Amyloid fibers formed from a peptide ubiquitous in human seminal fluid (SEVI) have been found to dramatically enhance the infectivity of the HIV virus (3-5 orders of magnitude by some measures). To complement these previous in vivo studies we have performed in vitro assays of PAP248-286, the most active precursor to SEVI, and other polycationic polymers to investigate the physical mechanisms by which the PAP248-286 promotes the interaction with lipid bilayers. At acidic, but not at neutral, pH freshly dissolved PAP248-286 catalyzes the formation of large lipid flocculates in a variety of membrane compositions which may be linked to the promotion of convective transport in the vaginal environment rather transport by a random Brownian motion. Furthermore, PAP248-286 is itself fusiogenic and weakens the integrity of the membrane in such a way that may promote fusion by the HIV gp41 protein. A partially α-helical conformation of PAP248-286, lying parallel to the membrane surface, is implicated in promoting bridging interactions between membranes by the screening of the electrostatic repulsion that occurs when two membranes are brought into close contact. This suggests non-specific binding of monomeric or small non-fibrillar oligomeric forms of SEVI to lipid membranes may be an additional mechanism by which SEVI enhances the infectivity of the HIV virus. In addition, we show that the curli protein, a strongly amyloidogenic protein used by bacteria to adhere to surfaces, seeds SEVI amyloid formation, indicating a possible role for bacterial infection in HIV transmission.

A peptide ubiquitous in human seminal fluid has been recently described that dramatically enhances the infectivity of the HIV virus (3-5 orders of magnitude by some measures). Previous studies have shown that this peptide, a fragment of human Prostatic Acid Phosphatase (PAP248-286) referred to as SEVI (Semen-derived Enhancer of Viral Infection), is amyloidogenic and the enhancement of viral infectivity is dependent on the aggregation state of the peptide. To complement these previous in vivo studies we have performed in vitro assays to investigate the physical mechanisms by which the PAP248-286 promotes the interaction with lipid bilayers. Our results indicate a strong interaction of freshly dissolved PAP248-286 with lipid bilayers but a weaker interaction with the amyloid form of PAP248-286, as measured by the tendency of freshly dissolved PAP248-286 to induce aggregation of lipid vesicles and membrane fusion. The amyloid form of PAP248-286 had little effect on either vesicle aggregation or fusion. To further investigate this effect we have solved the structure of PAP248-286 in SDS micelles. A largely α-helical conformation of PAP248-286, lying parallel to the membrane surface, is implicated in promoting bridging interactions between membranes by the screening of the electrostatic repulsion that occurs when two membranes are brought into close contact. This suggests non-specific binding of small oligomeric forms of SEVI in an α-helical conformation to lipid membranes may be an additional mechanism by which SEVI enhances the infectivity of the HIV virus.

The growing problem of bacterial resistance to conventional antibiotic compounds and the need for new antibiotics have stimulated interest in the development of antimicrobial peptides (AMPs) as human therapeutics. Development of topically applied agents, such as pexiganan (also known as MSI-78, an analog of the naturally occurring magainin2, extracted from the skin of the African frog Xenopus laevis) has been the focus of pharmaceutical development largely because of the relative safety of topical therapy and the uncertainty surrounding the long-term toxicology of any new class of drug administered systemically. The main hurdle that has hindered the development of antimicrobial peptides is that many of the naturally occurring peptides (such as magainin), although active in vitro, are effective in animal models of infection only at very high doses, often close to the toxic doses of the peptide, reflecting an unacceptable margin of safety. Though MSI-78 did not pass the FDA approval, it is still the best-studied AMP to date for therapeutic purposes. Biophysical studies have shown that this peptide is unstructured in solution, forms an antiparallel dimer of amphipathic helices upon binding to the membrane, and disrupts membrane via toroidal-type pore formation. This article covers functional, biophysical, biochemical and structural studies on pexiganan.

In previous in vivo studies, amyloid fibers formed from a peptide ubiquitous in human seminal fluid (semen-derived enhancer of viral infection (SEVI)) were found to dramatically enhance the infectivity of the HIV virus (3-5 orders of magnitude by some measures). To complement those studies, we performed in vitro assays of PAP(248-286), the most active precursor to SEVI, and other polycationic polymers to investigate the physical mechanisms by which the PAP(248-286) promotes the interaction with lipid bilayers. At acidic (but not at neutral) pH, freshly dissolved PAP(248-286) catalyzes the formation of large lipid flocculates in a variety of membrane compositions, which may be linked to the promotion of convective transport in the vaginal environment rather than transport by a random Brownian motion. Furthermore, PAP(248-286) is itself fusiogenic and weakens the integrity of the membrane in such a way that may promote fusion by the HIV gp41 protein. An alpha-helical conformation of PAP(248-286), lying parallel to the membrane surface, is implicated in promoting bridging interactions between membranes by the screening of the electrostatic repulsion that occurs when two membranes are brought into close contact. This suggests that nonspecific binding of monomeric or small oligomeric forms of SEVI in a helical conformation to lipid membranes may be an additional mechanism by which SEVI enhances the infectivity of the HIV virus.

Protegrins are potent members of the beta-hairpin-forming class of antimicrobial peptides. Key to their antimicrobial activity is their assembly into oligomeric structures upon binding to the bacterial membrane. To examine the relationship between the physicochemical properties of the peptide and its biological activity, we have synthesized variants of protegrin-1 in which key residues in the hydrophobic core, valine-14 and -16, are changed to leucine and to the extensively fluorinated analogue hexafluoroleucine. These substitutions have the effect of making the peptide progressively more hydrophobic while minimally perturbing the secondary structure. The leucine-containing peptide was significantly more active than wild-type protegrin against several common pathogenic bacterial strains, whereas the hexafluoroleucine-substituted peptide, in contrast, showed significantly diminished activity against several bacterial strains. Isothermal titration calorimetry measurements revealed significant changes in the interaction of the peptides binding to small unilamelar vesicles that mimic the lipid composition of the bacterial membrane. The binding isotherms for wild-type and leucine-substituted protegrins indicate that electrostatic interactions dominate the membrane-peptide interaction, whereas the isotherm for the hexafluoroleucine-substituted protegrin suggests a diminished electrostatic component to binding. Notably both of these substitutions appear to alter the stoichiometry of the lipid-peptide interaction, suggesting that these substitutions may stabilize oligomerized forms of protegrin that are postulated to be intermediates in the assembly of the beta-barrel membrane pore structure.

To test the prediction that extensively fluorinated (fluorous) proteins should be more stable and exhibit novel self-segregating behavior, the properties of the de novo designed model 4-alpha-helix bundle protein, alpha 4F 6, in which the hydrophobic core is packed entirely with the extensively fluorinated amino acid l-5,5,5,5',5',5'-hexafluoroleucine, have been compared with its nonfluorinated counterpart, alpha 4H, in which the core is packed with leucine. alpha 4F 6 exhibits much greater resistance to proteolysis by either chymotrypsin or trypsin than alpha 4H and resists unfolding by organic solvents far better than alpha 4H. Whereas increasing concentrations of ethanol or 2-propanol cause the helices of the alpha 4H tetramer first to dissociate into monomeric helices and then to completely unfold, these solvents have little effect on the structure of alpha 4F 6. In contrast, increasing the concentrations of the fluorinated alcohol trifluoroethanol promotes dissociation of both alpha 4H and alpha 4F 6 to monomeric helices, whereas the secondary structure of both peptides remains intact. (19)F NMR experiments indicate that the two peptides can form mixed alpha-helical alpha 4F 6:alpha 4H bundles and thus do not exhibit the self-segregating behavior predicted by the fluorous effect. We conclude that the properties of alpha 4F 6 are best explained by the more hydrophobic nature of the hexafluoroleucine side chain, rather than the low solubility of fluorocarbons in hydrocarbon solvents that forms the basis of the fluorous effect.

Leap frog: Antimicrobial peptides based on the structure of the magainin peptides from the African clawed frog have shown great promise as therapeutic agents. However their efficacy in vivo is limited by their susceptibility to proteolysis. We show that incorporating the fluorous amino acid hexafluoroleucine into a magainin analogue (see scheme) dramatically improves stability to proteolysis while retaining biological activity. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2268/2008/z700643_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.