Conformational study of an Aib-rich peptide in DMSO by NMR.

The strong propensity of 2-amino-2-methyl propanoic acid (Aib)-rich peptides to form stable helical structures is well documented. NMR analysis of the short peptide Z-(Aib)5-L-Leu-(Aib)2-OMe indicates the presence of a well-characterized 3(10)-helix even in dimethylsulfoxide (DMSO), a solvent known to disrupt helical structures. The structure remains stable at least up to 348 K. Stereospecific assignment of the diastereotopic methyls of Aib was achieved, with the assumption of a specific helical screw sense. The methyl more eclipsed with respect to the CO vector resonates at a higher field in the carbon dimension. Molecular dynamics simulations successfully predict the 3J(CHNH) coupling constant of Leu6 and most of the H-bonding pattern. Discrepancies were found for Aib3 and Aib7 amide protons which can be explained by a higher sensitivity of the simulations to the helix fraying at the end of the peptide and by the presence of extended conformations for Leu6 during most of the simulations.     

Conformationally constrained alpha-helical peptide models for protein ion channels.

Recently we described the design, synthesis, and characterization of some simple amphiphilic alpha-helical models for protein ion channels. These peptides, composed of only Leu and Ser residues, are hypothesized to form helical bundles capable of passing ions across phospholipid bilayers. In an effort to demonstrate that the peptides are, in fact, helical in their active ion-conducting state, the conformationally constrained amino acid, C alpha, C alpha-dimethylglycine (alpha-aminoisobutyric acid, Aib), was introduced simultaneously at three positions into one of the model peptides, H2N-(Leu-Ser-Leu-Leu-Leu-Ser-Leu)3-CONH2, giving H2N-(Leu-Ser-Leu-Aib-Leu-Ser-Leu)3-CONH2. Examination of a tetrameric model for the channel suggested that this substitution should have a minimal effect on conductance. CD spectroscopy of the Aib-modified and original peptide in phospholipid vesicles indicated that both were highly alpha-helical. Furthermore, the Aib-containing peptide formed proton channels nearly identical in conductance to the original peptide.     

Tryptophan-containing peptide helices: interactions involving the indole side chain.

Two designed peptide sequences containing Trp residues at positions i and i + 5 (Boc-Leu-Trp-Val-Ala-Aib-Leu-Trp-Val-OMe, 1) as well as i and i + 6 (Boc-Leu-Trp-Val-Aib-Ala-Aib-Leu-Trp-Val-OMe, 2) containing one and two centrally positioned Aib residues, respectively, for helix nucleation, have been shown to form stable helices in chloroform solutions. Structures derived from nuclear magnetic resonance (NMR) data reveal six and seven intramolecularly hydrogen-bonded NH groups in peptides 1 and 2, respectively. The helical conformation of octapeptide 1 has also been established in the solid state by X-ray diffraction. The crystal structure reveals an interesting packing motif in which helical columns are stabilized by side chain-backbone hydrogen bonding involving the indole Nepsilon1H of Trp(2) as donor, and an acceptor C=O group from Leu(6) of a neighboring molecule. Helical columns also associate laterally, and strong interactions are observed between the Trp(2) and Trp(7) residues on neighboring molecules. The edge-to-face aromatic interactions between the indoles suggest a potential C-H...pi interaction involving the Czeta3H of Trp(2). Concentration dependence of NMR chemical shifts provides evidence for peptide association in solution involving the Trp(2) Nepsilon1H protons, presumably in a manner similar to that observed in the crystal.     

Promotion of peptide antimicrobial activity by fatty acid conjugation

Three peptides, YGAA[KKAAKAA](2) (AKK), KLFKRHLKWKII (SC4), and YG[AKAKAAKA](2) (KAK), were conjugated with lauric acid and tested for the effect on their structure, antibacterial activity, and eukaryotic cell toxicity. The conjugated AKK and SC4 peptides showed increased antimicrobial activity relative to unconjugated peptides, but the conjugated KAK peptide did not. The circular dichroism spectrum of AKK showed a significantly larger increase in its alpha-helical content in the conjugated form than peptide KAK in a solution containing phosphatidylethanolamine/phosphotidylglycerol vesicles, which mimics bacterial membranes. The KAK and AKK peptides and their corresponding fatty acid conjugates showed little change in their structure in the presence of phosphatidylcholine vesicles, which mimic the cell membrane of eukaryotic cells. The hemolytic activity of the KAK and AKK peptides and conjugates was low. However, the SC4 fatty acid conjugate showed a large increase in hemolytic activity and a corresponding increase in helical content in the presence of phosphatidylcholine vesicles. These results support the model of antimicrobial peptide hemolytic and antimicrobial activity being linked to changes in secondary structure as the peptides interact with lipid membranes. Fatty acid conjugation may improve the usefulness of peptides as antimicrobial agents by enhancing their ability to form secondary structures upon interacting with the bacterial membranes.     

The antimicrobial peptide trichogin and its interaction with phospholipid membranes.

The interaction of the antimicrobial peptide trichogin GA IV with phospholipid bilayers has been studied. A series of analogs of trichogin was synthesized in which the nitroxide spin label, 4-amino-4-carboxy-2,2,6,6-tetramethylpiperidino-1-oxyl (TOAC), replaced one of the three alpha-aminoisobutyric acid (Aib) residues in the sequence. These modified peptides were used to assess the location of different residues of the peptide in a phospholipid bilayer composed of egg phosphatidylcholine containing 0.4 mol% of a fluorescently labelled phospholipid. We demonstrate that the substitution of Aib residues with TOAC does not alter the manner in which the peptide affects membrane curvature or induces vesicle leakage. The proximity of the nitroxide group on the peptide to the 4,4-difluoro-4-bora-3a,4a-diaza-S-indacene (BODIPY) fluorophore attached to the phospholipid was estimated from the extent of quenching of the fluorescence. By this criterion it was concluded that the peptide penetrates into the bilayer and that Aib4 is the most deeply inserted of the Aib residues. The results suggest that the helix axis of the peptide is oriented along the plane of the membrane. All of the peptides were shown to raise the bilayer to the hexagonal phase transition temperature of dipalmitoleoylphosphatidylethanolamine, indicating that they promote positive membrane curvature. This is a property observed with peptides that do not penetrate deeply into the bilayer or are oriented along the bilayer normal. We also demonstrate trichogin-promoted leakage of the aqueous contents of liposomes. These results indicate that the peptides cause bilayer destabilization. The extent of leakage induced by trichogin is very sensitive to the peptide to lipid ratio over a narrow range.     

Conformation of peptides constructed from achiral amino acid residues Aib and DeltaZPhe: computational study of the effect of L/D- Leu at terminal positions

Conformational studies of the peptides constructed from achiral amino acid residues Aib and Delta(Z)Phe (I) Ac-Aib-Delta(Z)Phe-NHMe (II), and Ac-(Aib-Delta(Z)Phe)(3)-NHMe; peptides III-VI having L-Leu or D-Leu at either the N- or the C-terminal position and of peptides VII-X having Leu residues in different enantiomeric combinations at both the N- and the C-terminal positions in peptide II have been studied to design the peptide with the required helical sense. Peptide II, as expected, adopts degenerate left- and right-handed helical structures. It has been shown that the peptides IV and VI having D-Leu at either the N or the C terminus can be realized in the right-handed helical structure with the phi,psi values of -20 degrees and -60 degrees for the Aib/Delta(Z)Phe residues. L-Leu and D- Leu at both the terminals in peptides VII and VIII, respectively, have hardly any effect as both the left- and the right-handed structures are found to be degenerate. Peptides III and IX can be realized in right- and left-handed helical structures, respectively, in solvents of low polarity whereas peptides V and X are predicted to be in the right-handed helical structures stabilized by carbonyl-carbonyl interactions without the formation of hydrogen bonds. The conformational states with the phi,psi values of 0 degrees and -85 degrees in peptide V are characterized by rise per residue of 2.03 A, rotation per residue of 117.5 degrees , and 3.06 residues per turn. In all peptides having Leu residue at the N terminus, the methyl moiety of the acetyl group is involved in the CH/pi interactions with the Cepsilon--Cdelta edge of the aromatic ring of Delta(Z)Phe (3) and the amino group NH of Delta(Z)Phe is involved in the NH/pi interactions with its own aromatic ring. The CH(3) groups of the Aib residues are also involved in CH/pi interactions with the i + 1th and i + 3th Delta(Z)Phe's aromatic side chains.     

Cellular uptake of Aib-containing amphipathic helix peptide

Cell-penetrating peptides (CPPs) are useful tools for the delivery of hydrophilic bioactive molecules, such as peptides, proteins, and oligonucleotides, across the cell membrane. To realize the delivery of therapeutic macromolecules by CPPs, the CPPs are required to show resistance to protease and no cytotoxicity. In order to produce potent non-toxic and protease-resistant CPPs with high cellular uptake, we designed an amphipathic helix peptide using α-aminoisobutyric acid (Aib, U) and named it MAP(Aib). In the MAP(Aib) molecule, five Aib residues are aligned on the hydrophobic face of the helix and five lysine (K) residues are aligned on the hydrophilic face. MAP(Aib) showed potent resistance to trypsin and pronase compared with MAP, an amphipathic helix peptide formed by usual amino acids. Fluorescein-labeled MAP(Aib) efficiently traversed the A549 cell membrane, diffusing into the cytoplasm and slightly into the nucleus without exerting any cytotoxicity. In contrast, MAP was poorly taken up by the cell. These results indicate that the incorporation of Aib residues into CPPs markedly improves cellular uptake and MAP(Aib) may be a useful tool for the delivery of hydrophilic macromolecules.     

Synthesis and helical structure of lactam bridged BH3 peptides derived from pro-apoptotic Bcl-2 family proteins

Protein-protein interactions within the Bcl-2 family are mediated by the helical BH3 domains of pro-apoptotic family members. To study the mechanism of this BH3 domain-protein interaction, a series of cyclic lactam bridged BH3 peptide analogues were synthesized by a novel combined Fmoc/tBu/Bzl protections strategy. These peptide analogues were studied by circular dichroism spectroscopy and found to adopt highly helical structure. These helical peptides stabilized by a lactam bridge serve as useful models to analyze the structure-function relationship of the pro-apoptotic BH3 domains. Furthermore, the synthetic method for lactam bridge incorporation reported here may find application in studies of other helical structures and development of helix mimics.     

Conformational preferences of a short Aib/Ala‐based water‐soluble peptide as a function of temperature

The amino acid Aib predisposes a peptide to be helical with context‐dependent preference for either 3₁₀‐ or α‐ or a mixed helical conformation. Short peptides also show an inherent tendency to be unfolded. To characterize helical and unfolded states adopted by water‐soluble Aib‐containing peptides, the conformational preference of Ac‐Ala‐Aib‐Ala‐Lys‐Ala‐Aib‐Lys‐Ala‐Lys‐Ala‐Aib‐Tyr‐NH₂ was determined by CD, NMR and MD simulations as a function of temperature. Temperature‐dependent CD data indicated the contribution of two major components, each an admixture of helical and extended/polyproline II structures. Both right‐ and left‐handed helical conformations were detected from deconvolution of CD data and ¹³C NMR experiments. The presence of a helical backbone, more pronounced at the N‐terminal, and a temperature‐induced shift in α‐helix/3₁₀‐helix equilibrium, more pronounced at the C‐terminal, emerged from NMR data. Starting from polyproline II, the N‐terminal of the peptide folded into a helical backbone in MD simulations within 5 ns at 60°C. Longer simulations showed a mixed‐helical backbone to be stable over the entire peptide at 5°C while at 60°C the mixed‐helix was either stable at the N‐terminus or occurred in short stretches through out the peptide, along with a significant population of polyproline II. Our results point towards conformational heterogeneity of water‐soluble Aib‐based peptide helices and the associated subtleties. The problem of analyzing CD and NMR data of both left‐ and right‐handed helices are discussed, especially the validity of the ellipticity ratio [θ]₂₂₂/[θ]₂₀₇, as a reporter of α‐/3₁₀‐ population ratio, in right‐ and left‐handed helical mixtures. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Crystal-state conformation of Calpha,alpha-dialkylated peptides containing chiral beta-homo-residues.

Secondary structure formation and stability are essential features in the knowledge of complex folding topology of biomolecules. To better understand the relationships between preferred conformations and functional properties of beta-homo-amino acids, the synthesis and conformational characterization by X-ray diffraction analysis of peptides containing conformationally constrained Calpha,alpha-dialkylated amino acid residues, such as alpha-aminoisobutyric acid or 1-aminocyclohexane-1-carboxylic acid and a single beta-homoamino acid, differently displaced along the peptide sequence have been carried out. The peptides investigated are: Boc-betaHLeu-(Ac6c)2-OMe, Boc-Ac6c-betaHLeu-(Ac6c)2-OMe and Boc-betaHVal-(Aib)5-OtBu, together with the C-protected beta-homo-residue HCl.H-betaHVal-OMe. The results indicate that the insertion of a betaH-residue at position 1 or 2 of peptides containing strong helix-inducing, bulky Calpha,alpha-disubstituted amino acid residues does not induce any specific conformational preferences. In the crystal state, most of the NH groups of beta-homo residues of tri- and tetrapeptides are not involved in intramolecular hydrogen bonds, thus failing to achieve helical structures similar to those of peptides exclusively constituted of Calpha,alpha-disubstituted amino acid residues. However, by repeating the structural motifs observed in the molecules investigated, a beta-pleated sheet secondary structure, and a new helical structure, named (14/15)-helix, were generated, corresponding to calculated minimum-energy conformations. Our findings, as well as literature data, strongly indicate that conformations of betaH-residues, with the micro torsion angle equal to -60 degrees, are very unlikely.     

Stereochemical control of peptide folding

Stereochemically constrained amino acid residues that strongly favour specific backbone conformations may be used to nucleate and stabilize specific secondary structures in designed peptides. An overview of the use of alphaalpha-dialkyl amino acids in stabilizing helical structures in synthetic peptides is presented, with an emphasis on work carried out in the authors laboratory. Alpha-aminoisobutyric acid (Aib) and related achiral homologs facilitate stable helix formation in oligopeptides as exemplified by a large number of crystal structure determinations in the solid state. The ability to design conformationally rigid helical modules has been exploited in attempts to design structurally well characterized helix-linker helix, using potential nonhelical linking segments. Beta-hairpin design has been approached by exploiting the tendency of 'prime turns' to nucleate hairpin formation. The use of nucleating (D)Pro-Gly segments has resulted in the generation of several well characterized beta-hairpin structures, including the crystallographic observation of beta-hairpin in a synthetic apolar octapeptide. Extensions of this approach to three stranded beta-sheets and larger structures containing multiple (D)Pro-Gly segments appear readily possible.     

Effect of alpha,alpha-dialkyl amino acids on the protease resistance of peptides

A tryptic [EC 3.4.21.4] digestion assay of 2-aminoisobutyric acid (Aib)-containing peptides was carried out to investigate the effect of alpha,alpha-dialkyl amino acid residues on the protease resistance. The introduction of Aib residues to the P1' positions exhibited a 19-fold higher protease resistance than the peptide with Aib residues introduced to the P2 position or the non-Aib peptide. The peptide having Aib residues introduced to the P1' and P2 positions resulted in complete resistance.     

Investigating hydrophobic ligand–receptor interactions in parathyroid hormone receptor using peptide probes

With an increasing number of new chemical entities entering clinical studies, and an increasing share of the market, peptides and peptidomimetics constitute one of the most promising classes of therapeutics. The success of synthetic peptides as therapeutics relies on the lead optimization step in which the lead candidates are modified to improve drug‐like properties of peptides related to potency, pharmacokinetics, solubility, and stability, among others. Peptidomimetics based on the N‐terminal stretch of the first 11 amino acids of the PTH have been investigated as potential lead compounds for the treatment of osteoporosis. On the basis of a peptide reported in the literature, referred to here as the Parent Peptide (H‐Aib‐Val‐Aib‐Glu‐Ile‐Gln‐Leu‐Nle‐His‐Gln‐Har‐NH₂), we conducted systematic SAR analyses to investigate the effects of altering peptide hydrophobicity on PTH receptor functional potency as measured by the cAMP (cyclic adenosine monophosphate) accumulation and β‐arrestin recruitment assays. Among hydrophobic residues, we found that the Val2 position shows the least flexibility in terms of the SAR studies, whereas the Leu7 position appeared to be most flexible. Through circular dichroism and nuclear magnetic resonance spectroscopy studies, we were able to establish that changes in hydrophobic residues significantly change the extent of peptide helicity and that the helical character correlates well with receptor agonist activity. Here, we report several novel PTH 1–11 peptidomimetics that show comparable or enhanced potency to stimulate G_s‐signaling over β‐arrestin recruitment as compared with such properties of PTH 1–34 and the Parent Peptide. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Comparison of helix-stabilizing effects of alpha,alpha-dialkyl glycines with linear and cycloalkyl side chains

The ability of alpha, alpha-di-n-alkyl glycines with linear and cyclic alkyl side chains to stabilize helical conformations has been compared using a model heptapeptide sequence. The conformations of five synthetic heptapeptides (Boc-Val-Ala-Leu-Xxx-Val-Ala-Leu-OMe, Xxx = Ac8c, Ac7c, Aib, Dpg, and Deg, where Ac8c = 1-aminocyclooctane-1-carboxylic acid, Ac7c = 1-aminocycloheptane-1-carboxylic acid, Aib = alpha-aminoisobutyric acid, Dpg = alpha,alpha-di-n-propyl glycine, Deg = alpha,alpha-di-n-ethyl glycine) have been investigated. In crystals, helical conformations have been demonstrated by x-ray crystallography for the peptides, R-Val-Ala-Leu-Dpg-Val-Ala-Leu-OMe, (R = Boc and acetyl). Solution conformations of the five peptides have been studied by 1H-nmr. In the apolar solvent CDCl3, all five peptides favor helical conformations in which the NH groups of residues 3-7 are shielded from the solvent. Successive NiH<-->Ni + 1H nuclear Overhauser effects over the length of the sequence support a major population of continuous helical conformations. Solvent titration experiments in mixtures of CDCl3/DMSO provide evidence for solvent-dependent conformational transitions that are more pronounced for the Deg and Dpg peptides. Solvent-dependent chemical shift variations and temperature coefficients in DMSO suggest that the conformational distributions in the Deg/Dpg peptides are distinctly different from the Aib/Acnc peptides in a strongly solvating medium. Nuclear Overhauser effects provide additional evidence for the population of extended backbone conformations in the Dpg peptide, while a significant residual population of helical conformations is still detectable in the isomeric Ac7c peptide in DMSO.     

Effects of hydrogen-bond deletion on peptide helices: structural characterization of depsipeptides containing lactic acid

The insertion of alpha-hydroxy acids into peptide chains provides a convenient means for investigating the effects of hydrogen bond deletion on polypeptide secondary structures. The crystal structures of three oligopeptides containing L-lactic acid (Lac) residue have been determined. Peptide 1, Boc-Val-Ala-Leu-Aib-Val-Lac-Leu-Aib-Val-Ala-Leu-OMe (Boc: tert-butyloxycarbonyl; Aib: alpha- aminoisobutyric acid; OMe: methyl ester), and peptide 2, Boc-Val-Ala-Leu-Aib-Val-Lac-Leu-Aib-Val-Leu-OMe, adopt completely helical conformations in the crystalline state with the Lac(6) residue comfortably accommodated in the center of a helix. The distance between the O atoms of Leu(3) CO group and the Lac(6) O (ester) in both the structures is 3.1-3.3 A. The NMR and CD studies of peptide 1 and its all-amide analogue 4, Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe, provide firm evidence for a continuous helical conformation in solution in both the cases. In a 14-residue peptide 3, Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Val-Ala-Leu-Aib-Val-Lac-Leu-OMe, residues Val(1)-Leu(10) adopt a helical conformation. Aib(11) is the site of chiral reversal resulting in helix termination by formation of a Schellman motif. Residues 12-14 adopt nonhelical conformations. The loss of the hydrogen bond near the C-terminus appears to facilitate the chiral reversal at Aib(11). Published 2001 John Wiley & Sons, Inc. Biopolymers 59: 276-289, 2001     

Structure-function relationship of model Aib-containing peptides as ion transfer intermembrane templates

Peptaibols comprise a family of peptide antibiotics with high contents of 2-aminoisobutyric acid (Aib) residues and C-terminal amino alcohols. These peptides form alpha-helical structures leading to voltage-gated ion channels in lipid membranes. In the present study, amphiphilic helical Aib-containing peptides of various chain-lengths, Ac-(Aib-Lys-Aib-Ala)n-NH2 (n = 1-5), were designed to investigate the mechanisms of the aggregation and transmembrane orientation of helical motifs in lipid bilayer membranes. Peptide synthesis was performed by the conventional stepwise Fmoc solid-phase method. The crude peptides were obtained in high yields (66-85%) with high purities (69-95%). Conformational analysis of the synthetic peptides was performed by CD spectroscopy. It was found that these peptides take on highly helical structures, and the helicity of the peptides increases with an increase in chain-length. The longest peptide, Ac-(Aib-Lys-Aib-Ala)5-NH2, self-aggregates and adopts a barrel-stave conformation in liposomes. Ac-(Aib-Lys-Aib-Ala)5-NH2 exhibited potent antimicrobial activity against Gram-positive bacteria. Patch-clamp measurements revealed that this peptide can form well-defined ion channels with a long lifetime at relatively low transbilayer potentials and peptide concentrations. For this peptide, the single-channel conductance of the most frequent event is 227 pS, which could be related to a single-state tetrameric pore.     

A highly active chemotactic peptide analog incorporating the unusual residue 1-aminocyclohexanecarboxylic acid at position 2

Analogs of chemotactic peptides (Formyl-Met-X-Phe-OMe) containing the stereochemically constrained residues alpha-aminoisobutyric acid (Aib), 1-aminocyclopentanecarboxylic acid (Acc5) and 1-aminocyclohexanecarboxylic acid (Acc6) at position 2 are compared with the parent sequence (X = Leu) for their ability to induce lysozyme release in rabbit neutrophils. The Acc6 analog is about 78 times more active than the parent peptide, For-Met-Leu-Phe-OH, whereas Aib and Acc5 analogs are approximately 3 and 2 times, respectively, less active than the parent peptide. NMR and model building studies clearly favour a Met-Acc6 beta-turn solution conformation in the Acc6 analog, suggesting that the neutrophil receptor is capable of recognizing a folded peptide structure. The significant differences in the activities of the Acc5 and Acc6 analogs suggest an important role for the residue 2 sidechain in receptor interactions.     

Lipid-induced conformational changes in glucagon,secretin, and vasoactive intestinal peptide

The CD of glucagon, secretin, and vasoactive intestinal peptide has been studied as a function of temperature in water and in aqueous solutions of dodecyl sulfate, phosphatidyl glycerol, and L -α-phosphatidic acid (dipalmitoyl). The anionic detergent and lipids induce helix formation in all three peptides, with the amount of induced helical content increasing in the order glucagon < secretin < vasoactive intestinal peptide. These observations are subject to quantitative rationalization using a matrix formulation for the configuration partition function. In this formulation the major conformational consequences of the interaction with anionic lipids or detergents is an increase in the probability for helix formation by arginyl, histidyl, and lysyl residues. The region in which helix formation is maximal is found to be at amino acid residues 13–20 in all three peptides. Other studies have implicated this portion of the polypeptide chain in receptor binding. Thus, the helical segment induced by interaction with anionic lipids may play an important physiological role.