Publications

Lung infection is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients and is mainly dominated by Pseudomonas aeruginosa. Treatment of CF-associated lung infections is problematic because the drugs are vulnerable to multidrug-resistant pathogens, many of which are major biofilm producers like P. aeruginosa. Antimicrobial peptides (AMPs) are essential components in all life forms and exhibit antimicrobial activity. Here we investigated a series of AMPs (d,l-K6L9), each composed of six lysines and nine leucines but differing in their sequence composed of l- and d-amino acids. The d,l-K6L9 peptides showed antimicrobial and antibiofilm activities against P. aeruginosa from CF patients. Furthermore, the data revealed that the d,l-K6L9 peptides are stable and resistant to degradation by CF sputum proteases and maintain their activity in a CF sputum environment. Additionally, the d,l-K6L9 peptides do not induce bacterial resistance. Overall, these findings should assist in the future development of alternative treatments against resistant bacterial biofilms.

Antimicrobial peptides (AMPs), which can be modified to kill a broad spectrum of microoganisms or a specific microorganism, are considered as promising alternatives to combat the rapidly widespread, resistant bacterial infections. However, there are still several obstacles to overcome. These include toxicity, stability, and the ability to interfere with the immune response and bacterial resistance. To overcome these challenges, we herein replaced the regular peptide bonds with isopeptide bonds to produce new AMPs based on the well-known synthetic peptides Amp1L and MSI-78 (pexiganan). Two new peptides Amp1EP and MSIEP were generated while retaining properties such as size, sequence, charge, and molecular weight. These new peptides have reduced toxicity toward murine macrophage (RAW 264.7) cells, human monocytic (THP-1) cells, and human red blood cells (hRBCs) and enhanced the stability toward proteolytic degradation. Importantly, the new peptides do not repress the pro-inflammatory cytokine and hence should not modulate the immune response. Structurally, the new peptides, Amp1EP and MSIEP, have a structure of random coils in contrast to the helical structures of the parental peptides as revealed by circular dichroism (CD) analysis. Their mode of action, assessed by flow cytometry, includes permeabilization of the bacterial membrane. Overall, we present here a new approach to modulate AMPs to develop antimicrobial peptides for future therapeutic purposes.

Multidrug resistant bacteria possess various mechanisms that can sense environmental stresses such as antibiotics and antimicrobial peptides and rapidly respond to defend themselves. Two known defense strategies are biofilm formation and lipopolysaccharide (LPS) modification. Though LPS modifications are observed in biofilm-embedded bacteria, their effect on biofilm formation is unknown. Using biochemical and biophysical methods coupled with confocal microscopy, atomic force microscopy, and transmission electron microscopy, we show that biofilm formation is promoted in a Pseudomonas aeruginosa PAO1 strain with a loss of function mutation in the arnB gene. This loss of function prevents the addition of the positively charged sugar 4-amino-4-deoxy-l-arabinose to lipid A of LPS under restrictive magnesium conditions. The data reveal that the arnB mutant, which is susceptible to antimicrobial peptides, forms a biofilm that is more robust than that of the wild type. This is in line with the observations that the arnB mutant exhibits outer surface properties such as hydrophobicity and net negative charge that promote the formation of biofilms. Moreover, when grown under Mg
²⁺ limitation, both the wild type and the arnB mutant exhibited a reduction in the level of membrane-bound polysaccharides. The data suggest that the loss of polysaccharides exposes the membrane and alters its biophysical properties, which in turn leads to more biofilm formation. In summary, we show for the first time that blocking a specific lipid A modification promotes biofilm formation, suggesting a trade-off between LPS remodeling and resistance mechanisms of biofilm formation.

Multidrug-resistant bacteria are a growing problem worldwide. One extensively studied resistance mechanism is biofilm colonizationmicrobial colonies formed by many Gram-positive and Gram-negative bacteria species. Cationic antimicrobial peptides (AMPs) are innate immune system molecules serving as a first line of defense in fighting invading pathogens. The AMPs underlying mechanism and biophysical properties required for anti-biofilm activity are not fully known. Here we present protocols for investigating AMPs biological activity against major stages of biofilm life cycle, namely, planktonic stage (MIC assay), initial adhesion to surfaces (bacterial attachment assay), and formation or degradation of sessile microcolonies (biofilm formation and degradation assays). Furthermore, we demonstrate experiments that allow determination and comparison between peptide biophysical properties (secondary structure, hydrophobicity, and oligomerization) and how they affect their mechanism (peptide-binding assays) of anti-biofilm activity.

The development of antibacterial drugs to overcome various pathogenic species, which inhabit the oral cavity, faces several challenges, such as salivary flow and enzymatic activity that restrict dosage retention. Owing to their amphipathic nature, antimicrobial peptides (AMPs) serve as the first line of defense of the innate immune system. The ability to synthesize different types of AMPs enables exploitation of their advantages as alternatives to antibiotics. Sustained release of AMPs incorporated in biodegradable polymers can be advantageous in maintaining high levels of the peptides. In this study, four potent ultra-short lipopeptides, conjugated to an aliphatic acid chain (16C) were incorporated in two different biodegradable polymers: poly (lactic acid co castor oil) (PLACO) and ricinoleic acid-based poly (ester-anhydride) (P(SA-RA)) for sustained release. The lipopeptide and polymer formulations were tested for antibacterial activity during one week, by turbidometric measurements of bacterial outgrowth, anti-biofilm activity by live/dead staining, biocompatibility by hemolysis and XTT colorimetric assays, mode of action by fluorescence-activated cell sorting (FACS) and release profile by a fluorometric assay. The results show that an antibacterial and anti-biofilm effect, as well as membrane disruption, can be achieved by the use of a formulation of lipopeptide incorporated in biodegradable polymer.

The frog skin-derived antimicrobial peptide esculentin-1a(1-21)NH₂ [Esc(1-21)], and its diastereomer Esc(1-21)-1c (containing two D-amino acids at positions 14 and 17), have been recently found to neutralize the toxic effect of Pseudomonas aeruginosa lipopolysaccharide (LPS), although to different extents. Here, we studied the three-dimensional structure of both peptides in complex with P. aeruginosa LPS, by transferred nuclear Overhauser effect spectroscopy. Lack of NOE peaks revealed that both the peptides adopted a random coil structure in aqueous solution. However, Esc(1-21) adopted an amphipathic helical conformation in LPS micelles with 5 basic Lys residues forming a hydrophilic cluster. In comparison, the diastereomer maintained an alpha helical conformation only at the N-terminal region, whereas the C-terminal portion was quite flexible. Isothermal titration calorimetry (ITC) revealed that the interaction of Esc(1-21) with LPS is an exothermic process associated with a dissociation constant of ∼ 4 μM. In contrast, Esc(1-21)-1c had almost 8 times weaker binding affinity to LPS micelles. Moreover, STD NMR data supported by docking analysis have identified those amino acid residues responsible for the peptide's binding to LPS micelles. Overall, the data provide important mechanistic insights on the interaction of esculentin-derived peptides with LPS and the reason for their different anti-endotoxin activity. These data might also assist to further design more potent antimicrobial peptides with antisepsis properties, which are highly needed to overcome the widespread concern of the available anti-infective agents.

Many bacteria live as biofilms to cope with unfavourable surroundings. Biofilms start from (i) a planktonic stage, (ii) initial adhesion to surfaces and (iii) formation of sessile micro-colonies that secrete extracellular polymeric substance (EPS), leading to bacterial resistance to antibiotics. Antimicrobial peptides (AMPs) are extensively studied with regard to planktonic bacteria but much less so with regard to biofilm formation. In the present study, we investigated how the above three steps are affected by the properties of the AMPs using a series of peptides composed of six lysines and nine leucines, which differ in their sequences and hence their biophysical properties. Treatment with bactericidal peptides at non-inhibitory concentrations resulted in reduced biofilm growth, for some starting from 25 nM which is 0.2 and 0.4% of their minimum inhibitory concentration (MIC 6.3 and 12.5 μM, respectively), continuing in a dose-dependent manner. We suggest that reduced bacterial adhesion to surfaces and decreased biofilm growth are due to the peptide's ability to coat either the biomaterial surface or the bacterium itself. Degradation of established biofilms by bactericidal and nonbactericidal peptides, within 1 h of incubation, occurs by either killing of embedded bacteria or detachment of live ones. In addition to shedding light on the mechanism of biofilm inhibition and degradation, these datamay assist in the design of anti-biofilm AMPs.

Assembly of transmembrane domains (TMDs) is a critical step in the function of membrane proteins. In recent years, the role of specific amino acids in TMD-TMD interactions has been better characterized, with more emphasis on polar and aromatic residues. Despite the high abundance of proline residues in TMDs, contribution of proline to TMD-TMD association has not been intensively studied. Here, we evaluated statistically the frequency of appearance, and experimentally the contribution of proline, compared to other hydrophobic amino acids (Gly, Ala, Val, Leu, Ile, and Met), with regard to TMD-TMD self-assembly. Our model system is the assembly motif (²²QxxS²⁵) found previously in TMDs of the Escherichia coli aspartate receptor (Tar-1). Statistically, our data revealed that all different motifs, except PxxS (P/S), have frequencies similar to their theoretical random expectancy within a database of 41916 sequences of TMDs, while PxxS motif is underrepresented. Experimentally, using the ToxR assembly system, the SDS-gel running pattern of biotin-conjugated TMD peptides, and FRET experiments between fluorescence-labeled peptides, we found that only the P/S motif preserves the dimerization ability of wild-type Tar-1 TMD. Although proline is known as a helix breaker in solution, Circular Dichroism spectroscopy revealed that the secondary structure of the P/S and the wild-type peptides are similar. All together, these data suggest that proline can stabilize TM self-assembly when localized to the interaction interface of a transmembrane oligomer. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Lipid-conjugated peptides have advanced the understanding of membrane protein functions and the roles of lipids in the membrane milieu. These lipopeptides modulate various biological systems such as viral fusion. A single function has been suggested for the lipid, binding to the membrane and thus elevating the local concentration of the peptide at the target site. In the present paper, we challenged this argument by exploring in-depth the antiviral mechanism of lipopeptides, which comprise sphinganine, the lipid backbone of DHSM (dihydrosphingomyelin), and an HIV-1 envelope-derived peptide. Surprisingly, we discovered a partnership between the lipid and the peptide that impaired early membrane fusion events by reducing CD4 receptor lateral diffusion and HIV-1 fusion peptide-mediated lipid mixing. Moreover, only the joint function of sphinganine and its conjugate peptide disrupted HIV-1 fusion protein assembly and folding at the later fusion steps. Via imaging techniques we revealed for the first time the direct localization of these lipopeptides to the virus-cell and cell-cell contact sites. Overall, the findings of the present study may suggest lipid-protein interactions in various biological systems and may help uncover a role for elevated DHSM in HIV-1 and its target cell membranes.

Fusion of the HIV membrane with that of a target T cell is an essential first step in the viral infection process. Here we describe single-particle tracking (SPT) studies of a 16-amino-acid peptide derived from the HIV fusion protein (FP₁₆), as it interacts with a supported lipid bilayer. FP₁₆ was found to spontaneously insert into and move within the bilayer with two different modes of diffusion, a fast mode with a diffusion coefficient typical of protein motion in membranes and a much slower one. We observed transitions between the two modes: slow peptides were found to speed up, and fast peptides could slow down. Hidden Markov model analysis was employed as a method for the identification of the two modes in single-molecule trajectories and analysis of their interconversion rates. Surprisingly, the diffusion coefficients of the two modes were found to depend differently on solution viscosity. Thus, whereas the fast diffusive mode behaved as predicted by the Saffman-Delbrück theory, the slow mode behaved according to the Stokes-Einstein relation. To further characterize the two diffusive modes, FP₁₆ molecules were studied in bilayers cooled through their liquid crystalline-to-gel phase transition. Our analysis suggested that the slow diffusive mode might originate from the formation of large objects, such as lipid domains or local protrusions, which are induced by the peptides and move together with them.

The T-cell receptor-CD3 complex (TCR-CD3) serves a critical role in protecting organisms from infectious agents. The TCR is a heterodimer composed of α- and β-chains, which are responsible for antigen recognition. Within the transmembrane domain of the α-subunit, a region has been identified to be crucial for the assembly and function of the TCR. This region, termed core peptide (CP), consists of nine amino acids (GLRILLLKV), two of which are charged (lysine and arginine) and are crucial for the interaction with CD3. Earlier studies have shown that a synthetic peptide corresponding to the CP sequence can suppress the immune response in animal models of T-cell-mediated inflammation, by disrupting proper assembly of the TCR. As a step towards the understanding of the source of the CP activity, we focused on CP in egg phosphatidylcholine/cholesterol (9:1, mol/mol) model membranes and determined its secondary structure, oligomerization state, and orientation with respect to the membrane. To achieve this goal, 15-residue segments of TCRα, containing the CP, were synthesized and spin-labeled at different locations with a nitroxide derivative. Electron spin-echo envelope modulation spectroscopy was used to probe the position and orientation of the peptides within the membrane, and double electron-electron resonance measurements were used to probe its conformation and oligomerization state. We found that the peptide is predominantly helical in a membrane environment and tends to form oligomers (mostly dimers) that are parallel to the membrane plane. Core peptide membrane topology: We have characterized the core peptide (CP) derived from the TCRα trans-membrane domain by CD, ATR-FTIR, and EPR methods. The CP, in model membranes, was shown to be helical and to form oligomers (mostly dimers) that are parallel to the membrane plane.

One of the routes by which HIV-1 is able to escape the immune response is by immunosuppression. The gp41 fusion protein of the HIV-1 envelope mediates virus entry by membrane fusion and also functions as an inhibitor of T cell activation. Here, we review the recent studies suggesting that some of the gp41 immunosuppressive processes are initiated by novel motifs, located within the hydrophobic regions of the protein. This indicates that the immunosuppressive process mediated by gp41 is much more complex than initially thought. Additionally, we propose a model illustrating the interactions and interferences of these regions with the T cell receptor complex.

The organization and orientation of membrane-inserted helices is important for better understanding the mode of action of membrane-active peptides and of protein-membrane interactions. Here we report on the application of ESEEM (electron spin-echo envelope modulation) and DEER (double electron-electron resonance) techniques to probe the orientation and oligomeric state of an α-helical trans-membrane model peptide, WALP23, under conditions of negative mismatch between the hydrophobic cores of the model membrane and the peptide. Using ESEEM, we measured weak dipolar interactions between spin-labeled WALP23 and ²H nuclei of either the solvent (D₂O) or of lipids specifically deuterated at the choline group. The ESEEM data obtained from the deuterated lipids were fitted using a model that provided the spin label average distance from a layer of ²H nuclei in the hydrophilic region of the membrane and the density of the ²H nuclei in the layer. DEER was used to probe oligomerization through the dipolar interaction between two spin-labels on different peptides. We observed that the center of WALP23 does not coincide with the bilayer midplane and its N-terminus is more buried than the C-terminus. In addition, the ESEEM data fitting yielded a ²H layer density that was much lower than expected. The DEER experiments revealed the presence of oligomers, the presence of which was attributable to the negative mismatch and the electrostatic dipole of the peptide. A discussion of a possible arrangement of the individual helices in the oligomers that is consistent with the ESEEM and DEER data is presented.

Infrared (IR) spectroscopy has been shown to be very reliable for the characterization, identification and quantification of structural data. Particularly, the Attenuated Total Reflectance (ATR) technique which became one of the best choices to study the structure and organization of membrane proteins and membrane-bound peptides in biologically relevant membranes. An important advantage of IR spectroscopy is its ability to analyze material under a very wide range of conditions including solids, liquids and gases. This method allows elucidation of component secondary structure elements of a peptide or protein in a global manner, and by using site specific isotope labeling allows determination of specific regions. A few advantages in using ATR-FTIR spectroscopy include; a relatively simple technique, allow the determination of peptide orientation in the membrane, allow the determination of secondary structures of very small peptides, and importantly, the method is sensitive to isotopic labeling on the scale of single amino acids. Many studies were reported on the use of ATR-FTIR spectroscopy in order to study the structure and orientation of membrane bound hydrophobic peptides and proteins. The list includes native and de-novo designed peptides, as well as those derived from trans-membrane domains of various receptors (TMDs). The present review will focus on several examples that demonstrate the potential and the simplicity in using the ATR-FTIR approach to determine secondary structures of proteins and peptides when bound, inserted, and oligomerized within membranes. The list includes (i) a channel forming protein/peptide: the Ca^{2 +} channel phospholamban, (ii) a cell penetrating peptide, (iii) changes in the structure of a transmembrane domain located within ordered and non-ordered domains, and (iv) isotope edited FTIR to directly assign structure to the membrane associated fusion peptide in context of a Key gp41 Structural Motif. Importantly, a unique advantage of infrared spectroscopy is that it allows a simultaneous study of the structure of lipids and proteins in intact biological membranes without an introduction of foreign perturbing probes. Because of the long IR wavelength, light scattering problems are virtually non-existent. This allows the investigation of highly aggregated materials or large membrane fragments. This article is part of a Special Issue entitled: FTIR in membrane proteins and peptide studies.

Understanding the structural organization of lipids in the cell and viral membranes is essential for elucidating mechanisms of viral fusion that lead to entry of enveloped viruses into their host cells. The HIV lipidome shows a remarkable enrichment in dihydrosphingomyelin, an unusual sphingolipid formed by a dihydrosphingosine backbone. Here we investigated the ability of dihydrosphingosine to incorporate into the site of membrane fusion mediated by the HIV envelope (Env) protein. Dihydrosphingosine as well as cholesterol, fatty acid, and tocopherol was conjugated to highly conserved, short HIV-1 Env-derived peptides with no antiviral activity otherwise. We showed that dihydrosphingosine exclusively endowed nanomolar antiviral activity to the peptides (IC₅₀ as low as 120 nM) in HIV-1 infection on TZM-bl cells and on Jurkat T cells, as well as in the cell-cell fusion assay. These sphingopeptides were active against enfuvirtide-resistant and wild-type CXCR4 and CCR5 tropic HIV strains. The anti-HIV activity was determined by both the peptides and their dihydrosphingosine conjugate. Moreover, their mode of action involved accumulation in the cells and viruses and binding to membranes enriched in sphingomyelin and cholesterol. The data suggest that sphingopeptides are recruited to the HIV membrane fusion site and provide a general concept in developing inhibitors of sphingolipid-mediated biological systems.

Cationic antimicrobial peptides (CAMPs) serve as the first line of defense of the innate immune system against invading microbial pathogens. Gram-positive bacteria can resist CAMPs by modifying their anionic teichoic acids (TAs) with D-alanine, but the exact mechanism of resistance is not fully understood. Here, we utilized various functional and biophysical approaches to investigate the interactions of the human pathogen Group B Streptococcus (GBS) with a series of CAMPs having different properties. The data reveal that: (i) D-alanylation of lipoteichoic acids (LTAs) enhance GBS resistance only to a subset of CAMPs and there is a direct correlation between resistance and CAMPs length and charge density; (ii) resistance due to reduced anionic charge of LTAs is not attributed to decreased amounts of bound peptides to the bacteria; and (iii) Dalanylation most probably alters the conformation of LTAs which results in increasing the cell wall density, as seen by Transmission Electron Microscopy, and reduces the penetration of CAMPs through the cell wall. Furthermore, Atomic Force Microscopy reveals increased surface rigidity of the cell wall of the wild-type GBS strain to more than 20-fold that of the dltA mutant. We propose that D-alanylation of LTAs confers protection against linear CAMPs mainly by decreasing the flexibility and permeability of the cell wall, rather than by reducing the electrostatic interactions of the peptide with the cell surface. Overall, our findings uncover an important protective role of the cell wall against CAMPs and extend our understanding of mechanisms of bacterial resistance.

Protein-protein interactions within the membrane are involved in many vital cellular processes. Consequently, deficient oligomerization is associated with known diseases. The interactions can be partially or fully mediated by transmembrane domains (TMD). However, in contrast to soluble regions, our knowledge of the factors that control oligomerization and recognition between the membrane-embedded domains is very limited. Due to the unique chemical and physical properties of the membrane environment, rules that apply to interactions between soluble segments are not necessarily valid within the membrane. This review summarizes our knowledge on the sequences mediating TMD-TMD interactions which include conserved motifs such as the GxxxG, QxxS, glycine and leucine zippers, and others. The review discusses the specific role of polar, charged and aromatic amino acids in the interface of the interacting TMD helices. Strategies to determine the strength, dynamics and specificities of these interactions by experimental (ToxR, TOXCAT, GALLEX and FRET) or various computational approaches (molecular dynamic simulation and bioinformatics) are summarized. Importantly, the contribution of the membrane environment to the TMD-TMD interaction is also presented. Studies utilizing exogenously added TMD peptides have been shown to influence in vivo the dimerization of intact membrane proteins involved in various diseases. The chirality independent TMD-TMD interactions allows for the design of novel short d- and l-amino acids containing TMD peptides with advanced properties. Overall these studies shed light on the role of specific amino acids in mediating the assembly of the TMDs within the membrane environment and their contribution to protein function.

The activity of antimicrobial peptides (AMPs) that contain a large proportion of histidine residues (pK _a ∼ 6) depends on the physiological pH environment. Advantages of these AMPs include high activity in slightly acidic areas of the human body and relatively low toxicity in other areas. Also, many AMPs are highly active in a multivalent form, but this often increases toxicity. Here we designed pH dependent amphiphilic compounds consisting of multiple ultrashort histidine lipopeptides on a triazacyclophane scaffold, which showed high activity toward Aspergillus fumigatus and Cryptococcus neoformans at acidic pH, yet remained nontoxic. In vivo, treatment with a myristic acid conjugated trivalent histidine-histidine dipeptide resulted in 55% survival of mice (n = 9) in an otherwise lethal murine lung Aspergillus infection model. Fungal burden was assessed and showed completely sterile lungs in 80% of the mice (n = 5). At pH 5.5 and 7.5, differing peptide-membrane interactions and peptide nanostructures were observed. This study underscores the potential of unique AMPs to become the next generation of clinical antimicrobial therapy.

Studies of membrane peptide interactions at the molecular level are important for understanding essential processes such as membrane disruption or fusion by membrane active peptides. In a previous study, we combined several electron paramagnetic resonance (EPR) techniques, particularly continuous wave (CW) EPR, electron spin echo envelope modulation (ESEEM), and double electron-electron resonance (DEER) with Monte Carlo (MC) simulations to probe the conformation, insertion depth, and orientation with respect to the membrane of the membrane active peptide melittin. Here, we combined these EPR techniques with cryogenic transmission electron microscopy (cryo-TEM) to examine the effect of the peptide/phospholipid (P/PL) molar ratio, in the range of 1:400 to 1:25, on the membrane shape, lipids packing, and peptide orientation and penetration. Large unilamellar vesicles (LUVs) of DPPC/PG (7:3 dipalmitoylphosphatidylcholine/egg phosphatidylglycerol) were used as model membranes. Spin-labeled peptides were used to probe the peptide behavior whereas spin-labeled phspholipids were used to examine the membrane properties. The cryo-TEM results showed that melittin causes vesicle rupture and fusion into new vesicles with ill-defined structures. This new state was investigated by the EPR methods. In terms of the peptide, CW EPR showed decreased mobility, and ESEEM revealed increased insertion depth as the P/PL ratio was raised. DEER measurements did not reveal specific aggregates of melittin, thus excluding the presence of stable, well-defined pore structures. In terms of membrane properties, the CW EPR reported reduced mobility in both polar head and alkyl chain regions with increasing P/PL. ESEEM measurements showed that, as the P/PL ratio increased, a small increase in water content in the PL headgroup region took place and no change was observed in the alkyl chains part close to the hydrophilic region. In terms of lipid local density, opposite behavior was observed for the polar head and alkyl chain regions with increasing P/PL; while the DPPC density increased in the polar head region, it decreased in the alkyl chain region. These results are consistent with disruption of the lipid order and segregation of the PL constituents of the membrane as a consequence of the melittin binding. This work further demonstrates the applicability and potential of pulse EPR techniques for the study of peptide-membrane interactions.

The viral peptide fusion inhibitor Fuzeon (T-20/DP178/enfuvirtide) is an essential part of the drug combination that has significantly increased the quality of life and life span of many acquired immuno-deficiency syndrome (AIDS) patients. Its development as a drug preceded the elucidation of its precise inhibitory mechanism, as well as its molecular targets. The initial model was that Fuzeon inhibits human immunodeficiency virus (HIV) entry by targeting one site within the viral transmembrane envelope protein. Herein, we describe the emerging discoveries that extend this model towards a multifaceted mechanism for the drug in targeting HIV. This significantly advances the understanding of how viruses enter host cells and opens a new window of opportunity for designing future viral fusion inhibitors.

Fusion of human immunodeficiency virus (HIV-1) with target cells is mediated by the gp41 transmembrane envelope protein. The loop region within gp41 contains 2 crucial cysteines that play an unknown role in HIV-cell fusion. On the basis of cell-cell fusion assay, using human T-cell lines [Jurkat E6-1 and Jurkat HXBc2(4)], and virus-cell fusion assay, using fully infectious HIV-1 HXBc2 virus and TZM-bl human cell line, we provide evidence that the oxidation state of the disulfide bond within a loop domain peptide determines its activity. The oxidized (closed) form inhibits fusion, while the reduced (opened) form enhances hemifusion. These opposite activities reach 60% difference in viral fusion. Both forms of the loop domain interact with gp41: the opened form enhances gp41 folding into a bundle, whereas the closed form inhibits this folding. Therefore, the transformation of the cysteines from a reduced to an oxidized state enables the loop to convert from opened to closed conformations, which assists gp41 to fold and induces hemifusion. The significant conservation of the loop region within many envelope proteins suggests a general mechanism, which is exploited by viruses to enhance entry into their host cells.

We present high field DEER (double electron-electron resonance) distance measurements using Gd³⁺ (S = 7/2) spin labels for probing peptides' conformations in solution. The motivation for using Gd³⁺ spin labels as an alternative for the standard nitroxide spin labels is the sensitivity improvement they offer because of their very intense EPR signal at high magnetic fields. Gd³⁺ was coordinated by dipicolinic acid derivative (4MMDPA) tags that were covalently attached to two cysteine thiol groups. Cysteines were introduced in positions 15 and 27 of the peptide melittin and then two types of spin labeled melittins were prepared, one labeled with two nitroxide spin labels and the other with two 4MMDPA-Gd³⁺ labels. Both types were subjected to W-band (95 GHz, 3.5 T) DEER measurements. For the Gd³⁺ labeled peptide we explored the effect of the solution molar ratio of Gd ³⁺ and the labeled peptide, the temperature, and the maximum dipolar evolution time T on the DEER modulation depth. We found that the optimization of the [Gd³⁺]/[Tag] ratio is crucial because excess Gd³⁺ masked the DEER effect and too little Gd³⁺ resulted in the formation of Gd³⁺-tag₂ complexes, generating peptide dimers. In addition, we observed that the DEER modulation depth is sensitive to spectral diffusion processes even at Gd³⁺ concentrations as low as 0.2 mM and therefore experimental conditions should be chosen to minimize it as it decreases the DEER effect. Finally, the distance between the two Gd³⁺ ions, 3.4 nm, was found to be longer by 1.2 nm than the distance between the two nitroxides. The origin and implications of this difference are discussed.

Background: Streptococcus agalactiae (Group B Streptococcus) is a leading cause of sepsis and meningitis in newborns. Most bacterial pathogens, including gram-positive bacteria, have long filamentous structures known as pili extending from their surface. Although pili are described as adhesive organelles, they have been also implicated in many other functions including thwarting the host immune responses. We previously characterized the pilus-encoding operon PI-2a (gbs1479-1474) in strain NEM316. This pilus is composed of three structural subunit proteins: PilA (Gbs1478), PilB (Gbs1477), and PilC (Gbs1474), and its assembly involves two class C sortases (SrtC3 and SrtC4). PilB, the bona fide pilin, is the major component whereas PilA, the pilus associated adhesin, and PilC the pilus anchor are both accessory proteins incorporated into the pilus backbone. Methodology/Principal Findings: In this study, the role of the major pilin subunit PilB was tested in systemic virulence using 6-weeks old and newborn mice. Notably, the non-piliated ΔpilB mutant was less virulent than its wild-type counterpart in the newborn mice model. Next, we investigated the possible role(s) of PilB in resistance to innate immune host defenses, i.e. resistance to macrophage killing and to antimicrobial peptides. Phagocytosis and survival of wild-type NEM316 and its isogenic ΔpilB mutant in immortalized RAW 264.7 murine macrophages were not significantly different whereas the isogenic ΔsodA mutant was more susceptible to killing. These results were confirmed using primary peritoneal macrophages. We also tested the activities of five cationic antimicrobial peptides (AMP-1D, LL-37, colistin, polymyxin B, and mCRAMP) and found no significant difference between WT and ΔpilB strains whereas the isogenic dltA mutant showed increased sensitivity. Conclusions/Significance: These results question the previously described role of PilB pilus in resistance to the host immune defenses. Interestingly, PilB was found to be important for virulence in the neonatal context.

Viruses have evolved several strategies to modify cellular processes and evade the immune response in order to successfully infect, replicate, and persist in the host. By utilizing in-silico testing of a transmembrane sequence library derived from virus protein sequences, we have pin-pointed a nine amino-acid motif shared by a group of different viruses; this motif resembles the transmembrane domain of the α-subunit of the T-cell receptor (TCRα). The most striking similarity was found within the immunodeficiency virus (SIV and HIV) glycoprotein 41 TMD (gp41 TMD). Previous studies have shown that stable interactions between TCRα and CD3 are localized to this nine amino acid motif within TCRα, and a peptide derived from it (TCRα TMD, GLRILLLKV) interfered and intervened in the TCR function when added exogenously. We now report that the gp41 TMD peptide co-localizes with CD3 within the TCR complex and inhibits T cell proliferation in vitro. However, the inhibitory mechanism of gp41 TMD differs from that of the TCRα TMD and also from the other two known immunosuppressive regions within gp41.

Three Arg-rich nonapeptides, containing the same amino acid composition but different sequences, PFWRIRIRR-amide (PR-9), RRPFWIIRR-amide (RR-9) and PRFRWRIRI-amide (PI-9), are able to induce segregation of anionic lipids from zwitterionic lipids, as shown by changes in the phase transition properties of lipid mixtures detected by differential scanning calorimetry and freeze fracture electron microscopy. The relative Minimal Inhibitory Concentration (MIC) of these three peptides against several strains of Gram positive bacteria correlated well with the extent to which the lipid composition of the bacterial membrane facilitated peptide-induced clustering of anionic lipids. The lower activity of these three peptides against Gram negative bacteria could be explained by the retention of these peptides in the LPS layer. The membrane morphologies produced by PR-9 as well as by a cathelicidin fragment, KR-12 that had previously been shown to induce anionic lipid clustering, was directly visualized using freeze fracture electron microscopy. This work shows the insensitivity of phase segregation to the specific arrangement of the cationic charges in the peptide sequence as well as to their tendency to form different secondary structures. It also establishes the role of anionic lipid clustering in the presence of zwitterionic lipids in determining antimicrobial selectivity.

Fusion between viral and host cell membranes is the initial step of human immunodeficiency virus infection and is mediated by the gp41 protein, which is embedded in the viral membrane. The ∼ 20-residue N-terminal fusion peptide (FP) region of gp41 binds to the host cell membrane and plays a critical role in fusion catalysis. Key gp41 fusion conformations include an early pre-hairpin intermediate (PHI) characterized by extended coiled-coil structure in the region C-terminal of the FP and a final hairpin state with compact six-helix bundle structure. The large "N70" (gp41 1-70) and "FP-Hairpin" constructs of the present study contained the FP and respectively modeled the PHI and hairpin conformations. Comparison was also made to the shorter "FP34" (gp41 1-34) fragment. Studies were done in membranes with physiologically relevant cholesterol content and in membranes without cholesterol. In either membrane type, there were large differences in fusion function among the constructs with little fusion induced by FP-Hairpin, moderate fusion for FP34, and very rapid fusion for N70. Overall, our findings support acceleration of gp41-induced membrane fusion by early PHI conformation and fusion arrest after folding to the final six-helix bundle structure. FP secondary structure at Leu7 of the membrane-associated constructs was probed by solid-state nuclear magnetic resonance and showed populations of molecules with either β-sheet or helical structure with greater β-sheet population observed for FP34 than for N70 or FP-Hairpin. The large differences in fusion function among the constructs were not obviously correlated with FP secondary structure. Observation of cholesterol-dependent FP structure for fusogenic FP34 and N70 and cholesterol-independent structure for non-fusogenic FP-Hairpin was consistent with membrane insertion of the FP for FP34 and N70 and with lack of insertion for FP-Hairpin. Membrane insertion of the FP may therefore be associated with the early PHI conformation and FP withdrawal with the final hairpin conformation.

We have designed and chemically synthesized an artificial β-defensin based on a minimal template derived from the comparative analysis of over 80 naturally occurring sequences. This molecule has the disulfide-bridged β-sheet core structure of natural β-defensins and shows a robust salt-sensitive antimicrobial activity against bacteria and yeast, as well as a chemotactic activity against immature dendritic cells. An SAR (structure-activity relationship) study using two truncated fragments or a Cys → Ser point-mutated analogue, from which one or two of the three disulfide bridges were absent, indicated that altering the structure resulted in a different type of membrane interaction and a switch to different modes of action towards both microbial and host cells, and that covalent dimerization could favour antimicrobial activity. Comparison of the structural, aggregational and biological activities of the artificial defensin with those of three human β-defensins and their primate orthologues provided useful information on how their mode of action may relate to specific structural features.

Ribosomally synthesized antimicrobial peptides (AMPs) represent an essential component of the ancient and non-specific innate immune system in all forms of life, with the primary role of killing infectious microorganisms. Amphibian skin is one of the richest storehouses for them. Each frog species produces its own set of peptides with up to 10 isoforms, as in the case of the species Rana temporaria. Nowadays, human health is facing two major threats: (i) the increasing emergence of resistant pathogens to one or more available drugs, and (ii) the onset of septic shock, which is associated with the release of lipopolysaccharide (LPS) from the cell walls of Gram-negative bacteria, particularly upon antibiotic treatment. AMPs are considered as potential new anti-infective compounds with a novel mode of action, because many of them can kill bacteria and, at the same time, neutralize the toxic effects of LPS. Recent studies have suggested that the production of large number of structurally similar AMPs within the same animal is a strategy used by nature to increase the spectrum of antimicrobial activities, by using combinations of the peptide's isoforms. The biological rationale for their coexistence within the same organism is discussed. In addition, the distinctive and attractive synergistic effects of temporins in both antimicrobial and anti-endotoxin activities are reviewed, along with their plausible underlying molecular mechanism.

Membrane fusion between the human immunodeficiency virus (HIV) and the target cell plasma membrane is correlated with conformational changes in the HIV gp41 glycoprotein, which include an early exposed conformation (prehairpin) and a late low energy six helix bundle (SHB) conformation also termed hairpin. Peptides resembling regions from the exposed prehairpin have been previously studied for their interaction with membranes. Here we report on the expression, purification, SHB stability, and membrane interaction of the full-length ectodomain of the HIV gp41 and its two deletion mutants, all in their SHB-folded state. The interaction of the proteins with zwitterionic and negatively charged membranes was examined by using various biophysical methods including circular dichroism spectroscopy, differential scanning calorimetry, lipid mixing of large unilamellar vesicles, and atomic force microscopy (AFM). All experiments were done in an acidic environment in which the protein remains in its soluble trimeric state. The data reveal that all three proteins fold into a stable coiled-coil core in aqueous solution and retain a stable helical fold with reduced coiled-coil characteristics in a zwitterionic and negatively charged membrane mimetic environment. Furthermore, in contrast with the extended exposed N-terminal domain, the folded gp41 ectodomain does not induce lipid mixing of zwitterionic membranes. However, it disrupts and induces lipid mixing of negatively charged phospholipid membranes (∼100-fold more effective than fusion peptide alone), which are known to be expressed more in HIV-1-infected T cells or macrophages. The results support the emerging model in which one of the roles of gp41 folding into the SHB conformation is to slow down membrane disruption effects induced by early exposed gp41. However, it can further affect membrane morphology once exposed to negatively charged membranes during late stages.

Conformational changes in the HIV gp41 protein are directly correlated with fusion between the HIV and target cell plasma membranes, which is the initial step of infection. Key gp41 fusion conformations include an early extended conformation termed prehairpin which contains exposed regions and a final low-energy conformation termed hairpin which has a compact six-helix bundle structure. Current fusion models debate the roles of hairpin and prehairpin conformations in the process of membrane merger. In the present work, gp41 constructs have been engineered which correspond to fusion relevant parts of both prehairpin and hairpin conformations and have been analyzed for their ability to induce lipid mixing between membrane vesicles. The data correlate membrane fusion function with the prehairpin conformation and suggest that one of the roles of the final hairpin conformation is sequestration of membrane-perturbing gp41 regions with consequent loss of the membrane disruption induced earlier by the prehairpin structure. To our knowledge, this is the first biophysical study to delineate the membrane fusion potential of gp41 constructs modeling key fusion conformations.

One of the most extensively studied receptor tyrosine kinases is EGFR/ErbB1. Although our knowledge of the role of the extracellular domains and ligands in ErbB1 activation has increased dramatically based on solved domain structures, the exact mechanism of signal transduction across the membrane remains unknown. The transmembrane domains are expected to play an important role in the dimerization process, but the contribution of ErbB1 TM domain to dimer stability is not known, with published results contradicting one another. We address this controversy by showing that ErbB1 TM domain dimerizes in lipid bilayers and by calculating its contribution to stability as -2.5 kcal/mol. The stability calculations use two different methods based on Förster resonance energy transfer, which give the same result. The ErbB1 TM domain contribution to stability exceeds the change in receptor tyrosine kinases dimerization propensities that can convert normal signaling processes into pathogenic processes, and is thus likely important for biological function.

Aspergillus fumigatus is an opportunistic fungal pathogen responsible for invasive aspergillosis in immunocompromised individuals. The inefficiency of antifungal agents and high mortality rate resulting from invasive aspergillosis remain major clinical concerns. Recently, we reported on a new family of ultrashort cationic lipopeptides active in vitro against fungi. Mode of action studies supported a membranolytic or a detergent-like effect. Here, we screened several lipopeptides in vitro for their anti-A fumigatus activity. To investigate the therapeutic properties of the selected peptides in vivo, we challenged immunosuppressed C57BL/6 wild-type mice intranasals with DsRed-labeled A. fumigatus conidia and subsequently treated the animals locally with the lipopeptides. Confocal microscopic analysis revealed the degradation of DsRed-labeled hyphal forms and residual conidia in the lungs of the mice. The most efficient peptide was tested further using a survival assay and was found to significantly prolong the life of the treated animals, whereas no mice survived with the current standard antifungal treatment with amphotericin B. Moreover, as opposed to the drug-treated lungs, the peptide-treated lungs did not display any toxicity of the peptide. Our results highlight the potential of this family of lipopeptides for the treatment of pulmonary invasive aspergillosis.

Endotoxin [lipopolysaccharide (LPS)] covers more than 90% of the outer monolayer of the outer membrane of Gram-negative bacteria, and it plays a dual role in its pathogenesis: as a protective barrier against antibiotics and as an effector molecule, which is recognized by and activates the innate immune system. The ability of host-defense antimicrobial peptides to bind LPS on intact bacteria and in suspension has been implicated in their antimicrobial and LPS detoxification activities. However, the mechanisms involved and the properties of the peptides that enable them to traverse the LPS barrier or to neutralize LPS endotoxic activity are not yet fully understood. Here we investigated a series of antimicrobial peptides and their analogues with drastically altered sequences and structures, all of which share the same amino acid composition (K₆L₉). The list includes both all-L-amino acid peptides and their diastereomers (composed of both L- and D-amino acids). The peptides were investigated functionally for their antibacterial activity and their ability to block LPS-dependent TNF-α secretion by macrophages. Fluorescence spectroscopy and transmission electron microscopy were used to detect their ability to bind LPS and to affect its oligomeric state. Their secondary structure was characterized in solution, in LPS suspension, and in LPS multibilayers by using CD and FTIR spectroscopy. Our data reveal specific biophysical properties of the peptides that are required to kill bacteria and/or to detoxify LPS. Besides shedding light on the mechanisms of these two important functions, the information gathered should assist in the development of AMPs with potent antimicrobial and LPS detoxification activities.

The core complex is a structure involved in the fusion mechanism of many viruses, as well as in intracellular vesicle fusion. A powerful approach for studying the dynamic stages of HIV-1-cell fusion utilizes DP178, a core complex inhibitory peptide derived from the known sequence of the virus. Strikingly, we show that fatty acids can replace the entire C-terminal region of DP178, known to play a crucial role in the activity of the peptide. The inhibitory activity correlated with the length of the fatty acid, with the direction of fatty acid attachment (N- or C-terminus) and, as envisioned by a new triple staining assay, with the concentration of the peptides on cells. Our findings indicate, for the first time, the C-terminal boundary of the endogenous core structure in situ and establish that the C-terminal region of DP178 functions mainly as an anchor to the cell membrane. Apart from the mechanistic implications, such short lipopeptides provide new, promising fusion inhibitors. Because the fusion mechanism of HIV-1 is shared by other pathogen-enveloped viruses and by intracellular vesicle fusion, our results might influence the research and therapeutic efforts in these systems as well.

Plant diseases constitute an emerging threat to global food security. Many of the currently available antimicrobial agents for agriculture are highly toxic and nonbiodegradable and cause extended environmental pollution. Moreover, an increasing number of phytopathogens develop resistance to them. Recently, we have reported on a new family of ultrashort antimicrobial lipopeptides which are composed of only four amino acids linked to fatty acids (A. Makovitzki, D. Avrahami, and Y. Shai, Proc. Natl. Acad. Sci. USA 103:15997-16002, 2006). Here, we investigated the activities in vitro and in planta and the modes of action of these short lipopeptides against plant-pathogenic bacteria and fungi. They act rapidly, at low micromolar concentrations, on the membranes of the microorganisms via a lytic mechanism. In vitro microscopic analysis revealed wide-scale damage to the microorganism's membrane, in addition to inhibition of pathogen growth. In planta potent antifungal activity was demonstrated on cucumber fruits and leaves infected with the pathogen Botrytis cinerea as well as on corn leaves infected with Cochliobolus heterostrophus. Similarly, treatment with the lipopeptides of Arabidopsis leaves infected with the bacterial leaf pathogen Pseudomonas syringae efficiently and rapidly reduced the number of bacteria. Importantly, in contrast to what occurred with many native lipopeptides, no toxicity was observed on the plant tissues. These data suggest that the ultrashort lipopeptides could serve as native-like antimicrobial agents economically feasible for use in plant protection.

Low molecular weight compounds were isolated by high-performance liquid chromatography from the maggot or haemolymph extracts of Lucilia sericata (Meigen) (Diptera: Calliphoridae). Using gas chromatography-mass spectrometry analysis, three compounds were obtained: p-hydroxybenzoic acid (molecular weight 138 Da), p-hydroxyphenylacetic acid (molecular weight 152 Da) and octahydro-dipyrrolo[1,2-a;1,2-d] pyrazine-5,10-dione (molecular weight 194 Da), also known as the cyclic dimer of proline (or proline diketopiperazine or cyclo[Pro,Pro]). All three molecules revealed antibacterial activity when tested against Micrococcus luteus and/or Pseudomonas aeruginosa, and the effect was even more pronounced when these molecules were tested in combination and caused lysis of these bacteria.

Protein-protein interactions in the membrane are pivotal for the cellular response to receptor-sensed stimuli. Recently, it has been demonstrated that an all-D-amino acids analogue of the TCRα transmembrane peptide (CP) is recruited to the TCR complex and inhibits T-cell activation in vitro and in vivo, similarly to the wild-type CP peptide. Here we investigated the relative contributions of the secondary structure of CP compared to its side chains in the association of CP with the TCR. We disrupted the secondary structure of CP by replacing two positive residues, needed for the interaction of CP with the TCR complex, by their D-enantiomers (2D-CP). Structure disruption was demonstrated by CD and FTIR spectroscopy, and molecular dynamics simulation in a bilayer environment. In vitro, 2D-CP colocalized with the TCR (visualized with confocal microscopy), immunoprecipitated with TCR but not MHC I, and inhibited T-cell activation. The peptide was effective also in vivo: it inhibited adjuvant arthritis in rats and delayed type hypersensitivity in BALB/c mice. Moreover, 2D-CP manifested greater immunosuppressive activity than wild-type CP, both in vivo and in vitro, which can be attributed to the greater solubility and resistance to degradation of 2D-CP. In molecular terms, these findings suggest that, under certain conditions, protein-protein interactions within the membrane might be more dependent on side chain interactions than on a specific secondary structure. The new altered secondary structure probably determines how the Lys and the Arg are positioned with respect to each other, so they can interact with the TM domain of the receptor. In clinical terms, the increased solubility and resistance to degradation of D-stereoisomers might be exploited in the targeted inactivation of pathogenic signaling pathways such as those arising from TCR-triggered activation of T-cells in immune-mediated disorders.

Endogenous peptide antibiotics (termed also host-defense or antimicrobial peptides) are known as evolutionarily old components of innate immunity. They were found initially in invertebrates, but later on also in vertebrates, including humans. This secondary, chemical immune system provides organisms with a repertoire of small peptides that act against invasion (for both offensive and defensive purposes) by occasional and obligate pathogens. Each antimicrobial peptide has a broad but not identical spectrum of antimicrobial activity, predominantly against bacteria, providing the host maximum coverage against a rather broad spectrum of microbial organisms. Many of these peptides interact with the target cell membranes and increase their permeability, which results in cell lysis. A second important family includes lipopeptides. They are produced in bacteria and fungi during cultivation on various carbon sources, and possess a strong antifungal activity. Unfortunately, native lipopeptides are non-cell selective and therefore extremely toxic to mammalian cells. Whereas extensive studies have emerged on the requirements for a peptide to be antibacterial, very little is known concerning the parameters that contribute to antifungal activity. This review summarizes recent studies aimed to understand how antimicrobial peptides and lipopeptides select their target cell. This includes a new group of lipopeptides highly potent against pathogenic fungi and yeast. They are composed of inert cationic peptides conjugated to aliphatic acids with different lengths. Deep understanding of the molecular mechanisms underlying the differential cells specificity of these families of host defense molecule is required to meet the challenges imposed by the life-threatening infections.

Temporins are short and homologous antimicrobial peptides (AMPs) isolated from the frog skin of Rana genus. To date, very little is known about the biological significance of the presence of closely related AMPs in single living organisms. Here we addressed this question using temporins A, B, and L isolated from Rana temporaria. We found that temporins A and B are only weakly active toward Gram-negative bacteria. However, a marked synergism occurs when each is mixed with temporin L. To shed light on the underlying mechanisms involved in these activities, we used various experimental strategies to investigate: (i) the effect of the peptides' interaction on both the viability and membrane permeability of intact bacteria and spheroplasts; (ii) their interaction with lipopolysaccharides (LPS) and the effect of LPS on the oligomeric state of temporins, alone or combining one with another; (iii) their structure in solution and when bound to LPS, by using circular dichroism and ATR-FTIR spectroscopies. Our data reveal that temporin L synergizes with Aand B by preventing their oligomerization in LPS. This should promote their translocation across the outer membrane into the cytoplasmic membrane. To the best of our knowledge, this is the first study that explains how a combination of native AMPs from the same species can overcome bacterial resistance imposed by the LPS leaflet.

Central to our understanding of human immunodeficiency virus-induced fusion is the high resolution structure of fragments of the gp41 fusion protein folded in a low energy core conformation. However, regions fundamental to fusion, like the fusion peptide (FP), have yet to be characterized in the context of the cognate protein regardless of its conformation. Based on conformation-specific monoclonal antibody recognition, we identified the polar region consecutive to the N36 fragment as a stabilizer of trimeric coiled-coil assembly, thereby enhancing inhibitory potency. This tertiary organization is retained in the context of the hydrophobic FP (N70 fragment). Our data indicate that the N70 fragment recapitulates the expected organization of this region in the viral fusion intermediate (N-terminal half of the pre-hairpin intermediate (N-PHI)), which happens to be the prime target for fusion inhibitors. Regarding the low energy conformation, we show for the first time core formation in the context of the FP (N70 core). The α-helical and coiled-coil stabilizing polar region confers substantial thermal stability to the core, whereas the hydrophobic FP does not add further stability. For the two key fusion conformations, N-PHI and N70 core, we find that the FP adopts a nonhelical structure and directs higher order assembly (assembly of coiled coils in N-PHI and assembly of bundles in the N70 core). This supra-molecular organization of coiled coils or folded cores is seen only in the context of the FP. This study is the first to characterize the FP region in the context of the folded core and provides a basic understanding of the role of the elusive FP for key gp41 fusion conformations.

Human immunodeficiency virus 1 gp41 folds into a six-helix bundle whereby three C-terminal heptad repeat regions pack in an antiparallel manner against the coiled-coil formed by three N-terminal heptad repeats (NHR). Peptides that inhibit bundle formation contributed significantly to the understanding of the entry mechanism of the virus. DP178, which partially overlaps C-terminal heptad repeats, prevents bundle formation through an undefined mechanism; additionally it has been suggested to bind other ENV regions and arrest fusion in an unknown manner. We used two structurally altered DP178 peptides; in each, two sequential amino acids were substituted into their D configuration, D-SQ in the hydrophilic N-terminal region and D-LW in the hydrophobic C-terminal. Importantly, we generated an elongated NHR peptide, N54, obtaining the full N-helix docking site for DP178. Interestingly, D-LW retained wild type fusion inhibitory activity, whereas D-SQ exhibited significantly reduced activity. In correlation with the inhibitory data, CD spectroscopy and fluorescence studies revealed that all the DP178 peptides interact with N54, albeit with different stabilities of the bundles. We conclude that strong binding of DP178 N-terminal region to the endogenous NHR, without significant contribution of the C-terminal sequence of DP178 to core formation, is vital for DP178 inhibition. The finding that D-amino acid incorporation in the C terminus did not affect activity or membrane binding as revealed by surface plasmon resonance correlates with an additional membrane binding site, or membrane anchoring role, for the C terminus, which works synergistically with the N terminus to inhibit fusion.

Membrane-active peptides participate in many cellular processes, and therefore knowledge of their mode of interaction with phospholipids is essential for understanding their biological function. Here we present a new methodology based on electron spin-echo envelope modulation to probe, at a relatively high resolution, the location of membrane-bound lytic peptides and to study their effect on the water concentration profile of the membrane. As a first example, we determined the location of the N-terminus of two membrane-active amphipathic peptides, the 26-mer bee venom melittin and a de novo designed 15-mer D,L-amino acid amphipathic peptide (5D-L₉K₆C), both of which are antimicrobial and bind and act similarly on negatively charged membranes. A nitroxide spin label was introduced to the N-terminus of the peptides and measurements were performed either in H₂O solutions with deuterated model membranes or in D₂O solutions with nondeuterated model membranes. The lipids used were dipalmitoyl phosphatidylcholine (DPPC) and phosphatidylglycerol (PG), (DPPC/PG (7:3 w/w)), egg phosphatidylcholine (PC) and PG (PC/PG (7:3 w/w)), and phosphatidylethanolamine (PE) and PG (PE/PG, 7:3w/w). The modulation induced by the ²H nuclei was determined and compared with a series of controls that produced a reference "ruler". Actual estimated distances were obtained from a quantitative analysis of the modulation depth based on a simple model of an electron spin situated at a certain distance from the bottom of a layer with homogeneously distributed deuterium nuclei. The N-terminus of both peptides was found to be in the solvent layer in both the DPPC/PG and PC/PG membranes. For PE/PG, a further displacement into the solvent was observed. The addition of the peptides was found to change the water distribution in the membrane, making it "flatter" and increasing the penetration depth into the hydrophobic region.

The fusion peptide (FP) in the N terminus of the HIV envelope glycoprotein, gp41, functions together with other gp41 domains to fuse the virion with the host cell membrane. We now report that FP colocalizes with CD4 and TCR molecules, coprecipitates with the TCR, and inhibits antigen-specific T cell proliferation and proinflammatory cytokine secretion in vitro. These effects are specific: T cell activation by PMA/ionomycin or mitogenic antibodies is not affected by FPs, and FPs do not interfere with antigen-presenting cell function. In vivo, FPs inhibit the activation of arthritogenic T cells in the autoimmune disease model of adjuvant arthritis and reduce the disease-associated IFN-γ response. Hence, FPs might play 2 roles in HIV infection: mediating membrane fusion while downregulating T cell responses to itself that could block infection. Disassociated from HIV, however, the FP molecule provides a novel reagent for downregulating undesirable immune responses, exemplified here by adjuvant arthritis.

Assembly of transmembrane (TM) domains is a critical step in the function of membrane proteins, and therefore, determining the amino acid motifs that mediate this process is important. Studies along this line have shown that the GXXXG motif is involved in TM assembly. In this study we characterized the minimal dimerization motif in the bacterial Tar-1 homodimer TM domain, which does not contain a GXXXG sequence. We found that a short polar motif QXXS is sufficient to induce stable TM-TM interactions. Statistical analysis revealed that this motif appears to be significantly over-represented in a bacterial TM data base compared with its theoretical expectancy, suggesting a general role for this motif in TM assembly. A truncated short TM peptide (9 residues) that contains the QXXS motif interacted slightly with the wild-type Tar-1. However, the same short TM peptide regained wild-type-like activity when conjugated to an octanoyl aliphatic moiety. Biophysical studies indicated that this modification compensated for the missing hydrophobicity, stabilized α-helical structure, and enabled insertion of the peptide into the membrane core. These findings serve as direct evidence that even a short peptide containing a minimal recognition motif is sufficient to inhibit the proper assembly of TM domains. Interestingly, electron microscopy revealed that above the critical micellar concentration, the TM lipopeptide forms a network of nanofibers, which can serve for the slow release of the active lipopeptide.

The HIV gp41 protein mediates fusion with target host cells. The region primarily involved in directing fusion, the fusion peptide (FP), is poorly understood at the level of structure and function due to its toxic effect in expression systems. To overcome this, we used a synthetic approach to generate the N70 construct, whereby the FP is stabilized in context of the adjacent auto oligomerization domain. The amide I profile of unlabeled N70 in membranes reveals prominent α-helical contribution, along with significant β-structure. By truncating the N terminus (FP region) of N70, β-structure is eliminated, suggesting that the FP adopts a β-structure in membranes. To assess this directly, ¹³C Fourier-transformed infra-red analysis was carried out to map secondary structure of the 16 N-terminal hydrophobic residues of the fusion peptide (FP16). The ¹³C isotope shifted absorbance of the FP was filtered from the global secondary structure of the 70 residue construct (N70). On the basis of the peak shift induced by the ¹³C-labeled residues of FP16, we directly assign β-sheet structure in ordered membranes. A differential labeling scheme in FP16 allows us to distinguish the type of β-sheet structure as parallel. Dilution of each FP16-labeled N70 peptide, by mixing with unlabeled N70, shows directly that the FP16 β-strand region self-assembles. We discuss our structural findings in the context of the prevailing gp41 fusion paradigm. Specifically, we address the role of the FP region in organizing supramolecular gp41 assembly, and we also discuss the mechanism by which exogenous, free FP constructs inhibit gp41-induced fusion.

In the last decade intensive research has been conducted to determine the role of innate immunity host defense peptides (also termed antimicrobial peptides) in the killing of prokaryotic and eukaryotic cells. Many antimicrobial peptides damage the cellular membrane as part of their killing mechanism. However, it is not clear what makes cancer cells more susceptible to some of these peptides, and what the molecular mechanisms underlying these activities are. Two general mechanisms were suggested: (i) plasma membrane disruption via micellization or pore formation, and (ii) induction of apoptosis via mitochondrial membrane disruption. To be clinically used, these peptides need to combine high and specific anticancer activity with stability in serum. Although so far very limited, new studies have paved the way for promising anticancer host defense peptides with a new mode of action and with a broad spectrum of anticancer activity.

Protein-protein interactions within the membrane, partially or fully mediated by transmembrane (TM) domains, are involved in many vital cellular processes. Since the unique feature of the membrane environment enables protein-protein assembly that otherwise is not energetically favored in solution, the structural restrictions involved in the assembly of soluble proteins are not necessarily valid for the assembly of TM domains. Here we used the N-terminal TM domain (Tar-1) of the Escherichia coli aspartate receptor as a model system for examining the stereospecificity of TM-TM interactions in vitro and in vivo in isolated systems, and in the context of the full receptor. For this propose, we synthesized Tar-1 all-L and all-D amino acid TM peptides, a mutant TM peptide and an unrelated TM peptide. The data revealed: (i) Tar-1 all-D specifically associated with Tar-1 all-L within a model lipid membrane, as determined by using fluorescence energy transfer experiments; (ii) Tar-1 all-L and all-D, but not the control peptides, demonstrated a dose-dependant dominant negative effect on the Tar-1 TM homodimerization in the bacterial ToxR assembly system, suggesting a wild-type-like interaction; and most interestingly, (iii) both Tar-1 all-L and all-D showed a remarkable ability to inhibit the chemotaxis response of the full-length receptor, in vivo. Peptide binding to the bacteria was confirmed through confocal imaging, and Western blotting confirmed that ToxR Tar-1 chimera protein levels are not affected by the presence of the exogenous peptides. These findings present the first evidence that an all-D TM domain peptide acts in vivo similarly to its parental all-L peptide and suggest that the dimerization of the TM domains is mainly mediated by side-chain interactions, rather than geometrically fitted conformations. In addition, the study provides a new approach for modifying the function of membrane proteins by proteolysis-free peptides. (C) 2004 Elsevier Ltd. All rights

Gene-encoded host defense peptides are used as part of the innate immunity, and many of them act by directly lysing the cell membrane of the pathogen. A few of these peptides showed anticancer activity in vitro but could not be used in vivo because of their inactivation by serum. We designed a 15-amino acid peptide, composed of D- and L-amino acids (diastereomer), which targets both androgen-independent and androgen-dependent human prostate carcinoma cell lines (CL1, 22RV1, and LNCaP). Most importantly, we observed a complete arrest of growth in CL1 and 22RV1 xenografts treated intratumorally with the diastereomer. This was also accompanied by a lowering of prostate-specific antigen serum levels secreted by the 22RV1 xenograft. Furthermore, the diastereomer synergized with conventional chemotherapeutics. In contrast, the parental all L-amino acids peptide was highly active only in vitro and could not discriminate between tumor and nontumor cells. Fluorescent confocal microscopy, histopathologic examination, and cell permeability studies (depolarization of transmembrane potential and release of an encapsulated dye) suggest a necrotic mechanism of killing, after a threshold concentration of peptide has been reached. Its destructive killing effect and the simple sequence of the diastereomer make it an attractive chemotherapeutic candidate possessing a new mode of action, with potential to be developed additionally for the treatment of prostate carcinoma.

C-peptides derived from the HIV envelope glycoprotein transmembrane subunit gp41 C-terminal heptad repeat (C-HR) region are potent HIV fusion inhibitors. These peptides interact with the gp41 N-terminal heptad repeat (N-HR) region and block the gp41 six-helix bundle formation that is required for fusion. However, the parameters that govern this inhibition have yet to be elucidated. We address this issue by comparing the ability of C34, derived from HIV-1, HIV-2 and SIV gp41, to inhibit HIV-1, HIV-2 and SIV envelope-mediated fusion and the ability of these peptides to form stable six-helix bundles with N36 peptides derived from gp41 of these three viruses. The ability to form six-helix bundles was examined by circular dichroism spectroscopy, and HIV/SIV Env-mediated membrane fusion was monitored by a dye transfer assay. HIV-1 N36 formed stable helix bundles with HIV-1, HIV-2 and SIV C34, which all inhibited HIV-1 Env-mediated fusion at IC₅₀100nM), although HIV-2 and SIV N36 formed stable helix bundles with SIV C34. Priming experiments with sCD4 indicate that, in contrast to HIV-1, HIV-2 and SIV Env do not expose their N-HR region to SIV C34 following CD4 binding, but rapidly proceed to co-receptor engagement and six-helix bundle formation resulting in fusion. Our results suggest that several factors, including six-helix bundle stability and the ability of CD4 to destabilize the envelope glycoprotein, serve as determinants of sensitivity to entry inhibitors.

Protein assembly is a critical process involved in a wide range of cellular events and occurs through extracellular and/or transmembrane domains (TMs). Previous studies demonstrated that a GXXXG motif is crucial for homodimer formation. Here we selected the TMs of ErbB1 and ErbB2 as a model since these receptors function both as homodimers and as heterodimers. Both TMs contain two GXXXG-like motifs located at the C and N termini. The C-terminal motifs were implicated previously in homodimer formation, but the role of the N-terminal motifs was not clear. We used the ToxR system and expressed the TMs of both ErbB1 and ErbB2 containing only the N-terminal GXXXG motifs. The data revealed that the ErbB2 but not the ErbB1 construct formed homodimers. Importantly, a synthetic ErbB1 TM peptide was able to form a heterodimer with ErbB2, by displacing the ErbB2 TM homodimer. The specificity of the interaction was demonstrated by using three controls: (i) Two single mutations within the GXXXG-like motif of the ErbB1 peptide reduced or preserved its activity, in agreement with similar mutations in glycophorin A. (ii) A TM peptide of the bacterial Tar receptor did not assemble with the ErbB2 construct, (iii) The ErbB1 peptide had no effect on the dimerization of a construct containing the TM-1 domain of the Tar receptor. Fluorescence microscopy demonstrated that all the peptides localized on the membrane. Furthermore, incubation with the peptides had no effect on bacterial growth and protein expression levels. Our results suggest that the N-terminal GXXXG-like motif of the ErbB1 TM plays a role in heterodimerization with the ErbB2 transmembrane domain. To our knowledge, this is the first demonstration of a transmembrane domain with two distinct recognition motifs, one for homodimerization and the other for heterodimerization.

We report on the synthesis, biological function, and a plausible mode of action of a new group of lipopeptides with potent antifungal and antibacterial activities. These lipopeptides are derived from positively charged peptides containing D- and L-amino acids (diastereomers) that are palmitoylated (PA) at their N terminus. The peptides investigated have the sequence K ₄X₇W, where X designates Gly, Ala, Val, or Leu (designated D-X peptides). The data revealed that PA-D-G and PA-D-A gained potent antibacterial and antifungal activity despite the fact that both parental peptides were completely devoid of any activity toward microorganisms and model phospholipid membranes. In contrast, PA-D-L lost the potent antibacterial activity of the parental peptide but gained and preserved partial antifungal activity. Interestingly, both D-V and its palmitoylated analog were inactive toward bacteria, and only the palmitoylated peptide was highly potent toward yeast. Both PA-D-L and PA-D-V lipopeptides were also endowed with hemolytic activity. Mode of action studies were performed by using tryptophan fluorescence and attenuated total reflectance Fourier transform infrared and circular dichroism spectroscopy as well as transmembrane depolarization assays with bacteria and fungi. The data suggest that the lipopeptides act by increasing the permeability of the cell membrane and that differences in their potency and target specificity are the result of differences in their oligomeric state and ability to dissociate and insert into the cytoplasmic membrane. These results provide insight regarding a new approach of modulating hydrophobicity and the self-assembly of non-membrane interacting peptides in order to endow them with both antibacterial and antifungal activities urgently needed to combat bacterial and fungal infections.

Objectives: Increasing resistance of pathogenic bacteria to antibiotics is a severe problem in health care and has intensified the search for novel drugs. Cationic antibacterial peptides are the most abundant antibiotics in nature and have been frequently proposed as new anti-infective agents. Here, a group of diastereomeric (containing D- and L-amino acids) peptides is studied regarding their potency against multiply resistant clinical isolates and their modes of action against Gram-positive cocci. Methods: MIC determinations and chequerboard titrations followed established procedures. Mode of action studies included killing kinetics and a series of experiments designed to characterize the impact of the diastereomeric peptides on bacterial membranes. Results: The tested diastereomers displayed high antimicrobial and broad spectrum activity with amphipathic-2D being the most active peptide. Synergic activities were observed with individual strains. Mode of action studies clearly demonstrated that the cytoplasmic membrane is a primary target for the peptides and that membrane disruption constitutes a significant bactericidal activity for the major fraction of a bacterial population. However, depending on the indicator strain, the results also suggest that additional molecular events contribute to the overall activity.

We report on two new cyclic 17-residue peptides that we named ranacyclins E and T, the first isolated from Rana esculenta frog skin secretions and the second discovered by screening a cDNA library from Rana temporaria. Ranacyclins have a loop region that is homologous with that of an 18-mer peptide, pLR, isolated from the skin of the Northern Leopard frog, Rana pipiens, with no reported antimicrobial activity. Here we show that ranacyclins and pLR have antimicrobial and antifungal activity. However, despite the high structural similarity, they differ in their spectrum of activity. The data reveal that ranacyclins and pLR have several properties that differentiate them from most known antimicrobial peptides. These include the following: (i) they adopt a significant portion of random coil structure in the membrane as revealed by ATR-FTIR and CD spectroscopy (50% for ranacyclin T and 70% for both ranacyclin E and pLR); (ii) they bind similarly to both zwitterionic and negatively charged membranes as revealed by using tryptophan fluorescence and surface plasmon resonance (SPR; BIAcore biosensor); (iii) they insert into the hydrophobic core of the membrane and presumably form transmembrane pores without damage to the bacterial wall, as revealed by SPR, ATR-FTIR, and transmission electron microscopy (TEM); and (iv) despite being highly and equally active in permeating bacterial spheroplasts and negatively charged membranes, they differ significantly in their potencies against target cells. Furthermore, a significant fraction of a given secondary structure is not prerequisite for membrane permeation and antimicrobial activity. However, increasing the fraction of a secondary structure and reducing peptide assembly in the membrane make it easier for the peptide to diffuse through the cell wall, which is different for each microorganism, into the cytoplasmic membrane.

To address the structure-function relationship of discrete regions within the gp41 ectodomain, 70-residue peptide constructs corresponding to the N-terminal subdomain of the HIV-1 gp41 ectodomain were examined in a membrane-associated context. These fragments encompass both fusion peptide (FP) and N-terminal heptad repeat (NHR) regions, and model the N-terminal half of the pre-hairpin intermediate (PHI), which is believed to be the target of the potent entry inhibitor DP-178, recently approved by the FDA. Using mutants, we attempted to map the structural organization of the N-terminal subdomain. Our results suggest that the N-terminal subdomain contains two discrete structural regions: the FP adopts a β-sheet conformation and the NHR is α-helical. This structural make-up is essential for fusogenic function, since loss of function mutants exhibit both a significant reduction in region-specific secondary structure as well as significant impairment in lipid mixing of large unilamellar vesicles. Our results, delineating membrane-associated structure of the FP region differ from previous ones by inclusion of the autonomous oligomerization domain (NHR), which likely contributes to stabilization of the FP structure. Correspondingly, the α-helical structure for the NHR, in context of the FP, correlates with structural predictions for this region in both the hairpin and PHI conformations during fusion. Based on our results, we postulate how oligomerization of regions in this sub-domain is essential for fusion pore formation.

T20, a synthetic peptide corresponding to a C-terminal segment of the envelope glycoprotein (gp41) of human and simian immunodeficiency viruses, is a potent inhibitor of viral infection. We report here that C-terminal octylation of simian immunodeficiency virus gp41-derived T20 induces a significant increase in its inhibitory potency. Furthermore, when C-terminally octylated, an otherwise inactive mutant in which the C-terminal residues GNWF were replaced by ANAA has potency similar to that of the wild type T20. This effect cannot be explained by a trivial inhibitory effect of the octyl group added to the peptides, because the N-terminally octylated peptides have the same activity as the non-octylated parent peptides. The effects caused by octylation on the oligomerization, secondary structure, and membrane-interaction properties of the peptides were investigated. Our results shed light on the mechanism of inhibition by T20 and provide experimental support for the existence of a pre-hairpin intermediate.

gp41 is the protein responsible for the process of membrane fusion that allows primate lentiviruses (HIV and SIV) to enter into their host cells. gp41 ectodomain contains an N-terminal and a C-terminal heptad repeat region (NHR and CHR) connected by an immunodominant loop. In the absence of membranes, the NHR and CHR segments fold into a protease-resistant core with a trimeric helical hairpin structure. However, when the immunodominant loop is not present (either in a complex formed by HIV-1 gp41-derived NHR and CHR peptides or by mild treatment with protease of recombinant constructs of HIV-1 gp41 ectodomain, which also lack the N-terminal fusion peptide and the C-terminal Trp-rich region) membrane binding induces a conformational change in the gp41 core structure. Here, we further investigated whether covalently linking the NHR and CHR segments by the immunodominant loop affects this conformational change. Specifically, we analyzed a construct corresponding to a fragment of SIVmac239 gp41ectodomain (residues 27-149, named e-gp41) by means of surface plasmon resonance, Trp and rhodamine fluorescence, ATR-FTIR spectroscopy, and differential scanning calorimetry. Our results suggest that the presence of the loop stabilizes the trimeric helical hairpin both when e-gp41 is in aqueous solution and when it is bound to the membrane surface. Bearing in mind possible differences between HIV-1 and SIV gp41, and considering that the gp41 ectodomain constructs analyzed to date lack the N-terminal fusion peptide and the C-terminal Trp-rich region, we discuss our observations in relation to the mechanism of virus-induced membrane fusion.

The search for antibiotics with a new mode of action led to numerous studies on antibacterial peptides. Most of the studies were carried out with L-amino acid peptides possessing amphipathic α-helix or β-sheet structures, which are known to be important for biological activities. Here we compared the effect of significantly altering the sequence of an amphipathic α-helical peptide (15 amino acids long) and its diastereomer (composed of both L- and D-amino acids) regarding their structure, function, and interaction with model membranes and intact bacteria. Interestingly, the effect of sequence alteration on biological function was similar for the L-amino acid peptides and the diastereomers, despite some differences in their structure in the membrane as revealed by attenuated total reflectance Fourier-transform infrared spectroscopy. However, whereas the all L-amino acid peptides were highly hemolytic, had low solubility, lost their activity in serum, and were fully cleaved by trypsin and proteinase K, the diastereomers were nonhemolytic and maintained full activity in serum. Furthermore, sequence alteration allowed making the diastereomers either fully, partially, or totally protected from degradation by the enzymes. Transmembrane potential depolarization experiments in model membranes and intact bacteria indicate that although the killing mechanism of the diastereomers is via membrane perturbation, it is also dependent on their ability to diffuse into the inner bacterial membrane. These data demonstrate the advantage of the diastereomers over their all L-amino acid counterparts as candidates for developing a repertoire of new target antibiotics with a potential for systemic use.

For many different enveloped viruses the crystal structure of the fusion protein core has been established. A striking conservation in the tertiary and quaternary arrangement of these core structures is repeatedly revealed among members of diverse families. It has been proposed that the primary role of the core involves structural rearrangements which facilitate apposition between viral and target cell membranes. Forming the internal trimeric coiled coil of the core, the N-terminal heptad repeat (NHR) of HIV-1 gp41 was suggested to have additional roles, due to its ability to bind biological membranes. The NHR is adjacent to the N-terminal hydrophobic fusion peptide (FP), which alone can fuse biological membranes. To investigate the role of the NHR in membrane fusion, we synthesized and functionally characterized HIV-1 gp41 peptides corresponding to the FP and NHR alone, as well as continuous peptides made of both FP and NHR (wild type and mutant). We show here that a consecutive, 70-residue peptide consisting of both the FP and NHR (gp41/1-70) has dramatic fusogenic properties. The effect of including the complete NHR, as compared to shorter 23-, 33-, or 52-residue N-terminal peptides, is illustrated by a leap in lipid mixing of phosphatidylcholine (PC) large unilamellar vesicles (LUV) and clearly delineates the synergistic role of the NHR in the fusion event. Furthermore, a mutation in the NHR that renders the virus noninfectious is reflected by a significant reduction in in vitro lipid mixing induced by the mutant, gp41/1-70 (I62D). Additional spectroscopic studies, characterizing membrane binding and apposition induced by the peptides, help to clarify the role of the NHR in membrane fusion.

Stereospecificity in protein-protein recognition and docking is an unchallenged dogma. Soluble proteins provide the main source of evidence for stereospecificity. In contrast, within the membrane little is known about the role of stereospecificity in the recognition process. Here, we have reassessed the stereospecificity of protein-protein recognition by testing whether it holds true for the well-defined glycophorin A (GPA) transmembrane domain in vivo. We found that the all-D amino acid GPA transmembrane domain and two all-D mutants specifically associated with an all-L GPA transmembrane domain, within the membrane milieu of Escherichia coli. Molecular dynamics techniques reveal a possible structural explanation to the observed interaction between all-D and all-L transmembrane domains. A very strong correlation was found between amino acid residues at the interface of both the all-L homodimer structure and the mixed L/D heterodimer structure, suggesting that the original interactions are conserved. The results suggest that GPA helix-helix recognition within the membrane is chirality-independent.

Water-membrane soluble protein and peptide toxins are used in the defense and offense systems of all organisms, including plants and humans. A major group includes antimicrobial peptides, which serve as a nonspecific defense system that complements the highly specific cell-mediated immune response. The increasing resistance of bacteria to conventional antibiotics stimulated the isolation and characterization of many antimicrobial peptides for potential use as new target antibiotics. The finding of thousands of antimicrobial peptides with variable lengths and sequences, all of which are active at similar concentrations, suggests a general mechanism for killing bacteria rather than a specific mechanism that requires preferred active structures. Such a mechanism is in agreement with the "carpet model" that does not require any specific structure or sequence. It seems that when there is an appropriate balance between hydrophobicity and a net positive charge the peptides are active on bacteria. However, selective activity depends also on other parameters, such as the volume of the molecule, its structure, and its oligomeric state in solution and membranes. Further, although many studies support that bacterial membrane damage is a lethal event for bacteria, other studies point to a multihit mechanism in which the peptide binds to several targets in the cytoplasmic region of the bacteria.

The interaction of many lytic cationic antimicrobial peptides with their target cells involves electrostatic interactions, hydrophobic effects, and the formation of amphipathic secondary structures, such as α helices or β sheets. We have shown in previous studies that incorporating ≈ 30% D-amino acids into a short α helical lytic peptide composed of leucine and lysine preserved the antimicrobial activity of the parent peptide, while the hemolytic activity was abolished. However, the mechanisms underlying the unique structural features induced by incorporating D-amino acids that enable short diastereomeric antimicrobial peptides to preserve membrane binding and lytic capabilities remain unknown. In this study, we analyze in detail the structures of a model amphipathic α helical cytolytic peptide KLLLKWLL KLLK-NH₂ and its diastereomeric analog and their interactions with zwitterionic and negatively charged membranes. Calculations based on high-resolution NMR experiments in dodecylphosphocholine (DPCho) and sodium dodecyl sulfate (SDS) micelles yield three-dimensional structures of both peptides. Structural analysis reveals that the peptides have an amphipathic organization within both membranes. Specifically, the α helical structure of the L-type peptide causes orientation of the hydrophobic and polar amino acids onto separate surfaces, allowing interactions with both the hydrophobic core of the membrane and the polar head group region. Significantly, despite the absence of helical structures, the diastereomer peptide analog exhibits similar segregation between the polar and hydrophobic surfaces. Further insight into the membrane-binding properties of the peptides and their depth of penetration into the lipid bilayer has been obtained through tryptophan quenching experiments using brominated phospholipids and the recently developed lipid/polydiacetylene (PDA) colorimetric assay. The combined NMR, FTIR, fluorescence, and colorimetric studies shed light on the importance of segregation between the positive charges and the hydrophobic moieties on opposite surfaces within the peptides for facilitating membrane binding and disruption, compared to the formation of α helical or β sheet structures.

The viral envelope glycoprotein gp41 mediates membrane fusion in HIV/SIV infection, gp41 ectodomain (e-gp41, residues 27-149), which was shown to interact with phospholipid membranes, exists in an equilibrium between the monomeric and trimeric states. Here, we analyzed, by intrinsic Trp fluorescence and resonance energy transfer, whether SIV e-gp41-membrane interaction depends on the gp41 oligomeric state. We found that both gp41 monomers and trimers bind membranes, with the monomers' full binding being reached at substantially lower lipid to protein ratios. Furthermore, the different characteristics of the Trp fluorescence of monomers and trimers enabled us to detect binding of each form at concentrations at which both species were present. CD spectroscopy revealed that the secondary structure of gp41 monomers does not change upon membrane binding, suggesting that membrane-bound monomeric-gp41 is a possible target for DP-178, a potent peptide inhibitor of HIV infection. The consequences of the interaction between monomeric and trimeric gp41 with membranes in HIV/SIV infection, its inhibition, and its associated neuropathologies are discussed.

Keywords: THURINGIENSIS DELTA-ENDOTOXIN; GLYCOPHORIN-A DIMERIZATION; MAGNETIC-RESONANCE DATA; PEPTIDE CECROPIN P1; PORE-FORMING DOMAIN; MODEL ION CHANNELS; SHAKER K+ CHANNEL; ISK MINK PROTEIN; SEA MOSES SOLE; BACILLUS-THURINGIENSIS

The spore-forming bacterium Bacillus thuringiensis produces intracellular inclusions comprised of protoxins active on several orders of insects. These highly effective and specific toxins have great potential in agriculture and for the control of disease-related insect vectors. Inclusions ingested by larvae are solubilized and converted to active toxins in the midgut. There are two major classes, the cytolytic toxins and the δ-endotoxins. The former are produced by B. thuringiensis subspecies active on Diptera. The latter, which will be the focus of this review, are more prevalent and active on at least three orders of insects. They have a three-domain structure with extensive functional interactions among the domains. The initial reversible binding to receptors on larval midgut cells is largely dependent upon domains II and III. Subsequent steps involve toxin insertion into the membrane and aggregation, leading to the formation of gated, cation-selective channels. The channels are comprised of certain amphipathic helices in domain I, but the three processes of insertion, aggregation and the formation of functional channels are probably dependent upon all three domains. Lethality is believed to be due to destruction of the transmembrane potential, with the subsequent osmotic lysis of cells lining the midgut. In this review, the mode of action of these δ-endotoxins will be discussed with emphasis on unique features.

Living organisms of all types produce a large repertoire of gene-encoded, net positively charged, antimicrobial peptides as part of their innate immunity to microbial invasion. Despite significant variations in composition, length and secondary structure most antimicrobial peptides are active in micromolar concentrations, suggesting a common general mechanism for their mode of action. Many antimicrobial peptides bind bacterial phospholipid membranes up to a threshold concentration, followed by membrane permeation/disintegration (the "carpet" mechanism). Recent data suggest that the details of the permeation pathways may vary for different peptides and are assigned to different modes of action. Accumulating data reveal that the molecular basis for cell selectivity is the ability of peptides to specifically bind the negatively charged bacterial membrane, as well as their oligomeric state in solution and in the membrane. Based on the "carpet" mechanism and the role of the peptide oligomeric state, a novel group of diastereomeric (containing D- and L-amino acids) antimicrobial peptides were developed. These peptides may serve as promising templates for the future designs of antimicrobial peptides.

The human immunodeficiency virus type 1 gp41 ectodomain forms a three-hairpin protease-resistant core in the absence of membranes, namely, the putative gp41 fusion-active state. Here, we show that recombinant proteins corresponding to the ectodomain of gp41, but lacking the fusion peptide, bind membranes and consequently undergo a major conformational change. As a result, the protease-resistant core becomes susceptible to proteolytic digestion. Accordingly, synthetic peptides corresponding to the segments that construct this core bind the membrane. It is remarkable that the hetero-oligomer formed by these peptides dissociates upon binding to the membrane. These results are consistent with a model in which, after the three-hairpin conformation is formed, membrane binding induces opening of the gp41 core complex. We speculate that binding of the segments that constructed the core to the viral and cellular membranes could bring the membranes closer together and facilitate their merging. (C) 2000 Academic Press.

The amphipathic α-helical structure is considered to be a prerequisite for the lytic activity of a large group of cytolytic peptides. However, despite numerous studies on the contribution of various parameters to their structure and activity, the importance of linearity has not been examined. In the present study we functionally and structurally characterized a linear amphipathic α-helical peptide (wt peptide), its diastereomer, and cyclic analogues of both. Using analogues with the same sequence of hydrophobic and positively charged amino acids, but with different propensities to form a helical structure, we were able to examine the contribution of linearity to helix formation, bilogical function, and membrane binding and permeation. Importantly, we found that cyclization increases the selectivity between bacteria and human erythrocytes by substantially reducing the hemolytic activity of the cyclic peptides compared with the linear peptides. Moreover, whereas the wt peptide was highly active toward Gram⁺ bacteria, its cyclic counterpart is active toward both Gram⁺ and Gram^- bacteria. These findings are correlated with an impaired ability of the cyclic analogues to bind and permeate zwitterionic phospholipid membranes compared with their linear counterparts and an increase in the binding and permeating activity of the cyclic wt peptide toward negatively charged membranes. Furthermore, cyclization abolished the oligomerization of the linear wt peptide in solution and in SDS, suggesting an additional factor that may account for the difference in the spectrum of antibacterial activity between the linear and the cyclic wt peptides. Interestingly, attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy revealed that, despite cyclization and incorporation of 33% D-amino acids along the peptide backbone, the membrane environment can impose a predominantly helical structure on the peptides, which is required for their bilogical function. Overall, our results indicate that linearity is not a prerequisite for lytic activity of amphipathic α-helical peptides but rather affects the selectivity between Gram⁺ and Gram^- bacteria and between mammalian cells and bacteria. In addition, the combination of incorporating of D-amino acids into lytic peptides and their cyclization open the way for developing a new group of antimicrobial peptides with improved properties for treating infectious diseases.

The fusion peptide of HIV-1 gp41 is formed by the 16 N-terminal residues of the protein. This 16-amino acid peptide, in common with several other viral fusion peptides, caused a reduction in the bilayer to hexagonal phase transition temperature of dipalmitoleoylphosphatidylethanolamine (T(H)), suggesting its ability to promote negative curvature in membranes. Surprisingly, an elongated peptide corresponding to the 33 N-terminal amino acids raised T(H), although it was more potent than the 16-amino acid fusion peptide in inducing lipid mixing with large unilamellar liposomes of 1:1:1 dioleoylphosphatidylethanolamine/dioleoylphosphatidylcholine/cholesterol. The 17-amino acid C-terminal fragment of the peptide can induce membrane fusion by itself, if it is anchored to a membrane by palmitoylation of the amino terminus, indicating that the additional 17 hydrophilic amino acids contribute to the fusogenic potency of the peptide. This is not solely a consequence of the palmitoylation, as a random peptide with the same amino acid composition with a palmitoyl anchor was less potent in promoting membrane fusion and palmitic acid itself had no fusogenic activity. The 16- amino acid N-terminal fusion peptide and the longer 33-amino acid peptide were labeled with NBD. Fluorescence binding studies indicate that both peptides bind to the membrane with similar affinities, indicating that the increased fusogenic activity of the longer peptide was not a consequence of a greater extent of membrane partitioning. We also determined the secondary structure of the peptides using FTIR spectroscopy. We find that the amino- terminal fusion peptide is inserted into the membrane as a β-sheet and the 17 C-terminal amino acids lie on the surface of the membrane, adopting an α- helical conformation. It was further demonstrated with the use of rhodamine- labeled peptides that the 33-amino acid peptide self-associated in the membrane while the 16-amino acid N-terminal peptide did not. Thus, the 16- amino acid N-terminal fusion peptide of HIV inserts into the membrane and, like other viral fusion peptides, lowers T(H). In addition, the 17 consecutive amino acids enhance the fusogenic activity of the fusion peptide presumably by promoting its self-association.

The entry of enveloped viruses into host cells is accomplished by fusion of the viral envelope and target plasma membrane and is mediated by fusion proteins. Recently, several functional domains within fusion proteins from different viral families were identified. Some are directly involved in conformational changes after receptor binding, as suggested by the recent release of crystallographically determined structures of a highly stable core structure of the fusion proteins in the absence of membranes. However, in the presence of membranes, this core binds strongly to the membrane's surface and dissociates therein. Other regions, besides the N-terminal fusion peptide, which include the core region and an internal fusion peptide in paramyxoviruses, are directly involved in the actual membrane fusion event, suggesting an "umbrella" like model for the membrane induced conformational change of fusion proteins. Peptides resembling these regions have been shown to have specific antiviral activity, presumably because they interfere with the corresponding domains within the viruses. Overall, these studies shed light into the molecular mechanism of membrane fusion induced by envelope glycoproteins and suggest that fusion proteins from different viral families share common structural and functional motifs.

Some amphipathic bitter tastants and non-sugar sweeteners are direct activators of G proteins and stimulate transduction pathways in cells not related to taste. We demonstrate that the amphipathic bitter tastants quinine and cyclo(Leu-Trp) and the non-sugar sweetener saccharin translocate rapidly through multilamellar liposomes. Furthermore, when rat circumvallate (CV) taste buds were incubated with the above tastants for 30 s, their intracellular concentrations increased by 3.5- to 7-fold relative to their extracellular concentrations. The time course of this dramatic accumulation was also monitored in situ in rat single CV taste buds under a confocal laser-scanning microscope. Tastants were clearly localized to the taste cell cytosol. It is proposed that, due to their rapid permeation into taste cells, these amphipathic tastants may be available for activation of signal transduction components (e.g., G proteins) directly within the time course of taste sensation. Such activation may occur in addition to the action of these tastants on putative G protein-coupled receptors. This phenomenon may be related to the slow taste onset and lingering aftertaste typically produced by many bitter tastants and non-sugar sweeteners.

Permeation of the cell membrane leading to cell death is a mechanism used by a large number of membrane-lytic peptides. Some are linear, mostly helical, and others contain one or more disulfide bonds forming β-sheet or both β-sheet and α-helix structures. They are all soluble in solution but when they reach the target membrane, conformational changes occur which let them associate with and lyse the membrane. Some lytic peptides are not cell- selective and lyse different microorganisms and normal mammalian cells, while others are specific to either type of cells. Despite extensive studies, the mode of action of membrane-lytic peptides is not fully understood and the basis for their selectivity towards specific target cells is not known. Many studies have shown that peptide-lipid interactions leading to membrane permeation play a major role in their activity. Membrane permeation by amphipathic α-helical peptides has been proposed to occur via one of two general mechanisms: (i) transmembrane pore formation via a 'barrel-stave' mechanism; and (ii) membrane destruction/solubilization via a 'carpet' mechanism. This review, which is focused on the different stages of membrane permeation induced by representatives of amphipathic α-helical antimicrobial and cell non-selective lytic peptides distinguishes between the 'carpet' mechanism, which holds for antimicrobial peptides versus the 'barrel-stave' mechanism, which holds for cell non-selective lytic peptides.

Recent studies have demonstrated the importance of heptad repeat regions within envelope proteins of viruses in mediating conformational changes at various stages of viral infection. However, it is not clear if heptad repeats have a direct role in the actual fusion event. Here we have synthesized, fluorescently labeled and functionally and structurally characterized a wild-type 70 residue peptide (SV-117) composed of both the fusion peptide and the N-terminal heptad repeat of Sendai virus fusion protein, two of its mutants, as well as the fusion peptide and heptad repeat separately. One mutation was introduced in the fusion peptide (G119K) and another in the heptad repeat region (I154K). Similar mutations have been shown to drastically reduce the fusogenic ability of the homologous fusion protein of Newcastle disease virus. We found that only SV-117 was active in inducing lipid mixing of egg phosphatidylcholine/phospha tidyiglycerol (PC/PG) large unilamellar vesicles (LUV), and not the mutants nor the mixture of the fusion peptide and the heptad repeat. Functional characterization revealed that SV-117, and to a lesser extent its two mutants, were potent inhibitors of Sendai virus-mediated hemolysis of red blood cells, while the fusion peptide and SV-150 were negligibly active alone or in a mixture. Hemagglutinin assays revealed that none of the peptides disturb the binding of virions to red blood cells. Further studies revealed that SV-117 and its mutants oligomerize similarly in solution and in membrane, and have similar potency in inducing vesicle aggregation. Circular dichroism and FTIR spectroscopy revealed a higher helical content for SV-117 compared to its mutants in 40% tifluorethanol and in PC/PG multibilayer membranes, respectively, ATR-FTIR studies indicated that SV-117 lies more parallel with the surface of the membrane than its mutants. These observations suggest a direct role for the N-terminal heptad repeat in assisting the fusion peptide in mediating membrane fusion.

The antimicrobial peptide LL-37 belongs to the cathelicidin family and is the first amphipathic α-helical peptide isolated from human. LL-37 is considered to play an important role in the first line of defence against local infection and systemic invasion of pathogens at sites of inflammation and wounds. Understanding its mode of action may assist in the development of antimicrobial agents mimicking those of the human immune system. In vitro studies revealed that LL-37 is cytotoxic to both bacterial and normal eukaryotic cells. To gain insight into the mechanism of its non-cell-selective cytotoxicity, we synthesized and structurally and functionally characterized LL-37, its N-terminal truncated form FF-33, and their fluorescent derivatives (which retained structure and activity). The results showed several differences, between LL-37 and other native antimicrobial peptides, that may shed light on its in vivo activities. Most interestingly, LL-37 exists in equilibrium between monomers and oligomers in solution at very low concentrations. Also, it is significantly resistant to proteolytic degradation in solution, and when bound to both zwitterionic (mimicking mammalian membranes) and negatively charged membranes (mimicking bacterial membranes). The results also showed a role for the N-terminus in proteolytic resistance and haemolytic activity, but not in antimicrobial activity. The LL-37 mode of action with negatively charged membranes suggests a detergent-like effect via a 'carpel-like' mechanism. However, the ability of LL-37 to oligomerize in zwitterionic membranes might suggest the formation of a transmembrane pore in normal eukaryotic cells. To examine this possibility we used polarized attenuated total reflectance Fourier-transform infrared spectroscopy and found that the peptide is predominantly α-helical and oriented nearly parallel with the surface of zwitterionic-lipid membranes. This result does not support the channel-forming hypothesis, but rather it supports the detergent-like effect.

Peptides derived from conserved heptad-repeat regions of several viruses have been shown recently to inhibit virus-cell fusion. To find out their possible role in the fusion process, two biologically active heptad-repeat segments of the fusion protein (F) of Sendai virus, SV-150 (residues 150-186), and SV-473 (residues 473-495) were synthesized, fluorescently labeled and syectroscopically characterized for their structure and organization in solution and within the membrane. SV-150 was found to be 50-fold less active than SV-473 in inhibiting Sendai virus-cell fusion. Circular dichroism (CD) spectroscopy revealed that in aqueous solution, the peptides are self-associated and adopt low α-helical structure. However, when the two peptides are mixed together, their α-helical content significantly increases. Fluorescence studies, CD, and polarized attenuated total reflection infrared (ATR-FTIR) spectroscopy showed that both peptides, alone or as a complex, bind strongly to negatively charged and zwitterionic phospholipid membranes, dissociate therein into α-helical monomers, but do not perturb the lipid packing of the membrane. The ability of the peptides to interact with each other in solution may be correlated with antiviral activity, whereas their ability to interact with the membrane, together with their location near the fusion peptide and the transmembrane domain, suggests a revision to the currently accepted model for viral-induced membrane fusion. In the revised model, in the sequence of events associated with viral entry, the two heptad-repeat sequences may assist in bringing the viral and cellular membranes closer, thus facilitating membrane fusion.

A synthetic heptad repeat, SV-473, derived from Sendai virus fusion protein is a potent inhibitor of virus-cell fusion. In order to understand the mechanism of the inhibitory effect, we synthesized and fluorescently labeled SV-465, an extended version of SV-473 by one more heptad, its mutant peptide A^17,24-SV-465, in which two heptadic leucines were substituted with two alanines, and its enatiomer D-SV-465, composed entirely of D-amino acids. Similar mutations in the homologous fusion protein of the Newcastle disease virus drastically reduced its activity. The data revealed that SV- 465, but not A^17,24-SV-465 or its enantiomer, is highly active in inhibiting Sendai virus-induced hemolysis of red blood cells. None of the peptides interfere with the binding of virions to the target red blood cells as demonstrated by hemagglutinin assay. Fluorescence and circular dichroism (CD) spectroscopy indicated that: (i) only SV-465 could self-assemble in aqueous environment; (ii) only SV-465 could co-assemble with two other biologically active heptad repeats derived from Sendai virus fusion protein; (iii) SV-465 has a higher helical content than A^17,24-SV-465 in solution, and (iv) all the peptides bind strongly to zwitterionic and negatively charged phospholipids. Polarized attenuated total reflection infrared spectroscopy revealed that they bound as monomers onto the surface of i membranes with predominantly α-helical structures. The functional role of the amino acid 465-497 domain in Sendai virus-mediated membrane fusion is discussed in light of these findings.

Recent studies demonstrated that a synthetic fusion peptide of HIV-1 self-associates in phospholipid membranes and inhibits HIV-1 envelope glycoprotein-mediated cell fusion, presumably by interacting with the N-terminal domain of gp41 and forming inactive heteroaggregates [Kliger, Y., Aharoni, A., Rapaport, D., Jones, P., Blumenthal, R. & Shai, Y. (1997) J. Biol. Chem. 272, 13496-13505]. Here, we show that a synthetic all D-amino acid peptide corresponding to the N-terminal sequence of HIV-1 gp41 (D-WT) of HIV-1 associates with its enantiomeric wild-type fusion (WT) peptide in the membrane and inhibits cell fusion mediated by the HIV-1 envelope glycoprotein. D-WT does not inhibit cell fusion mediated by the HIV-2 envelope glycoprotein. WT and D-WT are equally potent in inducing membrane fusion. D-WT peptide but not WT peptide is resistant to proteolytic digestion. Structural analysis showed that the CD spectra of D-WT in trifluoroethanol/water is a mirror image of that of WT, and attenuated total reflectance-fourier transform infrared spectroscopy revealed similar structures and orientation for the two enantiomers in the membrane. The results reveal that the chirality of the synthetic peptide corresponding to the HIV-1 gp41 N-terminal sequence does not play a role in liposome fusion and that the peptides' chirality is not necessarily required for peptide-peptide interaction within the membrane environment. Furthermore, studies along these lines may provide criteria to design protease-resistant therapeutic agents against HIV and other viruses.

The antimicrobial activity of various naturally occurring microbicidal peptides was reported to result from their interaction with microbial membrane. In this study, we investigated the cytotoxicity of the hemolytic peptide dermaseptin S4 (DS4) and the nonhemolytic peptide dermaseptin S3 (DS3) toward human erythrocytes infected by the malaria parasite Plasmodium falciparum. Both DS4 and DS3 inhibited the parasite's ability to incorporate [³H]hypoxanthine. However, while DS4 was toxic toward both the parasite and the host erythrocyte, DS3 was toxic only toward the intraerythrocytic parasite. To gain insight into the mechanism of this selective cytotoxicity, we labeled the peptides with fluorescent probes and investigated their organization in solution and in membranes. In Plasmodium-infected cells, rhodamine-labeled peptides interacted directly with the intracellular parasite, in contrast to noninfected cells, where the peptides remained bound to the erythrocyte plasma membrane. Binding experiments to phospholipid membranes revealed that DS3 and DS4 had similar binding characteristics. Membrane permeation studies indicated that the peptides were equally potent in permeating phosphatidylserine/phosphatidylcholine vesicles, whereas DS4 was more permeative with phosphatidylcholine vesicles. In aqueous solutions, DS4 was found to be in a higher aggregation state. Nevertheless, both DS3 and DS4 spontaneously dissociated to monomers upon interaction with vesicles, albeit with different kinetics. In light of these results, we propose a mechanism by which dermaseptins permeate cells and affect intraerythrocytic parasites.

The increase in infectious diseases and bacterial resistance to antibiotics has resulted in intensive studies focusing on the use of linear, α-helical, cytolytic peptides from insects and mammals as potential drugs for new target sites in bacteria. Recent studies with diastereomers of the highly potent cytolytic peptides, pardaxin and melittin, indicate that α- helical structure is required for mammalian cells lysis but is not necessary for antibacterial activity. Thus, hydrophobicity and net positive charge of the polypeptide might confer selective antibacterial lytic activity. To test this hypothesis, a series of diastereomeric model peptides (12 amino acids long) composed of varying ratios of leucine and lysine were synthesized, and their structure and biological function were investigated. Peptide length and the position of D-amino acids were such that short peptides with stretches of only 1-3 consecutive L-amino acids that cannot form an α-helical structure were constructed. Circular dichroism spectroscopy showed that the peptides do not retain any detectable secondary structure in a hydrophobic environment. This enabled examination of the sole effect of hydrophobicity and positive charge on activity. The data reveal that modulating hydrophobicity and positive charge is sufficient to confer antibacterial activity and cell selectivity. A highly hydrophobic diastereomer that permeated both zwitterionic and negatively charged phospholipid vesicles, lysed eukaryotic and prokaryotic cells. In contrast, a highly positively charged diastereomer that only permeated slightly negatively charged phospholipid vesicles had low antibacterial activity and could not lyse eukaryotic cells. In the boundary between high hydrophobicity and high positive charge, the diastereomers acquired selective and potent antibacterial activity. Furthermore, they were completely resistant to human serum inactivation, which dramatically reduces the activity of native antibacterial peptides. In addition, a strong synergistic effect was observed at nonlethal concentrations of the peptides with the antibiotic tetracycline on resistant bacteria. The results are discussed in terms of proposed mechanisms of antibacterial activity, as well as a new strategy for the design of a repertoire of short, simple, and easily manipulated antibacterial peptides as potential drugs in the treatment of infectious diseases.

This review describes the numerous and innovative methods used to study the structure and function of viral fusion peptides. The systems studied include both intact fusion proteins and synthetic peptides interacting with model membranes. The strategies and methods include dissecting the fusion process into intermediate stages, comparing the effects of sequence mutations, electrophysiological patch clamp methods, hydrophobic photolabelling, video microscopy of the redistribution of both aqueous and lipophilic fluorescent probes between cells, standard optical spectroscopy of peptides in solution (circular dichroism and fluorescence) and attenuated total reflection-Fourier transform infrared spectroscopy of peptides bound to planar bilayers. Although the goal of a detailed picture of the fusion pore has not been achieved for any of the intermediate stages, important properties useful for constraining the development of models are emerging. For example, the presence of α-helical structure in at least part of the fusion peptide is strongly correlated with activity; whereas, β-structure tends to be less prevalent, associated with non-native experimental conditions, and more related to vesicle aggregation than fusion. The specific angle of insertion of the peptides into the membrane plane is also found to be an important characteristic for the fusion process. A shallow penetration, extending only to the central aliphatic core region, is likely responsible for the destabilization of the lipids required for coalescence of the apposing membranes and fusion. The functional role of the fusion peptides (which tend to be either nonpolar or aliphatic) is then to bind to and dehydrate the outer bilayers at a localized site; and thus reduce the energy barrier for the formation of highly curved, lipidic 'stalk' intermediates. In addition, the importance of the formation of specific, 'higher-order' fusion peptide complexes has also been shown. Recent crystallographic structures of core domains of two more fusion proteins (in addition to influenza haemagglutinin) has greatly facilitated the development of prototypic models of the fusion site. This latter effort will undoubtedly benefit from the insights and constraints of fusion peptides.

Studies on lipid-peptide interactions of cytolytic polypeptides tend to emphasize the importance of the amphipathic α-helical structure for their cytolytic activity. In this study, diasetereomers of the bee venom melittin (26 a.a.), a non-cell-selective cytolysin, were synthesized and investigated for their structure and cytolytic activity toward bacteria and mammalian cells. Similarly to the findings with the diastereomers of the less cytolytic peptide pardaxin (33 a.a.) (Shai and Oren, 1996), the melittin diastereomers lost their α-helical structure, which abrogated their hemolytic activity toward human erythrocytes. However, they retained their antibacterial activity and completely lysed both Gram-positive and Gram-negative bacteria, as revealed by transmission electron microscopy. To understand the molecular mechanism underlying this selectivity, binding experiments utilizing the intrinsic tryptophan of melittin, tryptophan quenching experiments using brominated phospholipids, and membrane destabilization studies were done. The data revealed that the melittin diastereomers bound to and destabilized only negatively-charged phospholipid vesicles, in contrast to native melittin, which binds strongly to both negatively-charged and zwitterionic phospholipids. However, the partition coefficient, the depth of penetration into the membrane, and the membrane-permeating activity of the diastereomers with negatively-charged phospholipids were similar to those obtained with melittin. The results obtained do not support the formation of transmembrane pores as the mode of action of the diastereomers, but rather suggest that these peptides bind to the surface of the bacterial membrane, cover it in a 'carpet-like' manner, and dissolve it like a detergent. The results presented here together with those obtained with the cytolytic peptide pardaxin suggest that the combination of hydrophobicity and net positive charge may be sufficient in the design of potent diastereomers of antibacterial polypeptides for the treatment of infectious diseases.

The mechanism of leakage induced by surface active peptides is not yet fully understood. To gain insight into the molecular events underlying this process, the leakage induced by the peptide pardaxin from phosphatidylcholine/phosphatidylserine/cholesterol large unilamellar vesicles was studied by monitoring the rate and extent of dye release and by theoretical modeling. The leakage occurred by an all-or-none mechanism: vesicles either leaked or retained all of their contents. We further developed a mathematical model that includes the assumption that certain peptides become incorporated into the vesicle bilayer and aggregate to form a pore. The current experimental results can be explained by the model only if the surface aggregation of the peptide is reversible. Considering this reversibility, the model can explain the final extents of calcein leakage for lipid/peptide ratios of >2000:1 to 25:1 by assuming that only a fraction of the bound peptide forms pores consisting of M = 6 ± 3 peptides. Interestingly, less leakage occurred at 43°C than at 30°C, although peptide partitioning into the bilayer was enhanced upon elevation of the temperature. We deduced that the increased leakage at 30°C was due to an increase in the extent of reversible surface aggregation at the lower temperature. Experiments employing fluorescein-labeled pardaxin demonstrated reversible aggregation of the peptide in suspension and within the membrane, and exchange of the peptide between liposomes. In summary, our experimental and theoretical results support reversible surface aggregation as the mechanism of pore formation by pardaxin.

Cecropins are positively charged antibacterial peptides that act by permeating the membrane of susceptible bacteria. To gain insight into the mechanism of membrane permeation, the secondary structure and the orientation within phospholipid membranes of the mammalian cecropin P1 (CecP) was studied using attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy and molecular dynamics simulations. The shape and frequency of the amide I and II absorption peaks of CecP within acidic PE/PG multibilayers (phosphatidylethanolamine/phosphatidylglycerol) in a 7:3 (w/w) ratio (a phospholipid composition similar to that of many bacterial membranes), indicated that the peptide is predominantly α-helical. Polarized ATR-FTIR spectroscopy was used to determine the orientation of the peptide relative to the bilayer normal of phospholipid multibilayers. The ATR dichroic ratio of the amide I band of CecP peptide reconstituted into oriented PE/PG phospholipid membranes indicated that the peptide is preferentially oriented nearly parallel to the surface of the lipid membranes. A similar secondary structure and orientation were found when zwitterionic phosphatidylcholine phospholipids were used. The incorporation of CecP did not significantly change the order parameters of the acyl chains of the multibilayer, further suggesting that CecP does not penetrate the hydrocarbon core of the membranes. Molecular dynamics simulations were used to gain insight into possible effects of transmembrane potential on the orientation of CecP relative to the membrane. The simulations appear to confirm that CecP adopts an orientation parallel to the membrane surface and does not insert into the bilayer in response to a cis positive transmembrane voltage difference. Taken together, the results further support a 'carpet-like' mechanism, rather than the formation of transmembrane pores, as the mode of action of CecP. According to this model, formation of a layer of peptide monomers on the membrane surface destablizes the phospholipid packing of the membrane leading to its eventual disintegration.

An amphipathic α-helical structure is considered to be a prerequisite for the lytic activity of most short linear cytolytic polypeptides that act on both mammalian cells and bacteria. This structure allows them also to exert diverse pathological and pharmacological effects, presumably by mimicking protein components that are involved in membrane-related events. In this study D-amino acid-incorporated analogues (diastereomers) of the cytolysin pardaxin, which is active against mammalian cells and bacteria, were synthesized and structurally and functionally characterized. We demonstrate that the diastereomers do not retain the α-helical structure, which in turn abolishes their cytotoxic effects on mammalian cells. However, they retain a high antibacterial activity, which is expressed in a complete lysis of the bacteria, as revealed by negative staining electron microscopy. The disruption of the α-helical structure should prevent the diastereomer analogues from permeating the bacterial wall by forming transmembrane pores but rather by dissolving the membrane as a detergent. These findings open the way for a new strategy in developing a novel class of highly potent antibacterial polypeptides for the treatment of infectious diseases, due to the increasing resistance of bacteria to the available antibacterial drugs.

Cecropins are positively charged antibacterial polypeptides that were originally isolated from insects. Later on a mammalian homologue, cecropin PI (CecP), was isolated from pig intestines. While insect cecropins are highly potent against both Gram-negative and Gram-positive bacteria, CecP is as active as insect cecropins against Gram-negative but has reduced activity against Gram-positive bacteria. To gain insight into the mechanism of action of CecP and the molecular basis of its antibacterial specificity, the peptide and its proline incorporated analogue (at the conserved position found in insect cecropins), P²²-CecP, were synthesized and labeled on their N-terminal amino-acids with fluorescent probes, without significantly affecting their antibacterial activities. Fluorescence studies indicated that the N-terminal of CecP is located on the surface of phospholipid membranes. Binding experiments revealed that CecP binds acidic phosphatidylserine/phosphatidylcholine (PS/PC) vesicles better than zwitterionic PC vesicles, which correlates with its ability to permeate the former better than the latter. The shape of the binding isotherms suggest that CecP, like insect cecropin, binds phospholipids in a simple, noncooperative manner. However, resonance energy transfer (RET) measurements revealed that, unlike insect cecropins, CecP does not aggregate in the membrane even at relatively high peptide to lipid ratios. The stoichiometry of CecP binding to vesicles suggests that amount of CecP sufficient to form a monolayer causes vesicle permeation. In spite of the incorporation of the conserved proline in P²²-CecP, the analogue has reduced antibacterial activity, which correlates with its reduced a-helical structure and its lower partitioning and membrane permeating activity with phospholipid vesicles. Taken together, our results support a mechanism in which CecP disrupts the structure of the bacterial membrane by (i) binding of peptide monomers to the acidic surface of the bacterial membrane and (ii) disintegrating the bacterial membrane by disrupting the lipid packing in the bilayers. These results, combined with data reported for other antibacterial polypeptides, suggest that the organization of peptide monomers within phospholipid membranes contributes to Grampositive/ Gram-negative antibacterial specificity.

The pore-forming domain of Bacillus thuringiensis insecticidal CryIIIA δ-endotoxin contains two helices, α5 and α7, that are highly conserved within all different Cry 5-endotoxins. To gain information on the mode of action of δ-endotoxins, we have used a spectrofluorimetric approach and characterized the structure, the organization state, and the ability to self-assemble and to co-assemble within lipid membranes of α5 and α7. Circular dichroism (CD) spectroscopy revealed that α7 adopts a predominantly α-helical structure in methanol, similar to what has been found for α5, and consistent with its structure in the intact molecule. The hydrophobic moment of α7 is higher than that calculated for α5; however, α7 has a lesser ability to permeate phospholipids as compared to α5. Binding experiments with 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD)-labeled peptide demonstrated that α7 binds to phospholipid vesicles with a partition coefficient in the order of 10⁴ M^-1 similar to α5, but with reduced kinetics and in a noncooperative manner, as opposed to the fast kinetics and cooperativity found with α5. Resonance energy transfer measurements between fluorescently labeled pairs of donor (NBD)/acceptor (rhodamine) peptides revealed that, in their membrane-bound state, α5 self-associates but α7 does not, and that α5 coassembles with α7 but not with an unrelated membrane bound α-helical peptide. Furthermore, resonance energy transfer experiments, using α5 segments, specifically labeled in either the N-or C-terminal sides, suggest a parallel organization of α5 monomers within the membranes. Taken together the results are consistent with an umbrella model suggested for the pore forming activity of δ-endotoxin (Li, J., Caroll, J., and Ellar, D. J. (1991) Nature 353, 815-821), where α5 has transmembrane localization and may be part of the pore lining segment(s) while α7 may serve as a binding sensor that initiates the binding of the pore domain to the membrane.

Dermaseptins are 2734 amino acid antimicrobial peptides that irreversibly inhibit growth of pathogenic filamentous fungi, in addition to their ability to inhibit the growth of bacteria, yeasts, and protozoa. Synthetic peptides, with sequences corresponding to dermaseptin-b (DS-b) and its N-terminal extended precursor form dermaseptin-B (DS-B), were synthesized and investigated with respect to their spectrum of antimicrobial activity and their mode of interaction with model membranes composed of PS or PC/PS phospholipids. We found that DS-B is much more potent than DS-b against all microorganisms tested. Furthermore, despite significant structural identity between DS-B and DS-S (Pouny et al., 1992), only the former is highly effective at inhibiting the growth of filamentous fungi. The peptides were labeled selectively at their N-terminal amino acid with either 7-nitrobenz-2-oxa-1, 3-diazol-4-yl (NBD) or rhodamine fluorescent probes, which facilitated the determination of their partition coefficients with phospholipid membranes and their organization in their membrane-bound state. The partition coefficients of DS-B are 10-fold higher than those of DS-b and DS-S, with both acidic and zwitterionic phospholipid vesicles. This may explain the ability of DS-B to permeate both types of vesicles efficiently. Furthermore, while both DS-b and DS-B interact with phospholipid membranes in a noncooperative manner, they are self-associated in their membrane-bound state. This noncooperative binding probably prevents aggregation of the peptides on the surface of outer bacterial membranes, and assists them in efficiently diffusing into the inner target membranes. The exceptional property of DS-B to bind strongly to phospholipid membranes and to form small bundles correlates with its high potential to kill yeast and filamentous fungi. As a molecular model, dermaseptins may be of potential interest in drug design, particularly in antifungal warfare.

Current models of voltage-activated K⁺ channels predict that the channels are formed by the coassembly of four polypeptide monomers, each of which consists of six transmembrane segments (S1S6) and long terminal domains. The aqueous pores are thought to be composed of the conserved H-5 regions contributed by four monomers. In this study, two putative membrane-embedded segments of the Shaker K⁺ channel were synthesized. One segment corresponds to the putative, transmembrane helix S-2 (amino acids 275300), and the other corresponds to the highly conserved 12 amino acid residues within the H-5 region [amino acids 432443, designated (12)H-5)]. Structural and functional characterization at elevated lipid/peptide molar ratios (>3000:1) was performed on the two segments, as well as on a previously synthesized 21 amino acid long peptide with a sequence resemblimg the entire H-5 region (designated (21)H-5) (Peled & Shai, 1993). Circular dichroism spectroscopy revealed that S-2 adopts predominantly α-helical structure in both trifluoroethanol and 35 mM SDS (78% or 99%, respectively), while (12)H-5 and (21)H-5 adopt low α-helical structure only in the presence of 35 mM SDS. Functional characterization demonstrated that S-2 and (12)H-5 segments bind to zwitterionic phospholipids, with partition coefficients on the order of 10⁴ M⁻¹. Resonance energy transfer measurements, between donor/acceptor-labeled pairs of peptides, revealed that the peptides self-associate in their membrane-bound state, which may correlate with the existence of functional interactions between the conserved (12)H-5 regions of different subunits of K⁺ channels (Kirsch et al., 1993). Furthermore, membrane-bound (12)H-5 or (21)H-5 associates with membrane-bound S-2, but does not associate with unrelated, membrane-bound α-helical peptides. These results demonstrate molecular recognition between transmembrane segments of the Shaker K⁺ channel that might contribute to the oligomerization and correct assembly of the monomers, which result in the formation of a functional channel.

A peptide representing the NH₂-terminal (33 amino acid residues) of the fusion protein (F) of Sendai virus, as well as its Gly¹² → Ala¹² mutant, were synthesized, fluorescently labeled, and spectroscopically and functionally characterized. Peptide-induced vesicle fusion was demonstrated by a combination of increased visible absorbance, lipid mixing assay, and electron microscopy. Both peptides, with the mutant peptide being significantly more potent, were shown to induce membrane fusion and bilayer perturbation of negatively charged phospholipid vesicles. These results are consistent with a previous study that showed that a similar mutation in the homologous NH₂-terminal segment of simian virus 5 greatly enhanced syncytium formation (Horvath, C. M., and Lamb, R. A. (1992) J. Virol. 66, 2443-2455). Circular dichroism spectroscopy revealed similar high α-helical contents of both peptides in methanol and in trifluoroethanol. Using fluorescently labeled peptide analogues we found that (i) the peptides' membrane partition coefficients are in the range of 10⁵ M^-1; (ii) the NH₂ terminus of the wild-type peptide is located within the lipid bilayer, whereas that of the variant peptide lies on the surface; and (iii) both peptides tend to self- associate in their membrane-bound state. The results support a model in which an α-helical secondary structure and self-aggregation of peptides are necessary conditions for membrane fusion. The observed differences in the peptides' fusogenic abilities are hypothesized to result from differences in the peptides' degree of penetration into the membrane, induction of membrane destabilization, and ability to cause vesicles to aggregate. The data support Sendai virus-cell fusion models in which the fusion peptide plays a crucial role in fusion induction by destabilizing the bilayer and by triggering the association of viral fusion protein molecules.

The fusogenic properties of the neurotoxin pardaxin and eight of its analogues with small unilamellar vesicles (SUV), composed of egg phosphatidylcholine and phosphatidylserine (PC/PS), were investigated. Fusion was demonstrated by a lipid-mixing assay and by an increase in vesicle size as revealed by electron microscopy. The lipid-mixing assay was performed at either neutral (pH 6.8) or acidic (pH 4.5) conditions, in solutions containing either high or low salt concentrations. A low level of fusion could be induced at neutral pH only by pardaxin derivatives with amino groups at both the peptide's backbone and N-terminus. However, a marked enhancement in the fusogenic activity occurred when amino groups were present also in the C-terminus. Pardaxin analogues in which amino groups were substituted by carboxylic groups induced elevated levels of fusion only at high salt concentrations where enhancement of aggregation occurs, and acidic pH, which increased α-helicity. The influence of mutual interactions between pardaxin's analogues possessing complementary charges on the lipid-mixing process was also studied. At neutral pH and high salt, an inactive acidic analogue increased the fusogenic activity of a complementarycharged basic peptide. However, such mutual interactions at low salt concentrations reduced the fusogenic activity of the pardaxin analogues. Analogues containing D-amino acids were not fusogenic, thus demonstrating the structural specificity of these observations. The results indicate that the charge, α-helical structure, and aggregation of peptide monomers play an important role in the fusogenic ability of polypeptides.

The Bacillus thuringiensis var. israelensis (Bti) cytolytic toxin is hypothesized to exert its toxic activity via pore formation in the cell membrane as a result of the aggregation of several monomers. To gain insight into the toxin's mode of action, 2 putative hydrophobic 22 amino acid peptides were synthesized and characterized spectroscopically and functionally. One peptide corresponded to the putative amphiphilic α-helical region (amino acids 110131, termed helix-2), and the other to amino acids 5071 (termed helix-1) [Ward, E. S., Ellar, D. J., & Chilcott, C. N. (1988) J. Mol. Biol. 202, 527535] of the toxin. Circular dichroism spectroscopy revealed that both segments adopt high α-helical content in a hydrophobic environment, in agreement with previous models. To monitor peptide-lipid and peptide-peptide interactions, the peptides were labeled selectively with either 7-nitro-2, 1,3-benzoxadiazol-4-yl (NBD) (to serve as donor) or tetramethylrhodamine (to serve as an acceptor), at their N-terminal amino acids. Both segments bind strongly to small unilamellar vesicles, composed of zwitterionic phospholipids, with surface partition coefficients on the order of 10⁴ M⁻¹. The shape of the binding isotherms indicates that helix-2 forms large aggregates within phospholipid membranes. Resonance energy transfer experiments demonstrated that the segments self-associate and interact with each other, but do not associate with unrelated membrane-bound peptides. Functional characterization demonstrated that helix-2 permeates phospholipid SUV with a potency similar to that of naturally occurring pore-forming peptides. Thus, the results support a role for helices-1 and −2 in the assembly and in the pore formation by Bti toxin.

MinK (I_sk) is a voltage-dependent K⁺ channel whose gene has been recently cloned and which consists of 130 amino acids [Takumi, T., Ohkubo, H., & Nakanishi, S. (1988) Science 242, 10421045]. The protein contains one putative transmembrane segment by hydropathy analysis. Whether this putative transmembrane segment is involved in the function of the protein was studied. A 32 amino acid peptide (residues 4172) with the sequence SKLEALYILMVLGFFGFFTLGIMLSYIRSKKL, containing the hypothesized transmembrane domain, designated TM-minK, was synthesized and fluorescently labeled. The α-helical content of TM-minK, assessed in methanol using circular dichroism (CD), was 57%. The fluorescent emission spectrum of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled TM-minK displayed a blue shift upon binding to small unilamellar vesicles (SUV), reflecting a relocation of the fluorescent probe to an environment of increased apolarity, i.e., within the lipid bilayer. The increase in NBD's fluorescence upon mixing NBD-labeled TM-minK with small unilamellar vesicles (SUV) was used to generate a binding isotherm, from which was derived a surface partition coefficient of 5.5 × 10⁴ M⁻¹. Fluorescence energy transfer measurements between carboxyfluoresceine-labeled and rhodamine-labeled analogues suggest that TM-minK aggregates within membranes. In addition, single-channel experiments revealed that TM-minK can form single channels in planar lipid membranes only when a trans negative potential is applied. The findings herein experimentally support a role of the transmembrane segment of minK both in the assembly and as a constituent of the pore formed by the protein.

The influence of specific L- to D-amino acid substitutions on the interaction of pardaxin, a shark repellent neurotoxin polypeptide, with phospholipid vesicles and human erythrocytes is described. Twelve modified, truncated, or fluorescently labeled [with the fluorophore 7-nitrobenz-2-oxa-1, 3-diazole-4-yl (NBD) at their N-terminal amino acid] analogues of pardaxin were synthesized by a solid-phase method. Fluorescence measurements were used to monitor the interaction of the analogues with membranes [Rapaport, D., & Shai, Y. (1991)J. Biol. Chem. 266, 23769-23775]. Upon titration of solutions containing the NBD-labeled peptides with small unilamellar vesicles, the fluorescent emission spectra of all NBD-labeled peptides displayed similar blue-shifts, in addition to enhanced intensities, upon relocation of the probe to the more apolar environment. Binding isotherms were constructed from which surface partition constants, in the range of 10⁴ M^-1 were derived. The existence of an aggregation process, suggested by the shape of the binding isotherms, could be associated only with those analogues in which the N-helix (residues 1-9) was not perturbed. The α-helical content of the analogues was estimated by circular dichroism (CD) spectroscopy, both before and after binding to vesicles at neutral pH. The ability of the peptides to dissipate a diffusion potential and to cause calcein release, as well as to lyse human erythrocytes, served to functionally characterize the peptides. The results support a two ケ-helix model, with a bend at position 13, as best describing pardaxin in its membrane-bound state. The study also demonstrates that local configurational changes of amino acids, including prolines, do not affect the abilities of the analogues to bind to phospholipid membranes, but do have some effect on their membrane-permeating activities.

Protonligand stability constants (pKd) and metalligand stability constants for six oximes of 4substituted2acetyl phenol in H₂O: Dioxane (1:3) are obtained by the spectrophotometric method of Magnusson et al. as modified by Patel et al.