Small changes in the primary structure of transportan 10 alter the thermodynamics and kinetics of its interaction with phospholipid vesicles

The kinetics and thermodynamics of binding of transportan 10 (tp10) and four of its variants to phospholipid vesicles, and the kinetics of peptide-induced dye efflux, were compared. Tp10 is a 21-residue, amphipathic, cationic, cell-penetrating peptide similar to helical antimicrobial peptides. The tp10 variants examined include amidated and free peptides, and replacements of tyrosine by tryptophan. Carboxy-terminal amidation or substitution of tryptophan for tyrosine enhance binding and activity. The Gibbs energies of peptide binding to membranes determined experimentally and calculated from the interfacial hydrophobicity scale are in good agreement. The Gibbs energy for insertion into the bilayer core was calculated using hydrophobicity scales of residue transfer from water to octanol and to the membrane/water interface. Peptide-induced efflux becomes faster as the Gibbs energies for binding and insertion of the tp10 variants decrease. If anionic lipids are included, binding and efflux rate increase, as expected because all tp10 variants are cationic and an electrostatic component is added. Whether the most important effect of peptide amidation is the change in charge or an enhancement of helical structure, however, still needs to be established. Nevertheless, it is clear that the changes in efflux rate reflect the differences in the thermodynamics of binding and insertion of the free and amidated peptide groups.     

A thermodynamic approach to the mechanism of cell-penetrating peptides in model membranes

We report a first test of the hypothesis that the mechanism of antimicrobial, cytolytic, and amphipathic cell-penetrating peptides in model membranes is determined by the thermodynamics of insertion of the peptide into the lipid bilayer from the surface-associated state. Three peptides were designed with minimal mutations relative to the sequence of TP10W, the Y3W variant of transportan 10, which is a helical, amphipathic cell-penetrating peptide previously studied. Binding to 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) membranes and release of dye from those vesicles were assessed by stopped-flow fluorescence, and the secondary structure of the peptides on the membrane was determined by circular dichroism. The Gibbs energy of binding determined experimentally was in excellent agreement with that calculated using the Wimley-White interfacial hydrophobicity scale, taking into account the helical content of the membrane-associated peptide. Release of dye from POPC vesicles remained graded, as predicted by the hypothesis. More significantly, as the Gibbs energy of insertion into the bilayer became more unfavorable, which was estimated using the Wimley-White octanol hydrophobicity scale, dye release became slower, in quantitative agreement with the prediction.     

The role of spontaneous lipid curvature in the interaction of interfacially active peptides with membranes

Research on antimicrobial peptides is in part driven by urgent medical needs such as the steady increase in pathogens being resistant to antibiotics. Despite the wealth of information compelling structure–function relationships are still scarce and thus the interfacial activity model has been proposed to bridge this gap. This model also applies to other interfacially active (membrane active) peptides such as cytolytic, cell penetrating or antitumor peptides. One parameter that is strongly linked to interfacial activity is the spontaneous lipid curvature, which is experimentally directly accessible. We discuss different parameters such as H-bonding, electrostatic repulsion, changes in monolayer surface area and lateral pressure that affect induction of membrane curvature, but also vice versa how membrane curvature triggers peptide response. In addition, the impact of membrane lipid composition on the formation of curved membrane structures and its relevance for diverse mode of action of interfacially active peptides and in turn biological activity are described. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Studies of the minimum hydrophobicity of alpha-helical peptides required to maintain a stable transmembrane association with phospholipid bilayer membranes

The effects of the hydrophobicity and the distribution of hydrophobic residues on the surfaces of some designed alpha-helical transmembrane peptides (acetyl-K2-L(m)-A(n)-K2-amide, where m + n = 24) on their solution behavior and interactions with phospholipids were examined. We find that although these peptides exhibit strong alpha-helix forming propensities in water, membrane-mimetic media, and lipid model membranes, the stability of the helices decreases as the Leu content decreases. Also, their binding to reversed phase high-performance liquid chromatography columns is largely determined by their hydrophobicity and generally decreases with decreases in the Leu/Ala ratio. However, the retention of these peptides by such columns is also affected by the distribution of hydrophobic residues on their helical surfaces, being further enhanced when peptide helical hydrophobic moments are increased by clustering hydrophobic residues on one side of the helix. This clustering of hydrophobic residues also increases peptide propensity for self-aggregation in aqueous media and enhances partitioning of the peptide into lipid bilayer membranes. We also find that the peptides LA3LA2 [acetyl-K2-(LAAALAA)3LAA-K2-amide] and particularly LA6 [acetyl-K2-(LAAAAAA)3LAA-K2-amide] associate less strongly with and perturb the thermotropic phase behavior of phosphatidylcholine bilayers much less than peptides with higher L/A ratios. These results are consistent with free energies calculated for the partitioning of these peptides between water and phospholipid bilayers, which suggest that LA3LA2 has an equal tendency to partition into water and into the hydrophobic core of phospholipid model membranes, whereas LA6 should strongly prefer the aqueous phase. We conclude that for alpha-helical peptides of this type, Leu/Ala ratios of greater than 7/17 are required for stable transmembrane associations with phospholipid bilayers.     

Structure-activity relationships of diastereomeric lysine ring size analogs of the antimicrobial peptide gramicidin S: mechanism of action and discrimination between bacterial and animal cell membranes

Structure-activity relationships were examined in seven gramicidin S analogs in which the ring-expanded analog GS14 [cyclo-(VKLKVdYPLKVKLdYP)] is modified by enantiomeric inversions of its lysine residues. The conformation, amphiphilicity, and self-association propensity of these peptides were investigated by circular dichroism spectroscopy and reversed phase high performance liquid chromatography. (31)P nuclear magnetic resonance spectroscopic and dye leakage experiments were performed to evaluate the capacity of these peptides to induce inverse nonlamellar phases in, and to permeabilize phospholipid bilayers; their growth inhibitory activity against the cell wall-less mollicute Acholeplasma laidlawii B was also examined. The amount and stability of beta-sheet structure, effective hydrophobicity, propensity for self-association in water, ability to disrupt the organization of phospholipid bilayers, and ability to inhibit A. laidlawii B growth are strongly correlated with the facial amphiphilicity of these GS14 analogs. Also, the magnitude of the parameters segregate these peptides into three groups, consisting of GS14, the four single inversion analogs, and the two multiple inversion analogs. The capacity of these peptides to differentiate between bacterial and animal cell membranes exhibits a biphasic relationship with peptide amphiphilicity, suggesting that there may only be a narrow range of peptide amphiphilicity within which it is possible to achieve the dual therapeutic requirements of high antibiotic effectiveness and low hemolytic activity. These results were rationalized by considering how the physiochemical properties of these GS14 analogs are likely to be reflected in their partitioning into lipid bilayer membranes.     

Counterion-mediated membrane penetration: Cationic cell-penetrating peptides overcome Born energy barrier by ion-pairing with phospholipids

Arginine-rich cell-penetrating peptides (CPPs) can enter cells non-endocytotically, despite that transport of charge across a membrane should be formally associated with an extremely high Born energy barrier. We studied partitioning of several derivatives of the CPP penetratin in a water-octanol two-phase system in presence of natural phospholipids to explore if solvation by ion-pairing to hydrophobic counter-ions may serve as a mechanism for cell internalisation. We demonstrate that anionic lipids can aid peptide partitioning into octanol. Particularly efficient partitioning into octanol is observed with an arginine-rich penetratin compared to a lysine-rich derivative. Substituting tryptophans for phenylalanines results in poor partitioning into octanol, due to decreased overall peptide hydrophobicity. Partitioning into octanol is dependent of phospholipid type and the peptides induced structural changes in the lipid assemblies found in octanol. Attachment of carboxyfluorescein as a model cargo was found to enhance peptide partitioning into octanol. We discuss our results with respect to theoretical electrostatic energies, empirical hydrophobicity scales and in terms of implications for CPP uptake mechanisms. An important improvement of the theoretical transfer energies is obtained when, instead of singular ions, the insertion of ion-paired dipolar species is considered.     

Identification of an apolipoprotein A-I structural element that mediates cellular cholesterol efflux and stabilizes ATP binding cassette transporter A1

Synthetic peptides were used in this study to identify a structural element of apolipoprotein (apo) A-I that stimulates cellular cholesterol efflux and stabilizes the ATP binding cassette transporter A1 (ABCA1). Peptides (22-mers) based on helices 1 (amino acids 44-65) and 10 (amino acids 220-241) of apoA-I had high lipid binding affinity but failed to mediate ABCA1-dependent cholesterol efflux, and they lacked the ability to stabilize ABCA1. The addition of helix 9 (amino acids 209-219) to either helix 1 (creates a 1/9 chimera) or 10 (9/10 peptide) endowed cholesterol efflux capability and ABCA1 stabilization activity similar to full-length apoA-I. Adding helix 9 to helix 1 or 10 had only a small effect on lipid binding affinity compared with the 22-mer peptides, indicating that helix length and/or determinants on the polar surface of the amphipathic alpha-helices is important for cholesterol efflux. Cholesterol efflux was specific for the structure created by the 1/9 and 9/10 helical combinations, as 33-mers composed of helices 1 and 3 (1/3), 2/9, and 4/9 failed to mediate cholesterol efflux in an ABCA1-dependent manner. Transposing helices 9 and 10 (10/9 peptide) did not change the class Y structure, hydrophobicity, or amphiphilicity of the helical combination, but the topography of negatively charged amino acids on the polar surface was altered, and the 10/9 peptide neither mediated ABCA1-dependent cholesterol efflux nor stabilized ABCA1 protein. These results suggest that a specific structural element possessing a linear array of acidic residues spanning two apoA-I amphipathic alpha-helices is required to mediate cholesterol efflux and stabilize ABCA1.     

Counterion-mediated membrane penetration: cationic cell-penetrating peptides overcome Born energy barrier by ion-pairing with phospholipids

Arginine-rich cell-penetrating peptides (CPPs) can enter cells non-endocytotically, despite that transport of charge across a membrane should be formally associated with an extremely high Born energy barrier. We studied partitioning of several derivatives of the CPP penetratin in a water-octanol two-phase system in presence of natural phospholipids to explore if solvation by ion-pairing to hydrophobic counter-ions may serve as a mechanism for cell internalisation. We demonstrate that anionic lipids can aid peptide partitioning into octanol. Particularly efficient partitioning into octanol is observed with an arginine-rich penetratin compared to a lysine-rich derivative. Substituting tryptophans for phenylalanines results in poor partitioning into octanol, due to decreased overall peptide hydrophobicity. Partitioning into octanol is dependent of phospholipid type and the peptides induced structural changes in the lipid assemblies found in octanol. Attachment of carboxyfluorescein as a model cargo was found to enhance peptide partitioning into octanol. We discuss our results with respect to theoretical electrostatic energies, empirical hydrophobicity scales and in terms of implications for CPP uptake mechanisms. An important improvement of the theoretical transfer energies is obtained when, instead of singular ions, the insertion of ion-paired dipolar species is considered.     

Is lipid bilayer binding a common property of inhibitor cysteine knot ion-channel blockers?

Recent studies of several ICK ion-channel blockers suggest that lipid bilayer interactions play a prominent role in their actions. Structural similarities led to the hypothesis that bilayer interactions are important for the entire ICK family. We have tested this hypothesis by performing direct measurements of the free energy of bilayer partitioning (DeltaG) of several peptide blockers using our novel quenching-enhanced fluorescence titration protocol. We show that various ICK peptides demonstrate markedly different modes of interaction with large unilamellar lipid vesicles. The mechanosensitive channel blocker, GsMTx4, and its active diastereomeric analog, D-GsMTx4, bind strongly to both anionic and zwitterionic membranes. One potassium channel gating modifier, rHpTx2gs, interacts negligibly with both types of vesicles at physiological pH, whereas another, SGTx1, interacts only with anionic lipids. The slope of DeltaG dependence on surface potential is very shallow for both GsMTx4 and D-GsMTx4, indicating complex interplay of their hydrophobic and electrostatic interactions with lipid. In contrast, a cell-volume regulator, GsMTx1, and SGTx1 exhibit a very steep DeltaG dependence on surface potential, resulting in a strong binding only for membranes rich in anionic lipids. The high variability of 5 kcal/mole in observed DeltaG shows that bilayer partitioning is not a universal property of the ICK peptides interacting with ion channels.     

Preferential binding of apolipoprotein E derived peptides with oxidized phospholipid

The physiological function of apolipoprotein E (apoE) includes transport and metabolism of lipids and its C-terminal domain harbors high affinity lipid-binding sites. Although the binding of apoE with non-oxidized phospholipid containing membranes has been characterized earlier, the interaction of apoE or its fragments with oxidized phospholipid containing membrane has never been studied. In this study we have compared the interaction of amphipathic helical peptide sequences derived from the C-terminal domain of apoE with membrane vesicles containing oxidized phospholipid, 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), with membrane vesicles without PazePC. The interaction was studied by monitoring (a) fluorescence emission maxima of the peptides, (b) acrylamide quenching of the peptides tryptophan residues and (c) by measuring the equilibrium binding constants by resonance energy transfer (RET) analysis. Our result shows that peptide sequence 202-223, 245-266 and 268-289 of apoE has higher affinity towards membrane containing PazePC, compared to membrane without PazePC. Presence of 1mM divalent cation or 50 mM NaCl in the buffer decreased the binding of peptides to PazePC containing membrane vesicles suggesting possible involvement of the electrostatic interaction in the binding. These observations suggest that the preferential binding of apoE to oxidized phospholipid containing membrane may play a role in the anti-oxidative properties of apoE.     

Properties and structures of the influenza and HIV fusion peptides on lipid membranes: implications for a role in fusion

The fusion peptides of HIV and influenza virus are crucial for viral entry into a host cell. We report the membrane-perturbing and structural properties of fusion peptides from the HA fusion protein of influenza virus and the gp41 fusion protein of HIV. Our goals were to determine: 1), how fusion peptides alter structure within the bilayers of fusogenic and nonfusogenic lipid vesicles and 2), how fusion peptide structure is related to the ability to promote fusion. Fluorescent probes revealed that neither peptide had a significant effect on bilayer packing at the water-membrane interface, but both increased acyl chain order in both fusogenic and nonfusogenic vesicles. Both also reduced free volume within the bilayer as indicated by partitioning of a lipophilic fluorophore into membranes. These membrane ordering effects were smaller for the gp41 peptide than for the HA peptide at low peptide/lipid ratio, suggesting that the two peptides assume different structures on membranes. The influenza peptide was predominantly helical, and the gp41 peptide was predominantly antiparallel beta-sheet when membrane bound, however, the depths of penetration of Trps of both peptides into neutral membranes were similar and independent of membrane composition. We previously demonstrated: 1), the abilities of both peptides to promote fusion but not initial intermediate formation during PEG-mediated fusion and 2), the ability of hexadecane to compete with this effect of the fusion peptides. Taken together, our current and past results suggest a hypothesis for a common mechanism by which these two viral fusion peptides promote fusion.     

Amphiphilicity is essential for mitochondrial presequence function.

We have shown earlier that a mitochondrial presequence peptide can form an amphiphilic helix. However, the importance of amphiphilicity for mitochondrial presequence function became doubtful when an artificial presequence, designed to be non-amphiphilic, proved to be active as a mitochondrial import signal. We now show experimentally that this 'non-amphiphilic' presequence peptide is, in fact, highly amphiphilic as measured by its ability to insert into phospholipid monolayers and to disrupt phospholipid vesicles. This result, and similar tests on three additional artificial presequences (two functionally active and one inactive), revealed that all active presequences were amphiphilic whereas the inactive presequence was non-amphiphilic. One of the active presequence peptides was non-helical in solution and in the presence of detergent micelles. We conclude that amphiphilicity is necessary for mitochondrial presequence function whereas a helical structure may not be essential.     

Contribution of the hydrophobicity gradient to the secondary structure and activity of fusogenic peptides

Fusogenic peptides belong to a class of helical amphipathic peptides characterized by a hydrophobicity gradient along the long helical axis. According to the prevailing theory regarding the mechanism of action of fusogenic peptides, this hydrophobicity gradient causes the tilted insertion of the peptides in membranes, thus destabilizing the lipid core and, thereby, enhancing membrane fusion. To assess the role of the hydrophobicity gradient upon the fusogenic activity, two of these fusogenic peptides and several variants were synthesized. The LCAT-(57-70) peptide, which is part of the sequence of the lipolytic enzyme lecithin cholesterol acyltransferase, forms stable beta-sheets in lipids, while the apolipoprotein A-II (53-70) peptide remains predominantly helical in membranes. The variant peptides were designed through amino acid permutations, to be either parallel, perpendicular, or to retain an oblique orientation relative to the lipid-water interface. Peptide-induced vesicle fusion was monitored by lipid-mixing experiments, using fluorescent probes, the extent of peptide-lipid association, the conformation of lipid-associated peptides and their orientation in lipids, were studied by Fourier Transformed Infrared Spectroscopy. A comparison of the properties of the wild-type and variant peptides shows that the hydrophobicity gradient, which determines the orientation of helical peptides in lipids and their fusogenic activity, further influences the secondary structure and lipid binding capacity of these peptides.     

Contribution of the hydrophobicity gradient to the secondary structure and activity of fusogenic peptides.

Fusogenic peptides belong to a class of helical amphipathic peptides characterized by a hydrophobicity gradient along the long helical axis. According to the prevailing theory regarding the mechanism of action of fusogenic peptides, this hydrophobicity gradient causes the tilted insertion of the peptides in membranes, thus destabilizing the lipid core and, thereby, enhancing membrane fusion. To assess the role of the hydrophobicity gradient upon the fusogenic activity, two of these fusogenic peptides and several variants were synthesized. The LCAT-(57-70) peptide, which is part of the sequence of the lipolytic enzyme lecithin cholesterol acyltransferase, forms stable beta-sheets in lipids, while the apolipoprotein A-II (53-70) peptide remains predominantly helical in membranes. The variant peptides were designed through amino acid permutations, to be either parallel, perpendicular, or to retain an oblique orientation relative to the lipid-water interface. Peptide-induced vesicle fusion was monitored by lipid-mixing experiments, using fluorescent probes, the extent of peptide-lipid association, the conformation of lipid-associated peptides and their orientation in lipids, were studied by Fourier Transformed Infrared Spectroscopy. A comparison of the properties of the wild-type and variant peptides shows that the hydrophobicity gradient, which determines the orientation of helical peptides in lipids and their fusogenic activity, further influences the secondary structure and lipid binding capacity of these peptides.     

Design and synthesis of cyclic disulfide-bonded antibacterial peptides on the basis of the alpha helical domain of Tenecin 1, an insect defensin

We synthesized cyclic disulfide-bonded (i, i+4) peptides with various net positive charges (+2-+5) from linear peptides derived from the alpha helical domain of Tenecin 1, an insect defensin, and investigated the effect of the intradisulfide bridge (i, i+4) on hydrophobicity, secondary structure, leakage activity and binding activity for large unilamellar vesicles, antimicrobial activity, and hemolytic activity. Intradisulfide bridge formation of the peptides resulted in the increase of amphiphilicity and hydrophobicity. Cyclic forms of the peptides did not deeply penetrate into PG/PC (1:1, mole ratio) large unilamellar vesicles and had a decreased lipid membrane perturbation activity for PG/PC LUVs. When the peptides interacted with PG/CL (2:1, mole ratio) LUVs, cyclic peptides with a high net positive charge (+4-+5) showed similar binding affinities and leakage activities for vesicles to those of linear forms, whereas cyclic peptides with a low net positive charge (+2-+3) exhibited lower leakage activity than their linear forms. CD spectra indicate that the intradisulfide bridge (i, i+4) provided little conformational constraint to linear peptides in buffer solution but resulted in the decrease of alpha helicity of the peptides in lipid membrane mimic conditions. The cyclic peptide with the highest net positive charge had a similar antibacterial activity to that of the linear peptide, whereas the cyclic peptides with a low net positive charge (+3-+4) exhibited lower antibacterial activity than their linear forms. The cyclic peptides of an appropriate net charge showed more potent activities against some bacteria than those of linear forms under high salt conditions.     

The helical hairpin structure of the influenza fusion peptide can be seen on a hydrophobic moment map

An assignment of the helical hairpin of the influenza fusion peptide has been made based on the hydrophobic moments, represented in a form of two-dimensional map. Such assignment holds for all serotypes, even for the cases of mutations altering the amino acid character. Similar results are obtained for the experimentally developed hydrophobicity scales, whose values reflect the transfer energies between aqueous and membrane environments. A distinct, however still structure-related hydrophobic map corresponds to a helical and contiguous HIV gp41 fp. The method may be used as a simple tool for sequence-based prediction of structures adopted by viral fusion peptides.     

Effect of multiple aliphatic amino acids substitutions on the structure, function, and mode of action of diastereomeric membrane active peptides

The initial stages leading to the binding and functioning of membrane-active polypeptides including hormones, signal sequences, and lytic peptides are mainly governed by electrostatic attraction and hydrophobic partitioning between water and lipid bilayers. Antimicrobial peptides serve as an important model for studying the details of these initial steps. However, a systematic analysis of the contribution of multiple hydrophobic amino acids to these steps have been hindered by the propensity of many peptides to aggregate and become inactivated in solution. To this end, we synthesized a series of model amphipathic all L-amino acid peptides and their diastereomers with the sequence KX(3)KWX(2)KX(2)K, where X = Gly, Ala, Val, Ile, or Leu. The effect of the aliphatic amino acids on the biological activity, binding, structure, membrane localization, and mode of action of these peptides was investigated. Most of the L-amino acid peptides oligomerized and adopted distinct structures in solution and in a membrane mimetic environment. Among this group only the Leu containing peptide was hemolytic and highly active on most bacteria tested. The Val- and Leu-containing peptides were hemolytic but inactive toward most bacteria tested. In contrast, the diastereomeric peptides were monomeric and unstructured in solution, but they adopted distinct structures upon membrane binding. While hemolytic activity was drastically reduced, the spectrum of antibacterial activity was preserved or increased. Importantly, we found a direct correlation with the diastereomers between hydrophobicity and propensity to form a helical/distorted-helix and activity (induced membrane leakage and antibacterial activity), despite the fact that they contained 30% D-amino acids. Furthermore, efficient increase in membrane permeability can proceed through different mechanisms. Specifically, the Leu-containing diastereomeric peptide micellized vesicles and possibly bacterial membranes while the Ile-containing diastereomeric peptide fused model membranes and irregularly disrupted bacterial membranes.     

Antibacterial mechanisms of GN-2 derived peptides and peptoids against Escherichia coli

Escherichia coli is the main etiological agent of urinary trait infections, able to form biofilms in indwelling devices, resulting in chronic infections which are refractory to antibiotics treatment. In this study, we investigated the antimicrobial and anti-biofilm properties exerted against E. coli ATCC 25922, by a set of peptoids and peptides modeled upon the peptide GN-2, previously reported as a valid antimicrobial agent. The putative antimicrobials were designed to evaluate the effect of cationicity, hydrophobicity and their partitioning on the overall properties against planktonic cells and biofilms as well as on LPS binding, permeabilization of Gram-negative bacteria membranes and hemolysis. The data demonstrated that peptides are stronger antimicrobials than the analogue peptoids which in return have superior anti-biofilm properties. In this study, we present evidence that peptides antimicrobial activity correlates with enhanced LPS binding and hydrophobicity but is not affected by partitioning. The data demonstrated that the enhanced anti-biofilm properties of the peptoids are associated with decreased hydrophobicity and increased penetration of the inner membrane, compared to that of their peptide counterpart, suggesting that the characteristic flexibility of peptoids or their lack of H-bonding donors in their backbone, would play a role in their ability to penetrate bacterial membranes.     

Transmembrane segment peptides with double D-amino acid replacements: helicity,hydrophobicity, and antimicrobial activity

The adoption of a helical conformation in a membrane environment effectively increases the "apparent hydrophobicity" of a peptide segment by satisfying the backbone H-bonding potential, thus stabilizing it in this environment. Here we sought to explore whether destabilizing the helical conformation would have a measurable effect on the apparent hydrophobicity of such segments in both aqueous and membrane-mimetic environments. In order to uncouple peptide hydrophobicity from helicity, we used the prototypic KKAAAAAAAAAAAAWAAAAAAKKKKNH(2) peptide as a template, and performed pairwise DD-scanning mutagenesis over the length of the sequence. Studies on this library of 13 peptides show that the DD replacements at positions near the center of peptide sequence had the most significant effects on the peptides' retention time in high performance liquid chromatography experiments. Decreased retention times correlate well with decreased helicity as measured by CD spectroscopy in the aqueous environment. Trp fluorescence measurements indicated that the peptides displayed a significant red shift in LPC (but not LPG) with peptides having DD replacements near the middle of the peptide sequence, emphasizing the importance of the anionic membrane in promoting peptide insertion. When tested against a laboratory strain of Escherichia coli, antimicrobial activity of the DD-peptides correlated with the apparent hydrophobicity but not with the overall micelle-based helical content of the peptides per se. Further analysis of the DD-positional dependence of the antimicrobial activity suggests that the presence of a local, uninterrupted stretch of helical structure (10-12 residues) may be a prerequisite for peptide biological activity. The overall findings support the notion that one should distinguish between the hydrophobicity of individual residues and the apparent hydrophobicity of the peptide as a whole, as the latter will ultimately have a greater influence on the properties of the full-length species.     

Exploring hydrophobicity limits of polyproline helix with oligomeric octahydroindole‐2‐carboxylic acid

The polyproline‐II helix is the most extended naturally occurring helical structure and is widely present in polar, exposed stretches and “unstructured” denatured regions of polypeptides. Can it be hydrophobic? In this study, we address this question using oligomeric peptides formed by a hydrophobic proline analogue, (2S,3aS,7aS)‐octahydroindole‐2‐carboxylic acid (Oic). Previously, we found the molecular principles underlying the structural stability of the polyproline‐II conformation in these oligomers, whereas the hydrophobicity of the peptide constructs remains to be examined. Therefore, we investigated the octan‐1‐ol/water partitioning and inclusion in detergent micelles of the oligo‐Oic peptides. The results showed that the hydrophobicity is remarkably enhanced in longer oligomeric sequences, and the oligo‐Oic peptides with 3 to 4 residues and higher are specific towards hydrophobic environments. This contrasts significantly to the parent oligoproline peptides, which were moderately hydrophilic. With these findings, we have demonstrated that the polyproline‐II structure is compatible with nonpolar media, whereas additional manipulations of the terminal functionalities feature solubility in extremely nonpolar solvents such as hexane.