disrupt the delicate side chain correlated motions, which in turn may alter the sequence of unfolding events.

Enhanced structural insights into the folding energy landscape of the N-terminal dimerization domain of Escherichia coli tryptophan repressor, [2-66]2 TR, were obtained from a combined experimental and theoretical analysis of its equilibrium folding reaction. Previous studies have shown that the three intertwined helices in [2-66]2 TR are sufficient to drive the formation of a stable dimer for the full-length protein, [2-107]2 TR. The monomeric and dimeric folding intermediates that appear during the folding reactions of [2-66]2 TR have counterparts in the folding mechanism of the full-length protein. The equilibrium unfolding energy surface on which the folding and dimerization reactions occur for [2-66]2 TR was examined with a combination of native-state hydrogen exchange analysis, pepsin digestion and matrix-assisted laser/desorption mass spectrometry performed at several concentrations of protein and denaturant. Peptides corresponding to all three helices in [2-66]2 TR show multi-layered protection patterns consistent with the relative stabilities of the dimeric and monomeric folding intermediates. The observation of protection exceeding that offered by the dimeric intermediate in segments from all three helices implies that a segment-swapping mechanism may be operative in the monomeric intermediate. Protection greater than that expected from the global stability for a single amide hydrogen in a peptide from the C-helix possibly and another from the A-helix may reflect non-random structure, possibly a precursor for segment swapping, in the urea-denatured state. Native topology-based model simulations that correspond to a funnel energy landscape capture both the monomeric and dimeric intermediates suggested by the HX MS data and provide a rationale for the progressive acquisition of secondary structure in their conformational ensembles.

This paper reports on studies of the fragmentation dynamics of multicharged (A+)55 Morse clusters, where the variation of the range of the Morse potential parameters induces cluster fission for a long-range potential and Coulomb explosion for a short-range potential. The multidimensional energy landscapes for these fragmentation processes were explored by constructing reduced coordinates utilizing the principal component analysis (PCA), which was previously applied for the energy landscapes and folding dynamics of biomolecules. The distance-matrix based PCA was applied to study the effects of the potential on the fragmentation dynamics and to explore the structural diversity of the fragmentation processes. The first principal coordinate (which captures 95% of the dynamic information content for each trajectory) constitutes an appropriate reaction coordinate for both fission and Coulomb explosion and was used to determine the temperature-dependent fragmentation rates. These obey the Arrhenius law, with the barrier for fission (0.36eV) being higher than for Coulomb explosion (0.22eV). Structural and energetic information on the radius of gyration and on the potential energy for small values of the reaction coordinate manifest considerably larger fluctuations for fission than for Coulomb explosion, indicating that in the former case the cluster shrinks and swells prior to dissociation. The joint projection of multiple trajectories for each fragmentation process allows for the description of the energy landscapes and fragmentation pathways in terms of two principal coordinates, which manifest a form of ski slopes. Different collective coordinates describe the spatially isotropic Coulomb explosion and the spatially unisotropic fission.

We studied the energetics and fragmentation patterns of multicharged (A⁺)_n Morse clusters (n = 55-321), with a total cluster charge Z = n. The Morse pair-potential parameters were characterized by the dissociation energy D = 1-10 eV, range parameter α = 1-3 Å^-1, and interatomic equilibrium separation R_e = 1-3 Å. The potential energies ε (per particle) of these multicharged Morse clusters at their equilibrium configuration (with bond length r ₀) were analyzed in terms of the liquid drop model. This resulted in the relation ε =(ā_C⁰/r₀)n ^2/3+(ā_v⁰D/αr₀) +[ā_s⁰D/(αr₀)^3/2]n ^-1/3, where the reduced parameters ā_C⁰ (for the Coulomb energy), ā_v⁰ (for the interior energy) and ā_s⁰ (for the surface energy) are independent of the Morse pair-potential parameters. The Rayleigh fissibility parameter X = E(Coulomb)/2E(surface), which determines the fragmentation pattern (i.e., X < 1 for cluster fission and X > 1 for Coulomb explosion), was expressed in the form X=(Z²/n)[(2ā_s ⁰/ā_C⁰)(D/α^3/2r ₀^1/2)]^-1. The application of this result to the Coulomb instability of multicharged globular proteins reveals that X < 1 for the currently available data. The dominating fragmentation channel is expected to involve spatially anisotropic protein fission into a small number of large fragments, rather than spatially isotropic protein Coulomb explosion into a large number of small fragments.

Many cellular functions rely on interactions among proteins and between proteins and nucleic acids. The limited success of binding predictions may suggest that the physical and chemical principles of protein binding have to be revisited to correctly capture the essence of protein recognition. In this Account, we discuss the power of reduced models to study the physics of protein assembly. Since energetic frustration is sufficiently small, native topology-based models, which correspond to perfectly unfrustrated energy landscapes, have shown that binding mechanisms are robust and governed primarily by the protein's native topology. These models impressively capture many of the binding characteristics found in experiments and highlight the fundamental role of flexibility in binding. The essential role of solvent molecules and electrostatic interactions in binding is also discussed. Despite the success of the minimally frustrated models to describe the dynamics and mechanisms of binding, the actual degree of frustration has to be explored to quantify the capacity of a protein to bind specifically to other proteins. We have found that introducing mutations can significantly reduce specificity by introducing an additional binding mode. Deciphering and quantifying the key ingredients for biological self-assembly is invaluable to reading out genomic sequences and understanding cellular interaction networks.

Minding your p's and q's has become as important to protein-folding theorists as it is for those being instructed in the rules of etiquette. To assess the quality of structural reaction coordinates in predicting the transition-state ensemble (TSE) of protein folding, we benchmarked the accuracy of four structural reaction coordinates against the kinetic measure P _fold, whose value of 0.50 defines the stochastic separatrix for a two-state folding mechanism. For two proteins that fold by a simple two-state mechanism, c-src SH3 and Cl-2, the Φ-values of the TSEs predicted by native topology-based reaction coordinates (including Q, the fraction of native contacts) are almost identical to those of the TSE based on P_fold, with correlation coefficients of >0.90. For proteins with complex folding mechanisms that have especially broad, asymmetrical free energy barriers such as the designed 3-ankyrin repeating protein (3ANK) or proteins with distinct intermediates such as cyanovirin-N (CV-N), we show that the ensemble of structures with P_fold = 0.50 generally does not include the chemically relevant transition states. This weakness of P_fold limits its usefulness in protein folding studies. For such systems, elucidating the essential features of folding mechanisms requires using multiple reaction coordinates, although the number is still rather small. At the same time, for simple folding mechanisms, there is no indication of superiority for P _fold over structurally chosen and thermodynamically relevant reaction coordinates that correctly measure the degree of nativeness.

Water is essential for life in many ways, and without it biomolecules might no longer truly be biomolecules. In particular, water is important to the structure, stability dynamics, and function of biological macromolecules. In protein folding, water mediates the collapse of the chain and the search for the native topology through a funneled energy landscape. Water actively participates in molecular recognition by mediating the interactions between binding partners and contributes to either enthalpic or entropic stabilization. Accordingly, water must be included in recognition and structure prediction codes to capture specificity. Thus water should not be treated as an inert environment, but rather as an integral and active component of biomolecular systems, where it has both dynamic and structural roles. Focusing on water sheds light on the physics and function of biological machinery and self-assembly and may advance our understanding of the natural design of proteins and nucleic acids.

We address unifying features of fragmentation channels driven by long-range Coulomb or pseudo-Coulomb forces in clusters, nuclei, droplets, and optical molasses. We studied the energetics, fragmentation patterns, and dynamics of multicharged (A+) n (n=55, 135, 321) clusters. In Morse clusters the variation of the range of the pair-potential induced changes in the cluster surface energy and in the fissibility parameter X=E (Coulomb) 2E (surface). X was varied in the range of X=1-8 for short-range interactions and of X=0.1-1.0 for long-range interactions. Metastable cluster configurations were prepared by vertical ionization of the neutral clusters and by subsequent structural equilibration. The energetics of these metastable ionic clusters was described in terms of the liquid drop model, with the coefficients of the volume and surface energies depending linearly on the Morse band dissociation energy. Molecular-dynamics simulations established two distinct fragmentation patterns of multicharged clusters that involve cluster fission into a small number of large, multicharged clusters for X

The prevalence of domain-swapping in nature is a manifestation of the principle of minimal frustration in that the interactions designed by evolution to stabilize the protein are also involved in this mode of binding. We previously demonstrated that the Symmetrized-Go potential accurately predicts the experimentally observed domain-swapped structure of Eps8 based solely on the structure of the monomer. There can be, however, multiple modes of domain-swapping, reflecting a higher level of frustration, which is a consequence of symmetry. The human prion and cyanovirin-N are too frustrated to form unique domain-swapped structures on the basis of the Symmetrized-Go potential. However, supplementing the completely symmetric model with intermolecular and intramolecular disulfide bonds in the prion and cyanovirin-N proteins, respectively, yielded unique domain-swapped structures with a remarkable similarity to the experimentally observed ones. These results suggest that the disulfide bonds may sometimes be critical in overcoming the intrinsic frustration of the symmetrized energy landscapes for domain-swapping. We also discuss the implications of intermolecular disulfide bonds in the formation of mammalian prion aggregates.

Many cellular functions rely on interactions between protein pairs and higher oligomers. We have recently shown that binding mechanisms are robust and owing to the minimal frustration principle, just as for protein folding, are governed primarily by the protein's native topology, which is characterized by the network of non-covalent residue-residue interacdons. The detailed binding mechanisms of nine dimers, a trimer, and a tetramer, each involving different degrees of flexibility and plasticity during assembly, are surveyed here using a model that is based solely on the protein topology, having a perfectly funneled energy landscape. The importance of flexibility in binding reactions is manifested by the fly-casting effect, which is diminished in magnitude when protein flexibility is removed. Many of the grosser and finer structural aspects of the various binding mechanisms (including binding of pre-folded monomers, binding of intrinsically unfolded monomers, and binding by domain-swapping) predicted by the native topology based landscape model are consistent with the mechanisms found in the laboratory. An asymmetric binding mechanism is often observed for the formation of the symmetric homodimers where one monomer is more structured at the binding transition state and serves as a template for the folding of the other monomer. phi values were calculated to show how the structure of the binding transition state ensemble would be manifested in protein engineering studies. For most systems, the simulated phi values are reasonably correlated with the available experimental values. This agreement suggests that the overall binding mechanism and the nature of the binding transition state ensemble can be understood from the network of interactions that stabilize the native fold. The phi values for the formation of an antibody-antigen complex indicate a possible role for solvation. of the interface in biomolecular association of large rigid proteins. (C) 2005 Elsevier Ltd. All rights reserved.

Protein folding has become one of the best understood biochemical reactions from a kinetic viewpoint. The funneled energy landscape, a consequence of the minimal frustration achieved by evolution in sequences, explains how most proteins fold efficiently and robustly to their functional structure and allows robust prediction of folding kinetics. The folding of Rop (repressor of primer) dimer is exceptional because some of its mutants with a redesigned hydrophobic core both fold and unfold much faster than the WT protein, which seems to conflict with a simple funneled energy landscape for which topology mainly determines the kinetics. We propose that the mystery of Rop folding can be unraveled by assuming a double-funneled energy landscape on which there are two basins that correspond to distinct but related topological structures. Because of the near symmetry of the molecule, mutations can cause a conformational switch to a nearly degenerate yet distinct topology or lead to a mixture of both topologies. The topology predicted to have the lower free-energy barrier height for folding was further found by all-atom modeling to give a better structural fit for those mutants with the extreme folding and unfolding rates. Thus, the non-Hammond effects can be understood within energy-landscape theory if there are in fact two different but nearly degenerate structures for Rop. Mutations in symmetric and regular structures may give rise to frustration and thus result in degeneracy.

Abstract Many cellular functions are carried out by proteins that are bound together in multiprotein complexes. The binding between two highly flexible proteins to form homodimers is studied here using energy landscape theory and simulations based on a perfectly funneled energy landscape. With the aim to survey the range of binding mechanisms, two sets of homodimers were selected based on the experimental knowledge of whether stable monomers are needed for binding to take place. We find that the binding mechanism can be predicted based on the structure of the complex subunits alone. On average, the theory predicts a lower stability for subunits that are less compact and less hydrophobic, indicating, in agreement with their experimental classification, that their folding will be coupled to their binding. On the other hand, when a monomeric intermediate is experimentally found, the predicted stability of the monomers is comparable to that of known folded proteins. Furthermore, when dimerization is coupled to monomer folding, the interface is more hydrophobic.

The same energy landscape principles associated with the folding of proteins into their monomeric conformations should also describe how these proteins oligomerize into domain-swapped conformations. We tested this hypothesis by using a simplified model for the epidermal growth factor receptor pathway substrate 8 src homology 3 domain protein, both of whose monomeric and domain-swapped structures have been solved. The model, which we call the symmetrized Gō-type model, incorporates only information regarding the monomeric conformation in an energy function for the dimer to predict the domain-swapped conformation. A striking preference for the correct domain-swapped structure was observed, indicating that overall monomer topology is a main determinant of the structure of domain-swapped dimers. Furthermore, we explore the free energy surface for domain swapping by using our model to characterize the mechanism of oligomerization.

HIV-1 protease (PR) is a major drug target in combating AIDS, as it plays a key role in maturation and replication of the virus. Six FDA-approved drugs are currently in clinical use, all designed to inhibit enzyme activity by blocking the active site, which exists only in the dimer. An alternative inhibition mode would be required to overcome the emergence of drug-resistance through the accumulation of mutations. This might involve ' inhibiting the formation of the dimer itself. Here, the folding of HIV-1 PR dimer is studied with several simulation models appropriate for folding mechanism studies. Simulations with an off-lattice Go-model, which corresponds to a perfectly funneled energy landscape, indicate that the enzyme is formed by association of structured monomers. All-atom molecular dynamics simulations strongly support the stability of an isolated monomer. The conjunction of results from a model that focuses on the protein topology and a detailed all-atom force-field model suggests, in contradiction to some reported equilibrium denaturation experiments, that monomer folding and dimerization are decoupled. The simulation result is, however, in agreement with the recent NMR detection of folded monomers of HIV-1 PR mutants with a destabilized interface. Accordingly, the design of dimerization inhibitors should not focus only on the flexible N and C termini that constitute most of the dimer interface, but also on other structured regions of the monomer. In particular, the relatively high phi values for residues 23-35 and 79-87 in both the folding and binding transition states, together with their proximity to the interface, highlight them as good targets for inhibitor design. (C) 2004 Elsevier Ltd. All rights reserved.

Protein recognition and binding, which result in either transient or long-lived complexes, play a fundamental role in many biological functions, but sometimes also result in pathologic aggregates. We use a simplified simulation model to survey a range of systems where two highly flexible protein chains form a homodimer. In all cases, this model, which corresponds to a perfectly funneled energy landscape for folding and binding, reproduces the macroscopic experimental observations on whether folding and binding are coupled in one step or whether intermediates occur. Owing to the minimal frustration principle, we find that, as in the case of protein folding, the native topology is the major factor that governs the choice of binding mechanism. Even when the monomer is stable on its own, binding sometimes occurs fastest through unfolded intermediates, thus showing the speedup envisioned in the fly-casting scenario for molecular recognition.

The flexibility and stability of both monomeric and dimeric HIV-1 PR were explored by 100 ns implicit solvent molecular dynamics simulation at 350 K with the aim to correlate the monomer stability with the dimerization mechanism. The principal component analysis (PCA) was applied to visualize the available regions in the conformational space of the two HIV-1 PR forms, to compare their structural diversity and to map the bottom of their underlying energy landscapes. It was found that whereas the flap tips (residues 45-55) are flexible and adopt close and open conformations in both monomeric and dimeric forms, the N-and C-termini (residues 1-4 and 96-99, respectively), which constitute the interface between the two subunits, are flexible only in the monomer. The different flexibility of the monomeric and dimeric HIV-1 PR is reflected in the different topography of their underlying energy landscape. Although the bottom of the monomer energy landscape is broad and rough, that of the dimer is narrower, deeper, and smoother, reflecting the enhanced flexibility of the monomer and the stabilizing interactions between the dimer subunits. Accordingly, blocking one or both terminals may prevent the formation of the active site. Despite the different flexibility of the termini in the monomeric and dimeric HIV-1 PR, their secondary structure contents are similar. The partial stability of the monomer together with the flexibility of its termini suggest that the HIV-1 PR is not a two-state dimer, as indicated by equilibrium denaturation experiments, but a three-state dimer with a marginally stable monomeric intermediate. This involves the swapping of the flexible termini across the two chains to form the dimer interface.

In this paper, the interbasin motion (IBM) approach is applied to studying dynamics of conformationally constrained peptides, being extended to a nonideal contact of the system with a thermal bath. The coupling of the system with the thermal bath is expressed in terms of a memory function. The aim of the present study is twofold. First, we present a dynamical diagnosis of the three hexapeptide variants with the main focus on the transitions between basins rather than between individual states. Second, the present study is intended to pinpoint a way for extracting useful information about the strength of the systemsolvent coupling and how this interaction affects the propensity of relaxation towards the native state. We show that a slight variation of the value of the memory friction parameter may induce a sizable modification of the relaxation time. In addition, the change of the memory friction parameter produces alterations on short time scales among the population distributions. Especially, high energy basins seem affected the most. In the Markovian limit, the basin populations computed within the IBM model are compared to those obtained by using state-to-state transition rates in the full master equation approach. The two methods yield similar results when the separation of time scales between intra- and interbasin dynamics is completely achieved.

We address the stability of multicharged finite systems driven by Coulomb forces beyond the Rayleigh instability limit. Our exploration of the nuclear dynamics of heavily charged Morse clusters enabled us to vary the range of the pair potential and of the fissibility parameter, which results in distinct fragmentation patterns and in the angular distributions of the fragments. The Rayleigh instability limit separates between nearly binary (or tertiary) spatially unisotropic fission and spatially isotropic Coulomb explosion into a large number of small, ionic fragments. Implications are addressed for a broad spectrum of dynamics in chemical physics, radiation physics of ultracold gases, and biophysics, involving the fission of clusters and droplets, the realization of Coulomb explosion of molecular clusters, the isotropic expansion of optical molasses, and the Coulomb instability of "isolated" proteins.

Conformational transitions are thought to be the prime mechanism of prion diseases. In this study, the energy landscapes of a wild-type prion protein (PrP) and the D178N and E200K mutant proteins were mapped, enabling the characterization of the normal isoforms (PrP^C) and partially unfolded isoforms (PrP^PU) of the three prion protein analogs. It was found that the three energy landscapes differ in three respects: (i) the relative stability of the PrP^C and the PrP^PU states, (ii) the transition pathways from Pr^PC to PrP^PU, and (iii) the relative stability of the three helices in the PrP^C state. In particular, it was found that although helix 1 (residues 144-156) is the most stable helix in wild-type PrP, its stability is dramatically reduced by both mutations. This destabilization is due to changes in the charge distribution that affects the internal salt bridges responsible for the greater stability of this helix in wild-type PrP. Although both mutations result in similar destabilization of helix 1, they a have different effect on the overall stability of PrP^C and of PrP^PU isoforms and on structural properties. The destabilization of helix 1 by mutations provides additional evidences to the role of this helix in the pathogenic transition from the PrP^C to the pathogenic isoform PrP^SC.

We explored the eigenvalue spectra of the kinetic matrix which defines the master equation for the complex kinetics of the analogous polypeptides ( linear Ala6, cyclic Ala6, and charged Ala6). For each system we obtained the entire eigenvalue spectrum as well as the histograms of the weighted eigenvalue spectra, where each relaxation mode is weighted by the overlap between the initial probability vector and the corresponding eigenvector. It was found that the spectra of the weighted eigenvalues were significantly filtered in comparison to those of the unweighted eigenvalues, indicating that the decay is described by a small number of eigenvalues. The important eigenvalues which are extracted from the weighted eigenvalues spectra are in good agreement with the characteristic lifetimes for the kinetics of each system, as found by the fitting of the energy relaxation temporal profiles to multiexponential functions. Moreover, a partial correlation is found between the relative heights of the contributions of the important eigenvalues and the preexponential factors obtained by the fitting. In addition, we applied the spectra of the weighted eigenvalues to study the effect of the initial population distribution on the dynamics and also to infer which minima provide the dominant contributions to a specific relaxation mode. From the latter results one can infer whether the multiexponential relaxations represent sequential or parallel processes. This analysis establishes the interrelationship between the topography and topology of the energy landscapes and the hierarchy of the relaxation channels.

In this paper we apply the master equation approach to study the effects of the energy landscape topology and topography on the kinetics of folding, and on kinetic transitions of three alanine-hexapeptides analogs which involve polypeptides with neutral and charged groups and a cyclized polypeptide. We rely on the potential-energy landscapes of these molecular systems, which have been constructed using both a topological mapping analysis and a principal component analysis. It was found that the different topology and topography of the energy landscapes result in different "folding" time scales and that the systems with geometrical constraints (cyclization and opposite charges at the termini) "fold" more slowly than the unconstrained peptide. In addition, for each of the three polypeptide systems, the kinetics is nonexponential at the temperature range 400-600 K. The relaxation kinetics is characterized by logarithmic oscillations, which indicate hierarchical dynamics characterized by multiple time scales of fast (few ps) and slow (few mus) events. At higher temperatures, successive relaxation channels with similar characteristic time scales collapse into a single relaxation channel. While the kinetics of the unconstrained peptide at 600 K can be reasonably well described by a single exponential time scale, the kinetics of the constrained hexapeptides are inherently hierarchical and featured by multiple time scales even at high temperatures. (C) 2001 American Institute of Physics.

Abstract A set of 34 molecular dynamic (MD) simulations totaling 305 ns of simulation time of the prion protein-derived peptide PrP106?126 was performed with both explicit and implicit solvent models. The objective of these simulations is to investigate the relative stability of the α-helical conformation of the peptide and the mechanism for conversion from the helix to a random-coil structure. At neutral pH, the wild-type peptide was found to lose its initial helical structure very fast, within a few nanoseconds (ns) from the beginning of the simulations. The helix breaks up in the middle and then unwinds to the termini. The spontaneous transition into the random coil structure is governed by the hydrophobic interaction between His111 and Val122. The A117V mutation, which is linked to GSS disease, was found to destabilize the helix conformation of the peptide significantly, leading to a complete loss of helicity approximately 1 ns faster than in the wild-type. Furthermore, the A117V mutant exhibits a different mechanism for helix-coil conversion, wherein the helix begins to break up at the C-terminus and then gradually to unwind towards the N-terminus. In most simulations, the mutation was found to speed up the conversion through an additional hydrophobic interaction between Met112 and the mutated residue Val117, an interaction that did not exist in the wild-type peptide. Finally, the ?-sheet conformation of the wild-type peptide was found to be less stable at acidic pH due to a destabilization of the His111?Val122, since at acidic pH this histidine is protonated and is unlikely to participate in hydrophobic interaction. Proteins 2001;45:382?396. ? 2001 Wiley-Liss, Inc.

The effect of a solvation on the thermodynamics and kinetics of polyalanine (Ala(12)) is explored on the basis of its energy landscapes in vacuum and in an aqueous solution. Both energy landscapes are characterized by two basins, one associated with alpha -helical structures and the other with coil and beta -structures of the peptide. In both environments, the basin that corresponds to the alpha -helical structure is considerably narrower than the basin corresponding to the beta -state, reflecting their different contributions to the entropy of the peptide. In vacuum, the alpha -helical state of Ala(12) constitutes the native state, in agreement with common helical propensity scales, whereas in the aqueous medium, the alpha -helical state is destabilized, and the beta -state becomes the native state. Thus solvation has a dramatic effect on the energy landscape of this peptide, resulting in an inverted stability of the two states. Different folding and unfolding time scales for Ala(12) in hydrophilic and hydrophobic chemical environments are caused by the higher entropy of the native state in water relative to vacuum. The concept of a helical propensity has to be extended to incorporate environmental solvent effects.

Conformation constraints are known to affect the flexibility and bioactivity of peptides. In this study we analyzed the effect of conformation constraints on the topography of the energy landscapes of three analogous hexapeptides. The three analogs vary in the degree of constraint imposed on their conformational motion: linear alanine hexapeptide with neutral terminals (Ala6), linear alanine hexapeptide with charged terminals (chrg-Ala6), and cyclic alanine hexapeptide (cyc-Ala6). It was found that significantly different energy landscapes characterize each of the three peptides, leading to different folding behaviors. Since all three analogs would be encoded by the same gene, these results suggest that nongenomic post-translational modifications may play an important role in determining the properties of proteins as well as of their folding pathways. In addition, the present study indicates that the complexity of those energy landscapes that are dominated by funnel topography can be captured by one or two reaction coordinates, such as conformational similarity to the native state. However, for more complex landscapes characterized by multiple basins such a description is insufficient. This study also shows that similar views of the landscape topography were obtained by principal component analysis (based only on local minima) and by topological mapping analysis (based on minima and barrier information). Both methods were able to resolve the complex landscape topographies for all three peptides. (C) 2001 American Institute of Physics.

Conformation constraints and molecular flexibility strongly affect the bioactivity of flexible molecules. The present study offers a new conceptual framework, as well as a practical quantitative procedure, for discussing and quantifying these effects. The theory is formulated in terms of weighted overlaps between the volume in conformation space occupied by the flexible ligand and the pre-prescribed conformational requirements imposed by the host molecule ("region of bioactivity"). From this theory a quantitative structure activity relationship (QSAR) type descriptor, which quantifies the effect of conformation constraints on bioactivity, was derived and the resulting model was shown to be in excellent correlation with the observed activity of the molecules. Three characteristic scenarios for the relationship between flexibility and bioactivity are outlined and demonstrated in realistic systems: conformationally constrained alanine hexapeptides, a series of substance P analogues, and a set of conformationally constrained Arg-Gly-Asp containing peptides.

The extent to which structural modifications affect the topography of complex molecular energy landscapes is largely unknown. Through quantitative mapping of the potential energy surfaces of two related hexapeptides, we find that a conformational constraint can completely alter the character of the molecular energy landscape. The linear peptide exhibits a single-funnel potential topography, while its cyclic analog exhibits a shallow energy landscape with competing basins and unevenly distributed roughness.