Control of the transmembrane orientation and interhelical interactions within membranes by hydrophobic helix length

We examined the effect of the length of the hydrophobic core of Lys-flanked poly(Leu) peptides on their behavior when inserted into model membranes. Peptide structure and membrane location were assessed by the fluorescence emission lambdamax of a Trp residue in the center of the peptide sequence, the quenching of Trp fluorescence by nitroxide-labeled lipids (parallax analysis), and circular dichroism. Peptides in which the hydrophobic core varied in length from 11 to 23 residues were found to be largely alpha-helical when inserted into the bilayer. In dioleoylphosphatidylcholine (diC18:1PC) bilayers, a peptide with a 19-residue hydrophobic core exhibited highly blue-shifted fluorescence, an indication of Trp location in a nonpolar environment, and quenching localized the Trp to the bilayer center, an indication of transmembrane structure. A peptide with an 11-residue hydrophobic core exhibited emission that was red-shifted, suggesting a more polar Trp environment, and quenching showed the Trp was significantly displaced from the bilayer center, indicating that this peptide formed a nontransmembranous structure. A peptide with a 23-residue hydrophobic core gave somewhat red-shifted fluorescence, but quenching demonstrated the Trp was still close to the bilayer center, consistent with a transmembrane structure. Analogous behavior was observed when the behavior of individual peptides was examined in model membranes with various bilayer widths. Other experiments demonstrated that in diC18:1PC bilayers the dilution of the membrane concentration of the peptide with a 23-residue hydrophobic core resulted in a blue shift of fluorescence, suggesting the red-shifted fluorescence at higher peptide concentrations was due to helix oligomerization. The intermolecular self-quenching of rhodamine observed when the peptide was rhodamine-labeled, and the concentration dependence of self-quenching, supported this conclusion. These studies indicate that the mismatch between helix length and bilayer width can control membrane location, orientation, and helix-helix interactions, and thus may mismatch control both membrane protein folding and the interactions between membrane proteins.     

The amphiphilic alpha-helix concept. Consequences on the structure of staphylococcal delta-toxin in solution and bound to lipids

Staphylococcal delta-toxin, a synthetic analogue and a fragment were studied in order to determine their structure in solution and bound in lipids. In solution, a self-association process is observed. Analytical ultracentrifuge and quasi-elastic light-scattering experiments suggest an isodesmic aggregation in the high concentration domain above 2 microM up to very large asymmetrical species. Decreasing concentrations below 2 microM of delta-toxin and the analogue allows dissociation, probably into monomers. The self-associated species are essentially alpha-helical (70%) with buried and highly immobilized Trp either at position 15 for natural delta-toxin or 16 for the analogue. At the lowest concentration studied, the alpha-helix content severely decreases down to 35% while Trp fluorescence shows that these residues are exposed to buffer. The fragment 11-26 is always monomeric and structureless. From all the data, a structural model of aggregated species is proposed with stacked antiparallel amphipathic rods. When bound to lipids, whatever their initial structure in solution, 26-residue long peptides mainly adopt an alpha-helix conformation (80%) while fragment 11-26 exhibits about 50% alpha-helix. The lipid-peptide interactions were quantitatively analysed. For fragment 11-26, a single-step mechanism fits the spectroscopic changes and defines a single monomeric bound structure. On the other hand, for the 26-residue-long analogue, multiple-step processes must occur. The data were analysed with a partition of tetramers into lipids followed by a partial dissociation. Finally, the affinity of fragment 11-26 severely decreases from micelles to fluid and gel-state bilayers. The partition coefficient of the delta-toxin analogue is higher than those of other more apolar peptides, such as melittin and alamethicin, correlating with Eisenberg's hydrophobic moments. It is therefore proposed that delta-toxin probably lies parallel to the surface, only penetrating weakly in lipids, depending on their packing.     

Effects of L- or D-Pro incorporation into hydrophobic or hydrophilic helix face of amphipathic alpha-helical model peptide on structure and cell selectivity

A synthetic amphipathic alpha-helical model peptide, KLW, displays non-cell selective cytotoxicity. To investigate the effects of L- or D-Pro kink incorporation into hydrophobic or hydrophilic helix face of KLW on structure, cell selectivity, and membrane-binding affinity, we designed a series of four peptides, in which Leu(9) and Lys(11) in the hydrophobic and hydrophilic helix face of KLW, respectively, are substituted with L- or D-Pro. A L- or D-Pro substitution (KLW-L9P or KLW-L9p) of Leu(9) at the hydrophobic helix face of KLW induced a more significant reduction in hemolytic activity with improved antibacterial activity than that (KLW-K11P or KLW-K11p) of Lys(11) in the hydrophilic helix face. In addition, D-Pro-containing peptides (KLW-L9p and KLW-K11p) displayed less hemolytic activity than L-Pro-containing peptides (KLW-L9P and KLW-K11P). Tryptophan fluorescence studies revealed that bacterial cell selectivity of KLW-L9P, KLW-L9p, and KLW-K11p is closely related to selective interactions with negatively charged phospholipids. CD analysis revealed that L- or D-Pro incorporation into KLW reduces the alpha-helicity of the peptide and D-Pro incorporation induces more significant disruption in alpha-helical structure than L-Pro incorporation. Our results collectively suggest that D-Pro incorporation into the hydrophobic helix face of non-cell selective amphipathic alpha-helical peptides may be useful for the design of novel antimicrobial peptides possessing high bacterial cell selectivity without hemolytic activity.     

Dynamics and orientation of amphipathic peptides in solution and bound to membranes: a steady-state and time-resolved fluorescence study of staphylococcal δ-toxin and its synthetic analogues

The environment of both the hydrophilic and hydrophobic sides of alpha-helical delta-toxin are probed by tryptophanyl (Trp) fluorescence, when self-association occurs in solution and on binding to membranes. The fluorescence parameters of staphylococcal delta-toxin (Trp15 on the polar side of the amphipathic helix) and synthetic analogues with single Trp at position 5 or 16 (on the apolar side) were studied. The time-resolved fluorescence decays of the peptides in solution show that the local environment of their single Trp is always heterogeneous. Although the self-association degree increases with concentration, as shown by fluorescence anisotropy decays, the lifetimes (and their statistical weight) of Trp16 do not change, contrary to what is observed for Trp15. The first step of self-association is then driven by hydrophobic interactions between apolar sides of alpha-helices, whilst further oligomerization involves their polar side (Trp15) via electrostatic interactions. This is supported by dissociation induced by salt. For all self-associated peptides, the polarity of the Trp microenvironment was not significantly modified upon binding to phospholipid vesicles, as indicated by the small shifts of the fluorescence emission spectra and lifetime values. However, the relative populations of the lifetime classes vary with bound-peptide density similar to the rates of their global motions in bilayers or smaller particles. Quenching experiments by water or lipid-soluble compounds show changes of the orientation of membrane-inserted peptides, from probably dimers lying flat at the interface at low peptide density, to oligomers spanning the membrane and inducing membrane fragmentation at high peptide density.     

Dermaseptin S9, an alpha-helical antimicrobial peptide with a hydrophobic core and cationic termini

The dermaseptins S are closely related peptides with broad-spectrum antibacterial activity that are produced by the skin of the South American hylid frog, Phyllomedusa sauvagei. These peptides are polycationic (Lys-rich), alpha-helical, and amphipathic, with their polar/charged and apolar amino acids on opposing faces along the long axis of the helix cylinder. The amphipathic alpha-helical structure is believed to enable the peptides to interact with membrane bilayers, leading to permeation and disruption of the target cell. We have identified new members of the dermaseptin S family that do not resemble any of the naturally occurring antimicrobial peptides characterized to date. One of these peptides, designated dermaseptin S9, GLRSKIWLWVLLMIWQESNKFKKM, has a tripartite structure that includes a hydrophobic core sequence encompassing residues 6-15 (mean hydrophobicity, +4.40, determined by the Liu-Deber scale) flanked at both termini by cationic and polar residues. This structure is reminiscent of that of synthetic peptides originally designed as transmembrane mimetic models and that spontaneously become inserted into membranes [Liu, L., and Deber, C. M. (1998) Biopolymers 47, 41-62]. Dermaseptin S9 is a potent antibacterial, acting on gram-positive and gram-negative bacteria. The structure of dermaseptin S9 in aqueous solution and in TFE/water mixtures was analyzed by circular dichroism and two-dimensional NMR spectroscopy combined with molecular dynamics calculations. Dermaseptin S9 is aggregated in water, but a monomeric nonamphipathic alpha-helical conformation, mostly in residues 6-21, is stabilized by the addition of TFE. These results, combined with membrane permeabilization assays and surface plasmon resonance analysis of the peptide binding to zwitterionic and anionic phospholipid bilayers, demonstrate that spatial segregation of hydrophobic and hydrophilic/charged residues on opposing faces along the long axis of a helix is not essential for the antimicrobial activity of cationic alpha-helical peptides.     

The spectroscopic analysis for binding of amphipathic and antimicrobial model peptides containing pyrenylalanine and tryptophan to lipid bilayer

The binding of basic amphipathic fluorescent peptides to lipid bilayers was studied in relation to their antimicrobial activity. Four fluorescent peptides containing pyrenylalanine or tryptophan in an amphipathic basic peptide (4(4] consisting of four repeated units of tetrapeptide, -L-Leu-L-Ala-L-Arg-L-Leu-, were found to have antimicrobial activities against Gram-positive bacteria and to take conformations with fairly high alpha-helical content both in aqueous solutions and liposomes. The fluorescence spectroscopic data suggested that the pyrenylalanine-peptide existed as a monomer in methanol or liposomes but as an oligomer in aqueous solutions to form an excimer between pyrenylalanyl residues. Upon binding with liposomes, the fluorescence spectra of the tryptophan-containing peptide shifted to a shorter wavelength, indicating the change in the state of tryptophan from hydrophilic environment to hydrophobic one. The analytical data for the quenching of tryptophan fluorescence by I- anion suggest that the tryptophan residue in the peptide is not deeply buried in the hydrophobic core of the bilayers. Based on these findings, it is suggested that the peptides may interact with liposomes in such a manner that they lie parallel to the surface of the lipid bilayers with their hydrophobic regions shallowly in the amphipathic moiety of the bilayers.     

Perturbation of the hydrophobic core of lipid bilayers by the human antimicrobial peptide LL-37

LL-37 is a cationic, amphipathic alpha-helical antimicrobial peptide found in humans that kills cells by disrupting the cell membrane. To disrupt membranes, antimicrobial peptides such as LL-37 must alter the hydrophobic core of the bilayer. Differential scanning calorimetry and deuterium ((2)H) NMR experiments on acyl chain perdeuterated lipids demonstrate that LL-37 inserts into the hydrophobic region of the bilayer and alters the chain packing and cooperativity. The results show that hydrophobic interactions between LL-37 and the hydrophobic acyl chains are as important for the ability of this peptide to disrupt lipid bilayers as its electrostatic interactions with the polar headgroups. The (2)H NMR data are consistent with the previously determined surface orientation of LL-37 (Henzler Wildman, K. A., et al. (2003) Biochemistry 42, 6545) with an estimated 5-6 A depth of penetration of the hydrophobic face of the amphipathic helix into the hydrophobic interior of the bilayer. LL-37 also alters the material properties of lipid bilayers, including the area per lipid, hydrophobic thickness, and coefficient of thermal expansion in a manner that varies with lipid type and temperature. Comparison of the effect of LL-37 on 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC-d(31)) and 1,2-dimyristoyl-phosphatidylcholine (DMPC-d(54)) at different temperatures demonstrates the importance of bilayer order in determining the type and extent of disordering and disruption of the hydrophobic core by LL-37. One possible explanation, which accounts for both the (2)H NMR data presented here and the known surface orientation of LL-37 under identical conditions, is that bilayer order influences the depth of insertion of LL-37 into the hydrophobic/hydrophilic interface of the bilayer, altering the balance of electrostatic and hydrophobic interactions between the peptide and the lipids.     

Cell selectivity and mechanism of action of antimicrobial model peptides containing peptoid residues

To develop a useful method for designing cell-selective antimicrobial peptides and to investigate the effect of incorporating peptoid residues into an alpha-helical model peptide on structure, function, and mode of action, we synthesized a series of model peptides incorporating Nala (Ala-peptoid) into different positions of an amphipathic alpha-helical model peptide (KLW). Incorporation of one or two Nala residues into the hydrophobic helix face of KLW was more effective at disrupting the alpha-helical structure and bacterial cell selectivity than incorporation into the hydrophilic helix face or hydrophobic/hydrophilic interface. Tryptophan fluorescence studies of peptide interaction with model membranes indicated that the cell selectivity of KLW-L9-a and KLW-L9,13-a is closely correlated with their selective interactions with negatively charged phospholipids. KLW-L9,13-a, which has two Nala residues in its hydrophobic helix face, showed a random structure in membrane-mimicking conditions. KLW-L9,13-a exhibited the highest selectivity toward bacterial cells, showing no hemolytic activity and no or less cytotoxicity compared with other peptides against four mammalian cell lines. Unlike other model peptides, KLW-L9,13-a caused no or little membrane depolarization in Staphylococcus aureus or lipid flip-flop in negatively charged vesicles. In addition, KLW-L9,13-a caused very little fluorescent dye leakage from negatively charged vesicles. Furthermore, confocal laser-scanning microscopy and DNA-binding assays showed that KLW-L9,13-a probably exerts its antibacterial action by penetrating the bacterial membrane and binding to cytoplasmic compounds (e.g., DNA), resulting in cell death. Collectively, our results demonstrate that the incorporation of two Nala residues into the central position of the hydrophobic helix face of noncell-selective alpha-helical peptides is a promising strategy for the rational design of intracellular, cell-selective antimicrobial peptides.     

Morphological behavior of acidic and neutral liposomes induced by basic amphiphilic alpha-helical peptides with systematically varied hydrophobic-hydrophilic balance

Lipid-peptide interaction has been investigated using cationic amphiphilic alpha-helical peptides and systematically varying their hydrophobic-hydrophilic balance (HHB). The influence of the peptides on neutral and acidic liposomes was examined by 1) Trp fluorescence quenched by brominated phospholipid, 2) membrane-clearing ability, 3) size determination of liposomes by dynamic light scattering, 4) morphological observation by electron microscopy, and 5) ability to form planar lipid bilayers from channels. The peptides examined consist of hydrophobic Leu and hydrophilic Lys residues with ratios 13:5, 11:7, 9:9, 7:11, and 5:13 (abbreviated as Hels 13-5, 11-7, 9-9, 7-11, and 5-13, respectively; Kiyota, T., S. Lee, and G. Sugihara. 1996. Biochemistry. 35:13196-13204). The most hydrophobic peptide (Hel 13-5) induced a twisted ribbon-like fibril structure for egg PC liposomes. In a 3/1 (egg PC/egg PG) lipid mixture, Hel 13-5 addition caused fusion of the liposomes. Hel 13-5 formed ion channels in neutral lipid bilayer (egg PE/egg PC = 7/3) at low peptide concentrations, but not in an acidic bilayer (egg PE/brain PS = 7/3). The peptides with hydrophobicity less than Hel 13-5 (Hels 11-7 and Hel 9-9) were able to partially immerse their hydrophobic part of the amphiphilic helix in lipid bilayers and fragment liposome to small bicelles or micelles, and then the bicelles aggregated to form a larger assembly. Peptides Hel 11-7 and Hel 9-9 each formed strong ion channels. Peptides (Hel 7-11 and Hel 5-13) with a more hydrophilic HHB interacted with an acidic lipid bilayer by charge interaction, in which the former immerses the hydrophobic part in lipid bilayer, and the latter did not immerse, and formed large assemblies by aggregation of original liposomes. The present study clearly showed that hydrophobic-hydrophilic balance of a peptide is a crucial factor in understanding lipid-peptide interactions.     

Experimental evidence for predicted transmembrane peptide topography: incorporation of hydrophobic peptide alpha-helical rods with an N-terminal positive charge having a length comparable to the thickness of lipid bilayers into the membranes

Liposomes consisting of egg yolk phosphatidylcholine and hydrophobic peptides Nps- and Cl-.+H2-(Met-Met-Leu)n-OEt (n = 6-10) with various polypeptide chain lengths were prepared by the sonication method. The conformation of the peptides incorporated into the liposomes was examined by CD spectroscopy. All the peptides incorporated assumed alpha-helical conformation. Quantitative analyses of the peptides and lipids in the membranes showed that the concentration of the peptides with a positive charge at the N-terminus in the liposomes decreased markedly as the peptide chain length increased, reaching zero for the peptides over n = 8. The peptides without a positive charge were hardly incorporated into the liposomes. Infrared attenuated reflection spectroscopy of multilayered membranes containing the peptides suggests that the axis of the alpha-helical peptide rods is oriented in parallel with the molecular axis of lipids in the membranes. These results suggest that the hydrophobic peptides can be incorporated into the lipid bilayers of the liposomes in the alpha-helical conformation, the rods of which have a length comparable to the thickness of the lipid bilayers, and the N-terminal positive charge of the peptides is essential for the stable peptide incorporated into the membranes.     

Structure of the HERG K+ channel S5P extracellular linker: role of an amphipathic alpha-helix in C-type inactivation

The HERG K+ channel has very unusual kinetic behavior that includes slow activation but rapid inactivation. These features are critical for normal cardiac repolarization as well as in preventing lethal ventricular arrhythmias. Mutagenesis studies have shown that the extracellular peptide linker joining the fifth transmembrane domain to the pore helix is critical for rapid inactivation of the HERG K+ channel. This peptide linker is also considerably longer in HERG K+ channels, 40 amino acids, than in most other voltage-gated K+ channels. In this study we show that a synthetic 42-residue peptide corresponding to this linker region of the HERG K+ channel does not have defined structural elements in aqueous solution; however, it displays two well defined helical regions when in the presence of SDS micelles. The helices correspond to Trp585-Ile593 and Gly604-Tyr611 of the channel. The Trp585-Ile593 helix has distinct hydrophilic and hydrophobic surfaces. The Gly604-Tyr611 helix corresponds to an N-terminal extension of the pore helix. Electrophysiological studies of HERG currents following application of exogenous S5P peptides show that the amphipathic helix in the S5P linker interacts with the pore region of the channel in a voltage-dependent manner.     

Structure and mechanism of action of the antimicrobial peptide piscidin

Piscidin, an antibacterial peptide isolated from the mast cells of striped bass, has potent antimicrobial activity against a broad spectrum of pathogens in vitro. We investigated the mechanism of action of this 22-residue cationic peptide by carrying out structural studies and electrophysiological experiments in lipid bilayers. Circular dichroism experiments showed that piscidin was unstructured in water but had a high alpha-helix content in dodecylphosphocholine (DPC) micelles. 1H NMR data in water and TFE confirmed these results and demonstrated that the segment of residues 8-17 adopted an alpha-helical structure in a micellar environment. This molecule has a marked amphipathic character, due to well-defined hydrophobic and hydrophilic sectors. This structure is similar to those determined for other cationic peptides involved in permeabilization of the bacterial membrane. Multichannel experiments with piscidin incorporated into azolectin planar bilayers gave reproducible I-V curves at various peptide concentrations and unambiguously showed that this peptide permeabilized the membrane. This pore forming activity was confirmed by single-channel experiments, with well-defined ion channels obtained at different voltages. The characteristics of the ion channels (voltage dependence, only one or two states of conductance) clearly suggest that piscidin is more likely to permeabilize the membrane by toroidal pore formation rather than via the "barrel-stave" mechanism.     

Examination of the peptide sequence requirements for lipid-binding. Alternative pathways for promoting the interaction of amphipathic alpha-helical peptides with phosphatidylcholine

To examine the relationship between peptide sequence and the interaction of amphipathic alpha-helical peptides with phosphatidylcholines, various methods of mixing the peptide and lipid were explored. A series of amphipathic alpha-helical peptides containing from 10 to 18 residues were synthesized by solid-phase techniques. An 18-residue peptide and two relatively hydrophobic 10-residue peptides did not disrupt dimyristoylphosphatidylcholine liposomes when added to the lipid in buffer. However, when the peptides were premixed with lipid in a suitable organic solvent and then reconstituted with aqueous buffer, clear micelles were formed, indicating association of the amphipathic alpha-helical peptide with lipid. In general, the best solvent for this purpose was trifluoroethanol. The circular dichroic and fluorescence spectra of peptides which readily formed clear mixtures when mixed in buffer with dimyristoylphosphatidylcholine liposomes were similar when prepared either by the alternative pathway technique using trifluoroethanol or by a cholate removal technique. For the peptides which did not clear liposomes in buffer, first mixing with dimyristoylphosphatidylcholine in trifluoroethanol resulted in an increase in the alpha-helicity of the peptides as judged by circular dichroic spectra and a blue-shift in the fluorescence emission maxima of the single tryptophan residue in each peptide. These data are consistent with formation of an amphipathic alpha-helix in lipid by peptides which based on mixing experiments with dimyristoylphosphatidylcholine liposomes in buffer at the phase transition temperature of the lipid would be considered ineffective in lipid binding. Thus, simple mixing of peptides with liposomes may give misleading results concerning the intrinsic affinity of a particular peptide sequence for lipid. In addition, the data demonstrate that relatively hydrophobic amphipathic alpha-helical peptides which do not form small micelles with dimyristoylphosphatidylcholine spontaneously in aqueous solution may interact with lipid as typical amphipathic alpha-helices when mixed by an alternative pathway.     

Influence of tryptophan on lipid binding of linear amphipathic cationic antimicrobial peptides

We recently demonstrated that a linear 18-residue peptide, (KIGAKI)(3)-NH(2), designed to form amphipathic beta-sheet structure when bound to lipid bilayers, possessed potent antimicrobial activity and low hemolytic activity. The ability of (KIGAKI)(3)-NH(2) to induce leakage from lipid vesicles was compared to that of the amphipathic alpha-helical peptide, (KIAGKIA)(3)-NH(2), which had equivalent antimicrobial activity. Significantly, the lytic properties of (KIGAKI)(3)-NH(2) were enhanced for mixed acidic-neutral lipid vesicles containing phosphatidylethanolamine instead of phosphatidylcholine as the neutral component, while the potency of (KIAGKIA)(3)-NH(2) was significantly reduced [Blazyk, J., et al. (2001) J. Biol. Chem. 276, 27899-27906]. In this paper, we measured the lytic properties of these peptides, as well as several fluorescent analogues containing a single tryptophan residue, by monitoring permeability changes in large unilamellar vesicles with varying lipid compositions and in Escherichia coli cells. The binding of these peptides to lipid bilayers with defined compositions was compared using surface plasmon resonance, circular dichroism, and fluorescence spectroscopy. Surprisingly large differences were observed in membrane binding properties, particularly in the case of KIGAKIKWGAKIKIGAKI-NH(2). Since all of these peptides possess the same charge and very similar mean hydrophobicities, the binding data cannot be explained merely in terms of electrostatic and/or hydrophobic interactions. In light of their equivalent antimicrobial and hemolytic potencies, some of these peptides may employ mechanisms beyond simply increasing plasma membrane permeability to exert their lethal effects.     

A novel linear amphipathic beta-sheet cationic antimicrobial peptide with enhanced selectivity for bacterial lipids

All known naturally occurring linear cationic peptides adopt an amphipathic alpha-helical conformation upon binding to lipids as an initial step in the induction of cell leakage. We designed an 18-residue peptide, (KIGAKI)3-NH2, that has no amphipathic character as an alpha-helix but can form a highly amphipathic beta-sheet. When bound to lipids, (KIGAKI)3-NH2 did indeed form a beta-sheet structure as evidenced by Fourier transform infrared and circular dichroism spectroscopy. The antimicrobial activity of this peptide was compared with that of (KIAGKIA)3-NH2, and it was better than that of GMASKAGAIAGKIAKVALKAL-NH2 (PGLa) and (KLAGLAK)3-NH2, all of which form amphipathic alpha-helices when bound to membranes. (KIGAKI)3-NH2 was much less effective at inducing leakage in lipid vesicles composed of mixtures of the acidic lipid, phosphatidylglycerol, and the neutral lipid, phosphatidylcholine, as compared with the other peptides. However, when phosphatidylethanolamine replaced phosphatidylcholine, the lytic potency of PGLa and the alpha-helical model peptides was reduced, whereas that of (KIGAKI)3-NH2 was improved. Fluorescence experiments using analogs containing a single tryptophan residue showed significant differences between (KIGAKI)3-NH2 and the alpha-helical peptides in their interactions with lipid vesicles. Because the data suggest enhanced selectivity between bacterial and mammalian lipids, linear amphipathic beta-sheet peptides such as (KIGAKI)3-NH2 warrant further investigation as potential antimicrobial agents.     

Cyclization of a cytolytic amphipathic alpha-helical peptide and its diastereomer: effect on structure, interaction with model membranes, and biological function

The amphipathic alpha-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 alpha-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 alpha-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.     

Identification of peptide hormones of the amphipathic helix class using the helical hydrophobic moment algorithm

Eisenberg's helical hydrophobic moment (less than mu H greater than) algorithm was applied to the analysis of the primary structure of amphipathic alpha-helical peptide hormones and an optimal method for identifying other peptides of this class determined. We quantitate and compare known amphipathic helical peptide hormones with a second group of peptides with proven nonamphipathic properties and determine the best method of distinguishing between them. The respective means of the maximum 11 residue less than mu H greater than for the amphipathic helical and control peptides were 0.46 (+/-/-0.07) and 0.33 (0.07) (P + 0.004). To better reflect the amphipathic potential of the entire peptide, the percent of 11 residue segments in each peptide above a particular less than mu H greater than was plotted vs less than mu H greater than. The resulting curves are referred to as HM-C. The mean HM-C (of the two groups) was highly significantly different such that the HM-C method was superior to others in its ability to distinguish amphipathic from nonamphipathic peptides. Several potential new members of this structural class were identified using this approach. Molecular modeling of a portion of one of these, prolactin inhibitory factor, reveals a strongly amphipathic alpha helix at residues 4-21. This computer-based method may enable rapid identification of peptides of the amphipathic alpha-helix class.     

Structural studies and model membrane interactions of two peptides derived from bovine lactoferricin.

The powerful antimicrobial properties of bovine lactoferricin (LfcinB) make it attractive for the development of new antimicrobial agents. An 11-residue linear peptide portion of LfcinB has been reported to have similar antimicrobial activity to lactoferricin itself, but with lower hemolytic activity. The membrane-binding and membrane-perturbing properties of this peptide were studied together with an amidated synthetic version with an added disulfide bond, which was designed to confer increased stability and possibly activity. The antimicrobial and cytotoxic properties of the peptides were measured against Staphylococcus aureus and Escherichia coli and by hemolysis assays. The peptides were also tested in an anti-cancer assay against neuroblastoma cell lines. Vesicle disruption caused by these LfcinB derivatives was studied using the fluorescent reporter molecule calcein. The extent of burial of the two Trp residues in membrane mimetic environments were quantitated by fluorescence. Finally, the solution NMR structures of the peptides bound to SDS micelles were determined to provide insight into their membrane bound state. The cyclic peptide was found to have greater antimicrobial potency than its linear counterpart. Consistent with this property, the two Trp residues of the modified peptide were suggested to be embedded deeper into the membrane. Although both peptides adopt an amphipathic structure without any regular alpha-helical or beta-sheet conformation, the 3D-structures revealed a clearer partitioning of the cationic and hydrophobic faces for the cyclic peptide.     

Systematic peptide engineering and structural characterization to search for the shortest antimicrobial peptide analogue of gaegurin 5

As part of an effort to develop new, low molecular mass peptide antibiotics, we searched for the shortest bioactive analogue of gaegurin 5 (GGN5), a 24-residue antimicrobial peptide. Thirty-one kinds of GGN5 analogues were synthesized, and their biological activities were analyzed against diverse microorganisms and human erythrocytes. The structural properties of the peptides in various solutions were characterized by spectroscopic methods. The N-terminal 13 residues of GGN5 were identified as the minimal requirement for biological activity. The helical stability, the amphipathic property, and the hydrophobic N terminus were characterized as the important structural factors driving the activity. To develop shorter antibiotic peptides, amino acid substitutions in an inactive 11-residue analogue were examined. Single tryptophanyl substitutions at certain positions yielded some active 11-residue analogues. The most effective site for the substitution was the hydrophobic-hydrophilic interface in the amphipathic helical structure. At this position, tryptophan was the most useful amino acid conferring favorable activity to the peptide. The introduced tryptophan played an important anchoring role for the membrane interaction of the peptides. Finally, two 11-residue analogues of GGN5, which exhibited strong bactericidal activity with little hemolytic activity, were obtained as property-optimized candidates for new peptide antibiotic development. Altogether, the present approach not only characterized some important factors for the antimicrobial activity but also provided useful information about peptide engineering to search for potent lead molecules for new peptide antibiotic development.     

The helical structure of surfactant peptide KL4 when bound to POPC: POPG lipid vesicles

KL 4 is a 21-residue peptide employed as a functional mimic of lung surfactant protein B, which successfully lowers surface tension in the alveoli. A mechanistic understanding of how KL 4 affects lipid properties has proven elusive as the secondary structure of KL 4 in lipid preparations has not been determined at high resolution. The sequence of KL 4 is based on the C-terminus of SP-B, a naturally occurring helical protein that binds to lipid interfaces. The spacing of the lysine residues in KL 4 precludes the formation of a canonical amphipathic alpha-helix; qualitative measurements using Raman, CD, and FTIR spectroscopies have given conflicting results as to the secondary structure of the peptide as well as its orientation in the lipid environment. Here, we present a structural model of KL 4 bound to lipid bilayers based on solid state NMR data. Double-quantum correlation experiments employing (13)C-enriched peptides were used to quantitatively determine the backbone torsion angles in KL 4 at several positions. These measurements, coupled with CD experiments, verify the helical nature of KL 4 when bound to lipids, with (phi, psi) angles that differ substantially from common values for alpha-helices of (-60, -45). The average torsion angles found for KL 4 bound to POPC:POPG lipid vesicles are (-105, -30); this deviation from ideal alpha-helical structure allows KL 4 to form an amphipathic helix at the lipid interface.