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.

Pseudomonas aeruginosa is the major microorganism colonizing the respiratory epithelium in cystic fibrosis (CF) sufferers. The widespread use of available antibiotics has drastically reduced their efficacy, and antimicrobial peptides (AMPs) are a promising alternative. Among them, the frog skin-derived AMPs, i.e., Esc(1-21) and its diastereomer, Esc(1-21)-1c, have recently shown potent activity against free-living and sessile forms of P. aeruginosa. Importantly, this pathogen also escapes antibiotics treatment by invading airway epithelial cells. Here, we demonstrate that both AMPs kill Pseudomonas once internalized into bronchial cells which express either the functional or the ΔF508 mutant of the CF transmembrane conductance regulator. A higher efficacy is displayed by Esc(1-21)-1c (90% killing at 15 μM in 1 h). We also show the peptides' ability to stimulate migration of these cells and restore the induction of cell migration that is inhibited by Pseudomonas lipopolysaccharide when used at concentrations mimicking lung infection. This property of AMPs was not investigated before. Our findings suggest new therapeutics that not only eliminate bacteria but also can promote reepithelialization of the injured infected tissue. Confocal microscopy indicated that both peptides are intracellularly localized with a different distribution. Biochemical analyses highlighted that Esc(1-21)-1c is significantly more resistant than the all-l peptide to bacterial and human elastase, which is abundant in CF lungs. Besides proposing a plausible mechanism underlying the properties of the two AMPs, we discuss the data with regard to differences between them and suggest Esc(1-21)-1c as a candidate for the development of a new multifunctional drug against Pseudomonas respiratory infections.

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.

For successful infection and propagation viruses must overcome many obstacles such as the immune system and entry into their host cells. HIV utilizes its trimeric envelope protein gp160, specifically the gp41 subunit, to enter its host cell. During this process, a gp41-central coiled coil is formed from three N- and three C-terminal heptad repeats, termed the six-helix bundle (SHB), which drives membrane fusion. Recently, T-cell suppression has been reported as an additional function for several regions of gp41 by interfering with the T-cell receptor (TCR) signalling cascade. One of these regions encompasses the conserved pocket binding domain (PBD) that is situated in the C-terminal heptad repeat (CHR) and stabilizes SHB formation. This could indicate that the PBD plays a role in T-cell suppression in addition to its role in membrane fusion. To investigate this dual function, we used two independent cell cultures coupled with biophysical techniques. The data reveal that the PBD mediates T-cell suppression by stabilizing a TCR-binding conformation in the membrane. Moreover, we show that the clinically used HIV fusion inhibitor T-20 did not show suppressive abilities, in contrast with the potent fusion inhibitor C34. In addition, by focusing on SHB conformation after its assembly, we shed light on a mechanism by which gp41's function alternates from membrane fusion facilitation to suppression of TCR activation.

Multidrug resistance bacteria are a major concern worldwide. These pathogens cannot be treated with conventional antibiotics and thus alternative therapeutic agents are needed. Antimicrobial peptides (AMPs) are considered to be good candidates for this purpose. Most AMPs are short and positively charged amphipathic peptides, which are found in all known forms of life. AMPs are known to kill bacteria by binding to the negatively charged bacterial surface, and in most cases cause membrane disruption. Resistance toward AMPs can be developed, by modification of bacterial surface molecules, secretion of protective material and up-regulation or elimination of specific proteins. Because of the general mechanisms of attachment and action of AMPs, bacterial resistance to AMPs often involves biophysical and biochemical changes such as surface rigidity, cell wall thickness, surface charge, as well as membrane and cell wall modification. Here we focus on the biophysical, surface and surrounding changes that bacteria undergo in acquiring resistance to AMPs. In addition we discuss the question of whether bacterial resistance to administered AMPs might compromise our innate immunity to endogenous AMPs. This article is part of a Special Issue entitled: Bacterial Resistance to Antimicrobial Peptides.

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.

Background:HIV-1 infectivity is decreased by specific mutations that alter the hydrophobicity level in the HIV-1 gp41 loop core. Results:Antibody recognition, disulfide-bond formation, and lipid mixing of loop domain peptides are strongly affected by these mutations. Conclusion:The hydrophobic core maintains proper function and structure of the loop region. Significance:A better understanding of the membrane fusion mechanism of HIV and similar viruses is provided.

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.

Modulation of T-cell responses by HIV occurs via distinct mechanisms, 1 of which involves inactivation of T cells already at the stage of virus-cell fusion. Hydrophobic portions of the gp41 protein of the viral envelope that contributes to membrane fusion may modulate T-cell responsiveness. Here we found a highly conserved sequence (termed "ISLAD") that is associated with the membranotropic gp41 loop region. We showed that ISLAD has the ability to bind the T-cell membrane and to interact with the T-cell receptor (TCR) complex. Furthermore, ISLAD inhibited T-cell proliferation and interferon-g secretion that resulted from TCR engagement through antigen-presenting cells. Moreover, administering ISLAD (10 mg per mouse) to an experimental autoimmune encephalomyelitis (EAE)model ofmultiple sclerosis reduced the severity of the disease. Thiswas related to the inhibition of pathogenic T-cell proliferation and to reduced pro-inflammatory cytokine secretion in the lymph nodes of ISLAD-treated EAE mice. The data suggest that T-cell inactivation by HIV during membrane fusion may lie in part in this conserved sequence associated with the gp41 loop region.

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.

The human immunodeficiency virus utilizes its gp41 fusion protein to mediate virus-cell membrane fusion. The conserved disulfide loop region in the gp41 hairpin conformation reverses the protein chain, such that the N-terminal heptad repeat and the C-terminal heptad repeat regions interact to form the six-helix bundle. Hence, it is conceivable that the sequential folded N- and C-terminal parts of the loop region also interact. We show that the N- and C-terminal parts of the loop preferably form disulfide-bonded heterodimers with slow oxidation kinetics. Furthermore, when the two parts were linked to a single polypeptide to form the full-length loop, only an intramolecular disulfide-bonded loop was formed. Fluorescence studies revealed that an interaction takes place between the N- and C-terminal parts of the loop in solution, which was sustained in membranes. Functionally, only a combination of the N- and C-loop parts induced lipid mixing of model liposomes, the level of which increased 8-fold when they were connected to a single polypeptide chain. In both cases, the activity was independent of the oxidation state of the cysteines. Overall, the data (i) provide evidence of a specific interaction between the N- and C-terminal parts of the loop, which can further stabilize gp41 hairpin conformation, and (ii) suggest that the interaction between the N- and C-terminal parts of the loop is sufficient to induce lipid merging without forming a disulfide bond.

Fusion of the human immunodeficiency virus (HIV) with target cells is mediated by the gp41 subunit of the envelope protein. Mutation and deletion studies within the transmembrane domain (TMD) of intact gp41 influenced its fusion activity. In addition, current models suggest that the TMD is in proximity with the fusion peptide (FP) at the late fusion stages, but there are no direct experimental data to support this hypothesis. Here, we investigated the TMD focusing on two regions: the N-terminal containing the GxxxG motif and the C-terminal containing the GLRI motif, which is conserved among the TMDs of HIV and the T-cell receptor. Studies utilizing the ToxR expression system combined with synthetic peptides and their fluorescent analogues derived from TMD revealed that the GxxxG motif is important for TMD self-association, whereas the C-terminal region is for its heteroassociation with FP. Functionally, all three TMD peptides induced lipid mixing that was enhanced significantly upon mixing with FP. Furthermore, the TMD peptides inhibited virus-cell fusion apparently through their interaction with their endogenous counterparts. Notably, the R2E mutant (in the GLRI) was significantly less potent than the two others. Overall, our findings provide experimental evidence that HIV-1 TMD contributes to membrane assembly and function of the HIV-1 envelope. Owing to similarities between functional domains within viruses, these findings suggest that the TMDs and FPs may contribute similarly in other viruses as well.

Pathogenic bacteria can resist their microenvironment by changing the expression of virulence genes. In Salmonella typhimurium, some of these genes are controlled by the two-component system PhoP-PhoQ. Studies have shown that activation of the system by cationic antimicrobial peptides (AMPs) results, among other changes, in outer membrane remodeling. However, it is not fully clear what characteristics of AMPs are required to activate the PhoP-PhoQ system and whether activation can induce resistance to the various AMPs. For that purpose, we investigated the ability of a broad repertoire ofAMPsto traverse the inner membrane, to activate the PhoP-PhoQ system, and to induce bacterial resistance. The AMPs differ in length, composition, and net positive charge, and the tested bacteria include two wild-type (WT) Salmonella strains and their corresponding PhoP-PhoQ knock-out mutants. A lacZ-reporting system was adapted to follow PhoP-PhoQ activation. The data revealed that: (i) a good correlation exists among the extent of the positive charge, hydrophobicity, and amphipathicity of an AMP and its potency to activate PhoP-PhoQ; (ii) a 1 charged peptide containing histidines was highly potent, suggesting the existence of an additional mechanism independent of the peptide charge; (iii) the WT bacteria are more resistant to AMPs that are potent activators of PhoP-PhoQ; (iv) only a subset of AMPs, independent of their potency to activate the system, is more toxic to the mutated bacteria compared with the WT strains; and (v) short term exposure of WT bacteria to these AMPs does not enhance resistance. Overall, this study advances our understanding of the molecular mechanism by which AMPs activate PhoP-PhoQ and induce bacterial resistance. It also reveals that some AMPs can overcome such a resistance mechanism.

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.

Due to the rapid emergence of resistant microbes to the currently available antibiotics, cationic antimicrobial peptides have attracted considerable interest as a possible new generation of anti-infective compounds. However, low cost development for therapeutic or industrial purposes requires, among other properties, that the peptides will be small and with simple structure. Therefore, considerable research has been devoted to optimizing peptide length combined with a simple design. This review focuses on the similarities and differences in the mode of action and target cell specificity of two families of small peptides: the naturally occurring temporins from the skin of amphibia and the engineered ultrashort lipopeptides. We will also discuss the finding that acylation of cationic peptides results in molecules with a more potent spectrum of activity and a higher resistance to proteolytic degradation. Conjugation of fatty acids to linear native peptide sequences is a powerful strategy to engineer novel successful anti-infective drugs.

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: Soft tissue sarcoma (STS) is an anatomically and histologically heterogeneous neoplasia that shares a putative mesenchymal cell origin. The treatment with common chemotherapeutics is still unsatisfying because of association with poor response rates. Although evidence is accumulating for potent oncolytic activity of host defense peptides (HDPs), their potential therapeutic use is often limited by poor bioavailability and inactivation in serum. Therefore, we tested the designer host defense-like lytic D,L-amino acid peptide [D]-K₃H₃L₉ on two STS cell lines in vitro and also in an athymic and syngeneic mouse model. In recent studies the peptide could show selectivity against prostate carcinoma cells and also an active state in serum. Methods: In vitro the human synovial sarcoma cell line SW982, the murine fibrosarcoma cell line BFS-1 and primary human fibroblasts as a control were exposed to [D]-K₃H₃L₉, a 15mer D,L-amino acid designer HDP. Cell vitality in physiological and acidic conditions (MTT-assay), cell growth (BrdU) and DNA-fragmentation (TUNEL) were investigated. Membrane damage at different time points could be analyzed with LDH assay. An antibody against the tested peptide and recordings using scanning electron microscopy could give an inside in the mode of action. In vivo [D]-K₃H₃L₉ was administered intratumorally in an athymic and syngeneic (immunocompetent) mouse model with SW982 and BFS-1 cells, respectively. After three weeks tumor sections were histologically analyzed. Results: The peptide exerts rapid and high significant cytotoxicity and antiproliferating activity against the malignant cell lines, apparently via a membrane disrupting mode of action. The local intratumoral administration of [D]-K₃H₃L₉ in the athymic and syngeneic mice models significantly inhibited tumor progression. The histological analyses of the tumor sections revealed a significant antiproliferative, antiangiogenic activity of the treatment group. Conclusion: These findings demonstrate the in vitro and in vivo oncolytic activity of [D]-K₃H₃L₉ in athymic and syngeneic mouse models.

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.

A new method is presented for patterning surfaces with gradient properties. The method is based on magnetolithography in which the surface patterning is performed by applying a gradient of a magnetic field on the substrate, using paramagnetic metal masks in the presence of a constant magnetic field. Superparamagnetic nanoparticles (NPs) are deposited on the substrate, and they assemble according to the field and its gradients induced by the mask. Once they pattern the substrate, they protect their sites on the substrate from interacting with any other species. The areas not protected by the NPs can be covered by molecules that chemically bind to the substrate. After these molecules are bound, the NPs are removed, and other molecules may be adsorbed on the newly exposed area. The new technique is based on a parallel process that can be carried out on a full wafer. It provides high resolution, it creates gradient continuously from submicrometers to milimeters, and it can be performed on surfaces that are not flat and that are even on the inside of a tube. The gradient that is formed is not limited to a specific property or type of substrate.

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 present a new approach to obtain details on the distribution and average structure and locations of membrane-associated peptides. The approach combines (i) pulse double electron-electron resonance (DEER) to determine intramolecular distances between residues in spin labeled peptides, (ii) electron spin echo envelope modulation (ESEEM) experiments to measure water exposure and the direct interaction of spin labeled peptides with deuterium nuclei on the phospholipid molecules, and (iii) Monte Carlo (MC) simulations to derive the peptide-membrane populations, energetics, and average conformation of the native peptide and mutants mimicking the spin labeling. To demonstrate the approach, we investigated the membrane-bound and solution state of the well-known antimicrobial peptide melittin, used as a model system. A good agreement was obtained between the experimental results and the MC simulations regarding the distribution of distances between the labeled amino acids, the side chain mobility, and the peptide's orientation. A good agreement in the extent of membrane penetration of amino acids in the peptide core was obtained as well, but the EPR data reported a somewhat deeper membrane penetration of the termini compared to the simulations. Overall, melittin adsorbed on the membrane surface, in a monomelic state, as an amphipatic helix with its hydrophobic residues in the hydrocarbon region of the membrane and its charged and polar residues in the lipid headgroup region.

A new family of synthetic, membrane-active, ultrashort lipopeptides composed of only four amino acids linked to fatty acids was tested for the ability to induce systemic resistance and defense responses in plants. We found that two peptides wherein the third residue is a D-enantiomer (italic), C ₁₆-KKKK and C₁₆-KLLK, can induce medium alkalinization of tobacco suspension-cultured cells and expression of defense-related genes in cucumber and Arabidopsis seedlings. Moreover, these compounds can prime systemic induction of antimicrobial compounds in cucumber leaves similarly to the plant-beneficial fungus Trichoderma asperellum T203 and provide systemic protection against the phytopathogens Botrytis cinerea B05, Pseudomonas syringae pv. lachrimans, and P. syringae pv. tomato DC3000. Thus, short cationic lipopeptides are a new category of compounds with potentially high utility in the induction of systemic resistance in plants.

Peptide inhibitors derived from HIV-gp41 envelope protein play a pivotal role in deciphering the molecular mechanism of HIV-cell fusion. According to accepted models, N-heptad repeat (NHR) peptides can bind two targets in an intermediate fusion conformation, thereby inhibiting progression of the fusion process. In both cases the orientation towards the endogenous intermediate conformation should be important. To test this, we anchored NHR to the cell membrane by conjugating fatty acids with increasing lengths to the N- or C-terminus of N36, as well as to two known N36 mutants; one that cannot bind C-heptad repeat (CHR) but can bind NHR (N36 MUTe,g), and the second cannot bind to either NHR or CHR (N36 MUTa,d). Importantly, the IC⁵⁰ increased up to 100-fold in a lipopeptide-dependent manner. However, no preferred directionality was observed for the wild type derived lipopeptides, suggesting a planar orientation of the peptides as well as the endogenous NHR region on the cell membrane. Furthermore, based on: (i) specialized analysis of the inhibition curves, (ii) the finding that N36 conjugates reside more on the target cells that occupy the receptors, and (iii) the finding that N36 MUTe,g acts as a monomer both in its soluble form and when anchored to the cell membrane, we suggest that anchoring N36 to the cell changes the inhibitory mode from a trimer which can target both the endogenous NHR and CHR regions, to mainly monomeric lipopetides that target primarily the internal NHR. Besides shedding light on the mode of action of HIV-cell fusion, the similarity between functional regions in the envelopes of other viruses suggests a new approach for developing potent HIV-1 inhibitors.

Previously, we reported that intratumor or systemic inoculation of a cationic 15-mer, innate immunity-like lytic peptide composed of d- and i-amino acids ([D]-K₆I₉) caused growth arrest of 22RV1 prostate carcinoma xenografts in a mouse model. However, despite its therapeutic potential, this peptide has significant systemic toxicity at concentrations slightly higher than the therapeutic one. Here, we used the acidic environment created by solid tumors as a trigger to activate anticancer lytic peptides by making them cationic only at low pH levels. We achieved this selectivity by substituting lysines (pKa, ~ 10.5) for histidines (pKa, ~ 6.1) in the parental peptide [D]-K₆L₉. Histidine is protonated below pH 7. For that purpose, we replaced either three or all six lysines in the parental peptide with histidines to obtain the peptides [DJ- K₃H ₃L₉ and [D]-H₆L₉. Interestingly, in vitro experiments showed pH-dependent activity only with [D]-H₆L ₉ mainly toward cancer cell lines. However, both peptides showed reduced systemic toxicity compared with the parental peptide. Intratumor and systemic inoculation of these peptides resulted in a significant decrease in the 22RV1 prostate cancer tumor volume and systemic secretion of prostate-specific antigen in a xenograft mice model. Moreover, histologic modifications revealed a significant reduction in new blood vessels selectively in tumor tissues after treatment with the peptides compared with the untreated tumors. The lytic mode of action of these new peptides, which makes it difficult for the cancer cells to develop resistance, and their selective and pH-dependent activity make them potential candidates for treatment of solid cancer tumors.

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.

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.

Conus venoms are estimated to comprise over 100,000 distinct pharmacologically active peptides, the majority probably targeting ion channels. Through the characterization of a cytolytic peptide from the venom of Conus mustelinus, conolysin-Mt, we expand the known conopeptide mechanisms to include association with and destruction of cellular membranes. A new 23AA conopeptide, conolysin-Mt has potent hemolytic activity when tested on human erythrocytes. At a concentration of 0.25 mu M, the peptide permeabilized both negatively charged prokaryotic (PE:PG) and zwitterionic eukaryotic (PC: cholesterol) model membranes. The affinity constants (K-A) of conolysin-Mt for PE:PG and PC:cholesterol model membranes were 0.9 +/- 0.3 x 10(7) and 3 +/- 1 1 x 10(7) M-1, respectively. In contrast, conolysin-Mt exhibited low antimicrobial activity (MIC > 50 mu M) against two Escherichia coli strains, with an MIC for the Gram-positive S. aureus of 25-50 mu M. The specificity of conolysin-Mt for native eukaryotic membranes is a novel feature of the peptide compared to other well-characterized cytolytic peptides such as melittin.

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.

HIV-1 entry into its host cell involves a sequential interaction whereby gp41 is in direct contact with the plasma membrane. Understanding the effect of membrane composition on the fusion mechanism can shed light on the unsolved phases of this complex mechanism. Here, we studied N36, a peptide derived from the N-heptad-repeat (NHR) of the gp41 ectodomain, its six helix bundle (SHB) forming counterpart C34, together with the N-terminal 70-mer wild-type peptide (N70), and additional gp41 ectodomain-derived peptides in the presence of two membranes, modeling inner and outer leaflets of the plasma membrane. Information on the structure of these peptides, their affinity towards phospholipids and their ability to induce vesicle fusion was gathered by a variety of fluorescence, spectroscopic and microscopy methods. We found that N36, having strong affinity towards phospholipids, prominently shifts conformation from α-helix in an outer leaflet-like zwitterionic membrane to β-sheet in a membrane mimicking the negatively charged inner leaflet environment, leading to pronounced fusion-activity. Real-time atomic force microscopy (AFM) was used to study the peptides' effect on the membrane morphology, revealing severe bilayer perturbation and extensive pore formation.We also found, that the N36/C34 core is destabilized by electronegative, but not zwitterionic phospholipids. Taken together, our data suggest that the fusion-active pore forming conformation of gp41 is extended, upstream of the SHB. In this manner, folding of the ectodomain into a SHB might also serve as a negative regulator of fusion by impeding gp41 fusion-active surfaces, thus preventing irreversible damage to the cell membrane. This assumption is supported by the finding that pre-incubation of large unilamellar vesicles (LUV) with C-heptad repeat (CHR)-derived fusion inhibitors reduces the fusogenic activity of N-terminal peptides in a dose-dependant manner, and suggests that CHR-derived fusion inhibitors inhibit HIV entry in an analogous mechanism.

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.

Binding of lipopolysaccharide (LPS) to macrophages results in proinflammatory cytokine secretion. In extreme cases it leads to endotoxic shock. A few innate immunity antimicrobial peptides (AMPs) neutralize LPS activity. However, the underlying mechanism and properties of the peptides are not yet clear. Toward meeting this goal we investigated four AMPs and their fluorescently labeled analogs. These AMPs varied in composition, length, structure, and selectivity toward cells. The list included human LL-37 (37-mer), magainin (24-mer), a 15-mer amphipathic α-helix, and its D,L-amino acid structurally altered analog. The peptides were investigated for their ability to inhibit LPS-mediated cytokine release from RAW264.7 and bone marrow-derived primary macrophages, to bind LPS in solution, and when LPS is already bound to macrophages (fluorescence spectroscopy and confocal microscopy), to compete with LPS for its binding site on the CD14 receptor (flow cytometry) and affect LPS oligomerization. We conclude that a strong binding of a peptide to LPS aggregates accompanied by aggregate dissociation prevents LPS from binding to the carrier protein lipopolysaccharide-binding protein, or alternatively to its receptor, and hence inhibits cytokine secretion.

Antimicrobial peptides and lipopeptides play an essential protective role in the innate immune system of all organisms. Despite many studies, the factors that dictate their cell-selectivity and pH-dependent activity are yet not clear. This is important because various organs of the human body have an acidic pH environment, for example, the vagina, gastric lumen, cryogenic dental foci, and lung-lining fluids in cystic fibrosis and asthma. In this study we synthesized a new group of lipopeptides by conjugating dodecanoic acid (DDA) to the N-termini of 12-mer peptides LXXLLXXLLXXL (L₆X₆, X = Lys, His, Arg, and all the leucines are D-amino acid enantiomers) and investigated their pH-dependent biological activity and a plausible mode of action by using model phospholipids mimicking bacterial, mammalian, and fungal membranes. The data revealed that, depending on the basic amino acid incorporated, the lipopeptides are active against both bacteria and fungi or solely toward fungi. Furthermore, their activity is expressed at an acidic pH alone, neutral pH alone, or at both environments. Determination of secondary structure, membrane leakage experiments, surface plasmon resonance (SPR) binding experiments, and transmission electron microscopy suggest the involvement of a membranolytic effect. This mode of action, which should make it hard for the microorganism to develop resistance, their selective and pH-dependent activity, as well as pharmacological advantages due to the presence of D-amino acids, make them potential candidates for the treatment of mycoses in organs, under various pH environments, especially in cases where the bacterial flora should not be harmed.

T cell activation requires the cross-talk between the CD3-signaling complex and the T cell receptor (TCR). A synthetic peptide coding for the TCR alpha. transmembrane domain (CP) binds CD3 molecules, interferes with the CD3/TCR cross-talk, and inhibits T cell activation. Intermolecular interactions are sterically constrained; accordingly no sequence-specific interactions are thought to occur between D- and L-stereoisomers. This argument was recently challenged when applied to intra-membrane protein assembly. In this paper we studied the ability of a D-stereoisomer of CP (D-CP) to inhibit T cell activation. L-CP and D-CP co-localized with the TCR in the membrane and inhibited T cell activation in a sequence-specific manner. In vivo, both L-CP and D-CP inhibited adjuvant arthritis. In molecular terms, these results suggest the occurrence of structural reorientation that facilitates native-like interactions between D-CP and CD3 within the membrane. In clinical terms, our results demonstrate that D-stereoisomers retain the therapeutic properties of their L-stereoisomers, while they benefit from an increased resistance to degradation.

The gp41 glycoprotein of HIV-1 is considered to be responsible for the actual fusion process between the virus and the host membranes. According to a prevailing model, gp41 trimer organization, directed by the N-terminal coiled-coil region (NHR), is essential for steps involved in the actual merging of viral and cellular membranes. This study addresses a major question: Is the specific sequence of the NHR obligatory for the fusion process, or can it be replaced by distant coiled coils that form different oligomeric states in solution? For this purpose we synthesized three known GCN4 coiled-coil mutants that oligomerize in solution into either dimers, trimers, or tetramers. These peptides were chemically ligated to the fusion peptide thereby creating three chimera peptides with different oligomeric tendencies in solution. These peptides were investigated, together with the 70-mer wild-type peptide (N70), regarding their structure in solution and membrane by using circular dichroism (CD) and FTIR spectroscopies, their ability to induce vesicle fusion, and their ability to bind phospholipid membranes by using surface plasmon resonance (SPR). Our results suggest that local assembly of fusion peptides, facilitated by coiled-coil oligomers, increases lipid mixing ability, probably by facilitating stronger binding of the fusion peptides to the opposing membrane as revealed by SPR. However, N70 is significantly more active than the other chimeras. Overall, the data indicate a correlation between the distinct conformation of N70 in solution and in membranes and its enhanced lipid mixing relative to the GCN4 chimeras.

Cationic antimicrobial peptides serve as the first chemical barrier between all organisms and microbes. One of their main targets is the cytoplasmic membrane of the microorganisms. However, it is not yet clear why some peptides are active against one particular bacterial strain but not against others. Recent studies have suggested that the lipopolysaccharide (LPS) outer membrane is the first protective layer that actually controls peptide binding and insertion into Gram-negative bacteria. In order to shed light on these interactions, we synthesized and investigated a 12-mer amphipathic α-helical antimicrobial peptide (K₅L₇) and its diastereomer (4D-K₅L₇) (containing four D-amino acids). Interestingly, although both peptides strongly bind LPS bilayers and depolarize bacterial cytoplasmic membranes, only the diastereomer kills Gram-negative bacteria. Attenuated total reflectance Fourier transform infrared, CD, and surface plasmon resonance spectroscopies revealed that only the diastereomer penetrates the LPS layer. In contrast, K₅L₇ binds cooperatively to the polysaccharide chain and the outer phosphate groups. As a result, the self-associated K₅L₇ is unable to traverse through the tightly packed LPS molecules, revealed by epifluorescence studies with LPS giant unilamellar vesicles. The difference in the peptides' modes of binding is further demonstrated by the ability of the diastereomer to induce LPS miscellization, as shown by transmission electron microscopy. In addition to increasing our understanding of the molecular basis of the protection off bacteria by LPS, this study presents a potential strategy to overcome resistance by LPS, and it should help in the design of antimicrobial peptides for future therapeutic purposes.

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

We report that intravenous injection (3 mg/kg of body weight twice daily) of a diastereomer (containing 33% D amino acids) of an antimicrobial peptide, K₆L₆, (LKLLKKLLKKLLKLL-NH₂), but not the all-L-amino-acid parental peptide, cures neutropenic mice infected with gentamicin-sensitive Pseudomonas aeruginosa and gentamicin-resistant Acinetobacter baumannii bacteria. Various biophysical experiments suggest a membranolytic-like effect.

The amphipathic α-helix is a common motif found in many cell lytic peptides including antimicrobial peptides. We have recently shown that significantly altering the amphipathic structure of a lytic peptide by reshuffling its sequence and/or replacing a few L-amino acids with their D-enantiomers did not significantly affect the antimicrobial activity of the peptides nor their ability to bind and permeate negatively charged (PE/PG) membranes. However, a pronounced effect was observed regarding their hemolytic activity and their ability to bind and permeate zwitterionic (PC/Cho) membranes. To shed light on these findings, here we used surface plasmon resonance (SPR) with mono- and bilayer membranes. We found that the L-amino acid (aa) peptides bound 10-25-fold stronger to PC/Cho bilayers compared with monolayers, whereas the diastereomers bound similarly to both membranes. A two-state reaction model analysis of the data indicated that this difference is due to the insertion of the L-aa peptides into the PC/Cho bilayers, whereas the diastereomers are surface-localized. In contrast, only an ∼2-fold difference was found with negatively charged membranes. Changes in the amphipathicity markedly affected only the insertion of the L-aa peptides into PC/Cho bilayers. Furthermore, whereas the all-L-aa peptides bound similarly to the PC/Cho and PE/PG membranes, the diastereomers bound ∼100-fold better to PE/PG compared with PC/Cho membranes, and selectivity was determined only in the first binding step. The effect of the peptides on the lipid order determined by using ATR-FTIR studies supported these findings. Besides shedding light on the mode of action of these peptides, the present study demonstrates SPR as a powerful tool to differentiate between non-cell-selective compared with bacteria-selective peptides, based on differences in their membrane binding behavior.

Protein-protein recognition is an essential process in life. The chemistry of these kind of interactions is predominately stereospecific (i.e. receptor-ligand, antibody-hapten binding). Here, we investigated whether the hydrophobic nature of the membrane affects this stereospecificity. To this end, we synthesized a diastereomer analogue (2D-GPA) of the glycophorin A transmembrane helix, with two L-valine residues replaced by their D-enantiomer. This ensures a disruption of the secondary structure. We investigated the ability of the diastereomer peptide to recognize the GPA chimera in the ToxR homodimer reporting system, in vivo. The peptide demonstrated a dose-dependent dominant negative effect on the GPA transmembrane in the bacterial ToxR system, suggesting a wild-type like interaction. This result was corroborated in vitro by fluorescence energy transfer between 2D-GPA and all-L GPA. Peptide binding to the bacteria was confirmed through confocal imaging, and Western blot confirmed that ToxR GPA receptor levels are not affected by the presence of the exogenous peptide. In order to understand the structural basis for heterodimer formation, homodimer and heterodimer structures, based on the NMR 3D structure of GPA, were subjected to a molecular dynamics simulation. The resulting heterodimer structure maintained most of the original inter-helical interactions, and its structure is similar to that of the homodimer. We postulate that the need to satisfy all H-bonds can compensate for the structural strain induced by the presence of the D-amino acid residues.

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.

The dramatically increased frequency of opportunistic fungal infections has prompted research to diversify the arsenal of antifungal agents. Antimicrobial peptides constitute a promising family for future antibiotics with a new mode of action. However, only a few are effective against fungal pathogens because of their ability to self-assemble. Recently, we showed that the conjugation of fatty acids to the potent antibacterial peptide magainin endowed it with antifungal activity concomitant with an increase in its oligomeric state in solution. To investigate whether a high potency of the parental peptide is prerequisite for antifungal activity, we conjugated undecanoic acid (UA) and palmitic acid (PA) to inactive diastereomers of magainin containing four D-amino acids ([D]-4-magainin), as well as to a weakly active diastereomeric lytic peptide containing Lys and Leu ([D]-K ₅L₇). All lipopeptides gained potent activity toward Cryptococcus neoformans. Most importantly, [D]-K₅L₇-UA was highly potent against all microorganisms tested, including bacteria, yeast, and opportunistic fungi. All lipopeptides increased the permeability of Escherichia coli spheroplasts and intact C. neoformans, as well as their corresponding membranes, phosphatidylethanol (PE)/phosphatidylglycerol (PG) and phosphatidylcholine (PC)/PE/phosphatidylinositol (PI)/ergosterol, respectively. The extent of membrane-permeating activity correlated with their biological function, suggesting that the plasma membrane was one of their major targets. Circular dichroism (CD) and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy revealed that their mode of oligomerization in solution, structure, and organization in membranes have important roles regarding their antibacterial and antifungal activities. Together with the advantage of using diastereomers versus all L-amino acid peptides, this study paves the way to the design of a new group of potent antifungal peptides urgently needed to combat opportunistic fungal infection.

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.

Antimicrobial peptides are a large group of gene-encoded, net positively charged polypeptides, produced by living organisms of all types including human and plants. They are mobilized shortly after infection as part of the innate immunity of these species and act rapidly to neutralize a broad range of microbes. Nowadays, thousands of native and de-novo designed antimicrobial peptides are available. They vary considerably in length, composition, charge and secondary structure. Despite these variations most antimicrobial peptides use a similar target, which is the bacterial phospholipid membrane. Many of them use a common general mechanism, the "carpet" mechanism, in which they accumulate on the bacterial membrane up to a threshold concentration, and then effect membrane permeation/disintegration. However, the structure of the permeation pathway may vary for different peptides and may include channel aggregates, toroidal pores or channels. Target specificity is determined by the negatively charged bacterial membrane, the net positive charge of the peptide, its hydrophobicity, oligomeric state in solution and in the membrane, and the stability of its secondary structure. A novel group of non hemolytic antimicrobial peptides were derived from diastereomers (containing D- and L-amino acids) of lytic peptides based on parameters required by the carpet mechanism. Because these disastereomers exhibit several advantages over their all-L amino acid counterparts, they have a potential to be developed for therapeutic use both in vitro and in vivo.

The N-terminal fusion peptide of Sendai virus F₁ envelope glycoprotein is a stretch of 14 amino acids, most of which are hydrophobic. Following this region, we detected a segment of 11 residues that are strikingly similar to the N-terminal fusion peptide. We found that, when anchored to the membrane by palmitoylation of its N-terminus, this segment (WT-palm-19-33) induces membrane fusion of large unilamellar liposomes to almost the same extent as a segment that includes the N-terminal fusion peptide. The activity of WT-palm-19-33 was dependent on its specific sequence, as a palmitoylated peptide with the same amino-acid composition but a scrambled sequence was inactive. Interestingly, two mutations (G7A and G12A) known to increase F₁-induced cell-cell fusion, also increased the homology between the N-terminal fusion peptide and WT-palm-19-33. The role of the amino-acid sequence on the fusogenicity, secondary structure, and mechanism of membrane fusion was analyzed by comparing a peptide comprising both homologous segments (WT 1-33), a G12A mutant (G12A 1-33), a G7A-G12A double mutant (G7A-G12A 1-33), and a peptide with a scrambled sequence (SC1-33). Based on these experiments, we postulate that replacement of Gly 7 and Gly12 by Ala increases the α helical content of the N-terminal region, with a concomitant increase in its fusogenic activity. Furthermore, the dissimilar abilities of the different peptides to induce membrane negative curvature as well as to promote isotropic ³¹P NMR signals, suggest that these mutations might also alter the extent of membrane penetration of the 33-residue peptide. Interestingly, our results serve to explain the effect of the G7A and G12A mutations on the fusogenic activity of the parent F₁ protein in vivo.

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

Paramyxoviruses penetrate into their host cells by fusing their membranes with the plasma membrane. The hydrophobic N terminus of their F1 protein, termed the 'fusion peptide', is thought to be responsible for this process. Recently, an additional internal fusion peptide, homologous in sequence to the N-terminal fusion peptide of HIV-1, was identified in the Sendai virus F1 protein. Here, we investigated whether the presence of an additional internal fusion peptide is a general feature of paramyxoviridae. To this end, we synthesized and structurally and functionally characterized three peptides: (i) MV-197, which corresponds to an internal segment of the F1 protein of the measles virus (amino acids 197-225), homologous in location but not in sequence to the internal fusion peptide of the Sendai virus, (ii) Mu-MV-197, a randomized version of MV-197, and (iii) the 33 amino acid N-terminal fusion peptide of the measles virus. Remarkably, only MV-197 was highly fusogenic toward large unilamellar vesicles composed of either zwitterionic (phosphatidylcholine or phosphatidylcholine/sphingomyelin/cholesterol, a composition similar to that of human cell membranes) or negatively charged phospholipids. Binding experiments, circular dichroism spectroscopy in phospholipid membranes, and homo energy-transfer studies with fluorescently labeled peptides revealed that MV-197 adopts a predominant α-helical structure and shares properties similar to those reported for known fusion peptides. These results suggest that the presence of two fusion peptides in the F1 protein is a general feature of paramyxoviruses.

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 pore-forming domain of Bacillus thuringiensis Cry1Ac insecticidal protein comprises of a seven α-helix bundle (α1-α7). According to the 'umbrella model,' α4 and α5 helices form a hairpin structure thought to be inserted into the membrane upon binding. Here, we have synthesized and characterized the hairpin domain, α4-loop-α5, its α4 and α5 helices, as well as mutant α4 peptides based on mutations that increased or decreased toxin toxicity. Membrane permeation studies revealed that the α4-loop-α5 hairpin is extremely active compared with the isolated helices or their mixtures, indicating the complementary role of the two helices and the need for the loop for efficient insertion into membranes. Together with spectrofluorometric studies, we provide direct evidence for the role of α4-loop-α5 as the membrane-inserted pore-forming hairpin in which α4 and α5 line the lumen of the channel and α5 also participates in the oligomerization of the toxin. Strikingly, the addition of the active α4 mutant peptide completely inhibits α-loop-α5 pore formation, thus providing, to our knowledge, the first example that a mutated helix within a pore can function as an 'immunity protein' by directly interacting with the segments that form the pore. This presents a potential means of interfering with the assembly and function of other membrane proteins as well.

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.

HIV-1 entry into its host cell is modulated by its transmembrane envelope glycoprotein (gp41). The core of the activated conformation of gp41 consists of a trimer of heterodimers comprising a leucine/isoleucine zipper sequence (represented here by the synthetic peptide N36 or by the longer N51 peptide) and a C-terminal highly conserved region (represented here by C34). A correlation was found between the action of DP178, which is a potent inhibitor of HIV-1 entry into its host cell, and its ability to interact with the leucine/isoleucine zipper sequence. This correlation was further tested and confirmed by circular dichroism spectroscopy. We found that whereas DP178 perturbs the partial α-helix nature of peptides corresponding to the leucine/isoleucine zipper sequence (N36 or N51), it cannot perturb the trimer of heterodimers conformation, modeled by the complex of N36 or N51 with C34. Therefore, we suggest that the already formed trimer of heterodimers is not the target of inhibition by DP178. Our results are consistent with a model in which DP178 acquires its inhibitory activity by binding to an earlier intermediate of gp41, in which the N and C peptide regions are not yet associated, thus allowing DP178 to bind to the leucine/isoleucine zipper sequence and consequently to inhibit transition to the fusion-active conformation.

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.

Recently, we reported on a new group of diastereomers of short-model peptides (12 amino acids long) composed of leucine and lysine with varying ratios, possessing several properties that make them potentially better than native or de novo-designed all L-amino acid antimicrobial peptides. Preliminary studies have revealed that modulating the hydrophobicity and positive charges of these diastereomers is sufficient to confer antibacterial activity and cell selectivity. However, the relationship between their biological function, structure, and mode of action was not investigated. Here we synthesized and investigated three types of linear model diastereomers (12 amino acids long) with varying lysine:leucine (or tryptophan) ratios (i.e., K₃L₈W, K₅L₆W, and K₇L₄W), which confer different levels of lyric activities. For each K:L ratio, tryptophan was introduced in the middle or the N- or C-terminus of the peptides, as an intrinsic fluorescent probe. Only the hemolytic peptide K₃L₈W binds to both negatively charged and zwitterionic phospholipid membranes. K₅L₆W and K₇L₄W bind similarly, but only to negatively charged membranes, despite the fact that K₅L₆W is substantially more lytic to bacteria than K₇L₄W. Interestingly, although K₃L₈W contains 33% D-amino acids, ATR-FTIR spectroscopy revealed a structure of ~90% α-helix in both types of membranes. In addition, K₅L₆W contains ~40% 3₁₀-helix and K₇L₄W is predominantly a random coil in membranes. Polarized ATR-FTIR and tryptophan-quenching experiments, using brominated phospholipids, revealed a similar depth of penetration and an orientation that was parallel to the membrane surface for all the peptides, but with K₃L₈W affecting the lipid order more than the others. The results provide insight into the mode of action of this group of diastereomeric peptides, and the effect of hydrophobicity and positive charges on their membrane structure, function, and cell selectivity. Moreover, this research should assist in the development of suitable diastereomeric peptide antibiotics for therapeutic use that would overcome the problem the increasing resistance of bacteria to conventional antibiotics.

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 fusion domain of the HIV-1 envelope glycoprotein (gp120- gp41) is a conserved hydrophobic region located at the N-terminus of the transmembrane subunit (gp41). A prominent feature of this domain is a conserved five-residue 'FLGFL' sequence at positions 8-12. Mutation of the highly conserved Phe¹¹ to Val (F11V), presumed not to significantly affect the hydrophobicity and the structure of this region, has been shown to decrease the level of syncytium formation and virus infectivity. Here we show that the substitution of Gly for Phe¹¹ (F11G) reduces cell-cell fusion activity by 80-90%. To determine the effect of these mutations on the properties of the fusion peptide, a 33-residue peptide (WT) identical to the extended fusion domain and its F11V and F11G mutants were synthesized, fluorescently labeled, and studied with respect to their function, structure, and organization in phospholipid membranes. The WT peptide alone induced fusion of both zwitterionic (PC/Chol) and negatively charged (PS/PC/Chol and POPG) vesicles, in contrast to a 23-mer fusion peptide lacking the C-terminal domain which has been shown to be inactive with PC vesicles but able to induce fusion of POPG vesicles which had been preaggragated with Ca²⁺ or Mg²⁺. The F11V peptide preserved 50% activity, and the F11G peptide was virtually inactive. ATR-FTIR spectroscopy indicated similar secondary structure of the peptides in multibilayers that was independent of membrane composition. Furthermore, all the peptides increased the extent of lipid disorder to a similar extent, but the kinetics of amide II H to D exchange was in the following order: F11G > F11V > WT. Fluorescence studies in the presence of membranes, as well as SDS-PAGE, revealed that the WT and F11V peptides self-associate to similar levels while F11G exhibited a decreased level of self-association. The data suggest that the FLGFL motif contributes to the functional organization of the HIV-1 fusion peptide and that the C-terminal domain following the fusion peptide contributes to the membrane fusion process.

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 CytA toxin exerts its activity by the formation of pores within target cell membranes. However, the exact mechanism of pore formation and the structural elements that are involved in the toxic activity are yet to be determined. Recently, the structure of the highly similar CytB toxin was solved (Li et al., 1996), and a β-barrel was suggested as a possible structure of the pores. Due to the similarity between the toxins, the existence and positioning of α-helices and β-sheets in CytA were predicted from the alignment of the sequences. Here peptides corresponding to β5, β6, and β7 strands, to a conserved nonhelical region of the CytA toxin (p^149-170), to helices B and D, and to an analogue of helix A were synthesized, fluorescently labeled, and characterized. We found that, unlike helices A and C (Gazit and Shai, 1993), neither the β-strand peptides nor helix B could interact with lipid membranes, whereas p^149-170 and helix D bind the membrane weakly. Membrane permeation experiments suggested that CytA toxin exerts its activity by aggregation of several monomers. To learn about the structural elements that may mediate CytA oligomerization, the ability of the synthetic peptides to interact with membrane-bound CytA was studied. Helices A and C, but not the β-strands, helix D, or a control peptide, caused a large increase in the fluorescence of membrane-bound fluorescein-labeled CytA, whereas helix B had only a slight effect. Moreover, the addition of rearranged helix A, a peptide with the same composition as helix A, but with only two pairs of amino acids rearranged, did not affect the fluorescence. The addition of unlabeled CytA also caused an increase in the fluorescence intensity, further demonstrating the interaction between CytA monomers within the membrane. Taken together, our results provide further support for the suggestion that the CytA toxin self-assembles within membrane and that helices A and C are major structural elements involved in the membrane interaction and intermolecular assembly of the toxin.

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.

Increasing evidence suggests that membrane-embedded hydrophobic segments can interact within the phospholipid milieu of the membrane with varying degrees of specificity and thus contribute to the folding and oligomerization of proteins. We have used synthetic peptides corresponding to segments from the hydrophobic core of the Shaker potassium channel as a model system to study interactions between membrane-embedded segments. Three synthetic segments of the Shaker K⁺ channel, comprising the hydrophobic S2, S3, and S4 sequences, were used, and their secondary structure, their interactions with, and orientation within phospholipid membranes were examined. Secondary structure studies revealed that though S3 and S4 both adopt certain fractions of α-helical structures in membrane mimetic environments, the α-helical content of S3 is lower. Both S3 and S4 bind strongly to zwitterionic phospholipids, with partition coefficients in the order of 10⁴ and 10⁵ M^-1. ATR-FTIR studies showed that while the S4 peptide is oriented parallel to the membrane surface, S3 tends to a more transmembranal orientation. Enzymatic cleavage experiments demonstrated that the presence of S3 induces some change in the proteolytic accessibility of the S4 segment. Resonance energy transfer measurements, done in high lipid/peptide molar ratios, revealed that S3 and S4 cannot self-associate in zwitterionic phospholipid vesicles but can associate with each other and with the S2 segment of the channel. Furthermore, S3 does not interact with the homologous S4 region from the first repeat of the eel sodium channel, demonstrating specificity in the interactions. These results arc in line with data indicating that functionally important interactions indeed exist between the negatively charged S2 and S3 regions and the positively charged S4 region [Papazian, D. M., et al. (1995) Neuron 14, 1293-1301; Planells-Cases, R., et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 9422-9426]. From a broader point of view, these results provide further support to the notion that interactions (either specific or nonspecific) may exist between transmembrane segments of integral membrane proteins and therefore can contribute to their assembly and organization.

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.

IsK, also referred to as minK, is a membrane protein consisting of 130 amino acids and localized mainly in epithelial cells but also in human T lymphocytes. Depending on the cRNA concentration that was injected into Xenopus oocytes, IsK and its truncated forms can induce either a K⁺ current alone or both K⁺ and Cl^- currents [Attali et al. (1993) Nature 365, 850852]. To obtain information on the secondary structure and the topology of IsK in a membrane-bound state, the synthesis, fluorescent-labeling, and structural and functional characterization of five polypeptides of 2063 amino acids within the rat IsK protein were examined. The α-helical content of the segments, assessed in methanol using circular dichroism, suggests that both the N-terminal and transmembrane segments of IsK adopt α-helical structures. Binding experiments and the blue shift of 7-nitrobenz-2-oxa-l,3-diazol-4-yl (NBD)-labeled peptides suggest that while both the α-helical transmembrane segment and the N-terminal of IsK are located within the lipid bilayer, the linking segment between the two segments lies on the surface of the membrane. The fluorescence energy transfer, between donor and acceptor-labeled truncated IsK, suggests that it aggregates within phospholipid membranes. Although a protein whose sequence is similar to that of truncated IsK can induce K⁺ channel activity when expressed in Xenopus oocytes, the inability of a truncated IsK to form functional K⁺ channels in planar lipid membranes supports increasing evidence that the protein alone cannot form a K⁺ 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.

One of the most conserved sequences in various δ-endotoxins is the 30 amino acid long block I. Block I of cryIIIA δ-endotoxin contains a 23 amino acid amphiphilic α-helix termed α5. The potential involvement of this α5 helix in the toxic mechanism of δ-endotoxin was examined. For this purpose, a peptide corresponding to the α5 segment and its proline incorporated analogue (P-α5) were synthesized and characterized. The α-helical content of the peptides, assessed in methanol by circular dichroism (CD), was 58% and 24% for α5 and P-α5, respectively. To monitor the interaction of α5 peptides with phospholipid membranes, they were selectively labeled at their N-terminal amino acids with the fluorescent probes 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) or carboxyfluorescein. Fluorometric studies allowed the calculation of membrane surface partition constants, which were about 10⁴ M^-1 for both α5 and P-α5, and revealed that their N-terminals are located within the lipid bilayers. The shape of the binding isotherms indicated that α5 aggregated in both zwitterionic and acidic vesicles. Functional characterization of the α5 peptides was determined by assessing their ability to dissipate a diffusion potential from sonicated small unilamellar vesicles (SUV) composed of zwitterionic or acidic phospholipids and to lyse human erythrocytes. α5 was much more active than P-α5 in both assays. Moreover, membrane-bound α5 was more protected from enzymatic proteolysis than P-α5. The results reveal a good correlation between the structure and function of α5 and experimentally support the hypothesis that α5 is a structural component of the pores formed by δ-endotoxins via aggregation of amphiphilic α-helices.

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 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.

A 24-amino acid peptide corresponding to the S4 segment of the sodium channel was synthesized. In order to perform fluorescence energy transfer measurements and to monitor the interaction of the peptide with lipid vesicles, the peptide was selectively labeled with fluorescence probes at either its N-or C-terminal amino acids. The fluorescent emission spectra of 7-nitrobenz-2-oxa-1, 3-diazol-4-yl-(NBD-)labeled analogues displayed blue shifts upon binding to small unilamellar vesicles (SUV), reflecting the relocation of the fluorescent probe to an environment of increased apolarity. The results revealed that both the N-and C-terminus of the S4 segment are located within the lipid bilayer. Titration of solutions containing NBD-labeled peptides with SUV was used to generate binding isotherms, from which surface partition constants, in the range of 10⁴M^-1, were derived. The shape of the binding isotherms as well as fluorescence energy transfer measurements suggest that aggregation of peptide monomers within the membrane readily occurs in acidic but not in zwitterionic vesicles. Furthermore, the results provide good correlation between the incidence of aggregation in PC/PS vesicles and the ability of the peptides to permeate the vesicle's membrane. However, a transmembrane diffusion potential had no detectable effect on the location of the peptide within the lipid bilayer or on its aggregation state. Taken together, these results provide experimental support for a transmembranal localization for the sodium channel S4 segment as well as for its potential in forming part of the channel's lining, both properties in agreement with the \u201cpropagating helix\u201d model, suggested by Guy and Conti [Guy, H.R., & Conti, F. (1990) Trends Neurosci. 13, 201-206].

A unified approach to the synthesis of chelating phase transfer agents which uses attachment of various sidechains to a single chelating molecular precursor (such as 2acetyl4chloromethyl phenol, 4chloromethyl salicylaldehyde, 5chloromethyl8hydroxyquinoline), by alkylation at carbon (on aromatic substrates), at oxygen (on alkanols, oligoethylene glycols) or at nitrogen (dialkylamines), is presented. The reaction of a model precursor, 2acetyl4chloromethyl phenol, is studied in detail. The protonligand and protonmetal stability constants and Cu(II) phase transfer properties is determined.