Noncovalent chiral domino effect on one-handed helix of nonapeptide containing a midpoint L-residue

We here clarify whether noncovalent chiral domino effect characterized by the terminal interaction of a helical peptide with a chiral small molecule can alter the helical stability of N-deprotected peptides containing an L-residue covalently incorporated into the inner position. Two nonapeptides consisting of the midpoint L-leucine (1) or L-phenylalanine (2) and the achiral helix-forming residues were employed. NMR and IR spectroscopy and energy calculation indicated that both peptides adopt a 3(10)-helical conformation in chloroform. They strongly preferred a right-handed screw sense because of the presence of the midpoint L-residue. These original right-handed screw senses were retained on addition of chiral Boc-amino acid, but their helical stabilities clearly depended on its added chirality. Here, Boc-L-amino acid stabilizes the original right-handed helix, whereas the corresponding Boc-D-amino acid tends to less stabilize or destabilize it. This tendency was not observed for the corresponding N-Boc-protected peptides 1 and 2, strongly suggesting that the N-terminal amino group is required for controlling the stabilization of the original right-handed helix. Therefore, noncovalent chiral domino effect in peptides 1 and 2 can contribute even to the helical stability of a chiral peptide prevailing one-handed helix strongly through the midpoint L-residue. In addition, the N-terminal moiety of a 3(10)-helical peptide was found to generate chiral discrimination in complexation process with racemic additives.     

Chiral interaction in peptide molecules: effects of chiral peptide species on helix-sense induction in an N-terminal-free achiral peptide

Noncovalent chiral domino effect (NCDE) has been proposed as terminal-specific interaction upon a 3(10)-helical peptide chain, of which the helix sense is manipulated by an external chiral stimulus (mainly amino acid derivatives) operating on the N-terminus (Inai, Y., et al. J Am Chem Soc 2000, 122, 11731-11732; ibid., 2002, 124, 2466-2473; ibid., 2003, 125, 8151-8162). We have investigated here a helix-sense induction in an optically inactive N-terminal-free nonapeptide (1) through the screening of several peptide species that differ in chiral sequence, chain length, and C-terminal group. Helix-sense induction in peptide 1 depends largely on both the C-terminal chirality and carboxyl group in the external peptide, in which N-carbonyl-blocked amino acids, "monopeptide acids," should be the minimum requirement for effective induction. N-Protected mono- to tetrapeptides of L-Leu residue commonly induce a right-handed helix. NMR study and theoretical computation reveal that the N-terminal segment of peptide 1 binds the N-protected dipeptide molecule through multipoint coordination to induce a right-handed helix preferentially. The present findings not only will improve our understanding of the chiral roles in peptide or protein helical termini, but also might demonstrate potential applications to chirality-responsive materials based on peptide helical fragments.     

Peptides from chiral C alpha,alpha-disubstituted glycines. Crystallographic characterization of conformation of C alpha-methyl, C alpha-isopropylglycine [(alpha Me)Val] in simple derivatives and model peptides

The molecular and crystal structures of one derivative and three model peptides (to the pentapeptide level) of the chiral C alpha,alpha-disubstituted glycine C alpha-methyl, C alpha-isopropylglycine [(alpha Me)Val] have been determined by X-ray diffraction. The derivative is mClAc-L-(alpha Me)Val-OH, and the peptides are Z-L-(alpha Me)Val-(L-Ala)2-OMe monohydrate, Z-Aib-L-(alpha Me)Val-(Aib)2-OtBu, and Ac-(Aib)2-L-(alpha Me)Val-(Aib)2OtBu acetonitrile solvate. The tripeptide adopts a type-I beta-turn conformation stabilized by a 1----4N--H...O = C intramolecular H-bond. The tetra- and pentapeptides are folded in regular right-handed 3(10)-helices. All four L-(alpha Me)Val residues prefer phi, psi angles in the right-handed helical region of the conformational map. The results indicate that: (i) the (alpha Me)Val residue is a strong type-I/III beta-turn and helix former, and (ii) the relationship between (alpha Me)Val chirality and helix screw sense is the same as that of C alpha-monosubstituted protein amino-acids. The implications for the use of the (alpha Me)Val residue in designing conformationally constrained analogues of bioactive peptides are briefly discussed.     

Chiral interaction in Gly-capped N-terminal motif of 3(10)-helix and domino-type induction in helix sense

Chiral interaction of helical peptide with chiral molecule, and concomitant induction in its helix sense have been demonstrated in optically inactive nonapeptide (1) possessing Gly at its N-terminus: H-Gly-(Delta(Z)Phe-Aib)(4)-OCH(3) (1: Delta(Z)Phe = Z-dehydrophenylalanine; Aib = alpha-aminoisobutyric acid). Spectroscopic measurements [mainly nuclear magnetic resonance (NMR) and circular diochroism (CD)] as well as theoretical simulation have been carried out for that purpose. Peptide 1 in the 3(10)-helix tends to adopt preferentially a right-handed screw sense by chiral Boc-L-amino acid (Boc: t-butoxycarbonyl). Induction in the helix sense through the noncovalent chiral domino effect should be derived primarily from the complex supported by the three-point coordination on the N-terminal sequence. Thus the 3(10)-helical terminus consisting of only alpha-amino acid residues enables chiral recognition of the Boc-amino acid molecule, leading to modulation of the original chain asymmetry. Dynamics in the helix-sense induction also have been discussed on the basis of a low-temperature NMR study. Furthermore, the inversion of induced helix sense has been achieved through solvent effects.     

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.     

Preferred solution conformation of peptides rich in the lipophilic, chiral, C(alpha)-methylated alpha-amino acid (alpha Me)Aoc.

The lipophilic, chiral, C(alpha)-methylated alpha-amino acid L-(alphaMe)Aoc (2-methyl-2-amino-octanoic acid) was prepared using a chemo-enzymatic approach. Two series of terminally protected model peptides, from dimer through to hexamer, containing L-(alphaMe)Aoc in combination with either Gly or Aib, were synthesized by solution methods and were fully characterized. A solution conformational analysis, based on FT-IR absorption, 1H-NMR and circular dichroism (CD) techniques, was performed with the aim at determining the preferred conformation of this novel amino acid and the relationship between chirality at its alpha-carbon atom and screw sense of the helix that is formed. The results obtained strongly support the view that L-(alphaMe)Aoc favours the formation of the right-handed 3(10)-helical conformation.     

Aib residues in peptaibiotics and synthetic sequences: analysis of nonhelical conformations

The alpha-aminoisobutyric (Aib) residue has generally been considered to be a strongly helicogenic residue as evidenced by its ability to promote helical folding in synthetic and natural sequences. Crystal structures of several peptide natural products, peptaibols, have revealed predominantly helical conformations, despite the presence of multiple helix-breaking Pro or Hyp residues. Survey of synthetic Aib-containing peptides shows a preponderance of 3(10)-, alpha-, and mixed 3(10)/alpha-helical structures. This review highlights the examples of Aib residues observed in nonhelical conformations, which fall 'primarily' into the polyproline II (P(II)) and fully extended regions of conformational space. The achiral Aib residue can adopt both left (alpha(L))- and right (alpha(R))-handed helical conformations. In sequences containing chiral amino acids, helix termination can occur by means of chiral reversal at an Aib residue, resulting in formation of a Schellman motif. Examples of Aib residues in unusual conformations are illustrated by surveying a database of Aib-containing crystal structures.     

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     

Photo-control of peptide helix content by an azobenzene cross-linker: steric interactions with underlying residues are not critical

Photo-control of protein conformation could prove useful for probing function in diverse biological systems. Recently, we reported photo-switching of helix content in a short peptide containing an azobenzene cross-linker between cysteine residues at positions i and i + 7 in the sequence. In the original sequence, underlying residues at positions i + 3 and i + 4 were made bulky as preliminary modelling suggested that this would enhance photo-control of helix content. To test this hypothesis, peptides with Val, Aib; Ile, Aib; and Ala, Ala at positions i + 3 and i + 4 were synthesized, cross-linked and characterized. Before cross-linking, the peptides show distinct conformational behaviours: two with differing helix/coil mixtures whereas the other has a circular dichroism (CD) spectrum characteristic of beta-sheet and a tendency to aggregate. However, upon cross-linking the peptides have very similar CD spectra: predominantly random coil in the dark but predominantly helical upon irradiation. These results refute the original hypothesis. Steric interactions between the linker and underlying residues do not appear to be critical for photo-switching behaviour. When the cross-linking bridge is lengthened by replacing the i, i + 7 cysteine residues with homocysteine, a lower degree of photo-control of helicity is observed. Furthermore, a non-cross-linking version of the azobenzene reagent is shown not to produce any photo-control of helicity. We conclude that the intramolecular cross-link is essential for photo-switching and that it should be applicable to a wide range of peptides and proteins.     

Prevention of peptide fibril formation in an aqueous environment by mutation of a single residue to Aib

The behavior of a number of 16 residue polypeptides with a sequence Acetyl-EACARXZAACEAAARQ-amide, where X = V or A and Z = A or Aib, is studied under aqueous conditions. It is shown that the substitution of a single alanine residue by alpha-aminoisobutyric acid (Aib) completely alters both the conformation and the aggregation properties of the peptides. The Ala-Ala (X,Z = A,A) peptide is shown by circular dichroism and FTIR methods to adopt a predominately beta-sheet conformation. Furthermore, the peptide has limited solubility and is shown to form fibrils by electron microscopy and thioflavin T binding assays. In contrast, a single substitution at the center of peptide of alanine to Aib (X,Z = A,Aib) completely abolishes fibril formation and alters the conformation to a mixture of random coil and alpha-helix. The results show that Aib is a strong beta-sheet disrupter that is also able to adopt a helical conformation. This is linked to its role in peptaibol antibiotics. Aib provides an attractive alternative to proline and other substitutions in producing peptide variants with a lower tendency to produce fibril aggregates.     

The crystal structure of Afc-containing peptides

A systematic structural analysis of Afc (9-amino-fluorene-9-carboxylic acid) containing peptides is here reported. The crystal structures of four fully protected tripeptides containing the Afc residue in position 2: Z-X(1)-Afc(2)-Y(3)-OMe (peptide a: X = Y = Gly; peptide b: X = Aib, C(alpha, alpha)-dimethylglycine, Y = Gly; peptide c: X = Gly, Y = Aib; peptide d: X = Y = Aib) have been solved by x-ray crystallography. All the results suggest that the Afc residue has a high propensity to assume an extended conformation. In fact, the Afc residue adopts an extended conformation in three peptides examined in this paper (peptides a-c). In contrast, Afc was found in a folded conformation, in the 3(10)-helical region, only in the peptide d, in which it is both preceded and followed by the strong helix promoting Aib.     

(alphaMe)Aun: a highly lipophilic, chiral, Calpha-tetrasubstituted alpha-amino acid. Incorporation into model peptides and preferred conformation.

Using a chemo-enzymatic approach we prepared the highly lipophilic, chiral, Calpha-methylated alpha-amino acid (alphaMe)Aun. Two series of terminally protected model peptides containing either D-(alphaMe)Aun in combination with Aib or L-(alphaMe)Aun in combination with Gly were synthesized using solution methods and fully characterized. A detailed solution conformational analysis, based on FT-IR absorption, 1H NMR and CD techniques, allowed us to determine the preferred conformation of this amino acid and the relationship between chirality at its alpha-carbon atom and screw sense of the helix that is formed. The results obtained strongly support the view that D-(alphaMe)Aun favors the formation of the left-handed 3(10)-helical conformation.     

(alphaMe)Nva: stereoselective syntheses and preferred conformations of selected model peptides.

Using different stereoselective chemical and chemoenzymatic approaches we synthesized the chiral, Calpha-methylated alpha-amino acid L-(alphaMe)Nva with a short, linear side-chain. A set of terminally protected model peptides to the pentamer level containing either (alphaMe)Nva or Nva in combination with Ala and/or Aib was prepared using solution methods and characterized fully. Two (alphaMe)Nva peptides were also synthesized using side-chain hydrogenation of the corresponding Calpha-methyl, Calpha-allylglycine (Mag) peptides. A detailed solution and crystal-state conformational analysis based on FT-IR absorption, 1H NMR and X-ray diffraction techniques allowed us to define that: (i) (alphaMe)Nva is an effective beta-turn and 3(10)-helix former; and (ii) the relationship between (alphaMe)Nva chirality and the screw sense of the turn/helix formed is that typical of protein amino acids, i.e. L-(alphaMe)Nva induces the preferential formation of right-handed folded structures. In more general terms, this study reinforced previous conclusions that peptides based on alpha-amino acids with a Calpha-methyl substituent and a Calpha-linear alkyl substituent are characterized by a strong tendency to fold into turn and helical structures.     

Stereospecific binding of diastereomeric peptides to salmon sperm deoxyribonucleic acid. Further evidence for partial intercalation

A series of diastereomeric dipeptide amides, containing an N-terminal L-lysyl residue and a C-terminal L- or D-amino acid with a derivatized aromatic ring on the side chain, was synthesized to determine the dependence of (1) the chirality of the N-terminal amino acid alpha-carbon and (2) the length of the N-terminal amino acid side chain for intercalation of the aromatic ring. The nature of the complex between the peptide and DNA (i.e., electrostatic, intercalative, or a combination of these) was determined by UV and CD studies, viscometric titrations, and 1H NMR studies. The results of these studies reveal distinct differences in the binding site of the aromatic rings of the various peptides. In particular, the results suggest that the alpha- and epsilon-amino groups of the lysyl residue bind electrostatically to adjacent phosphates on the DNA backbone in a stereospecific manner. As a result of this stereospecificity, the aromatic rings of the peptides with the L-L designation point toward the DNA helix, while those of the peptides of the L-D designation point away from the helix. This is completely consistent with previously reported work [Gabbay, E.J., Adawadkar, P. D., & Wilson, W. D. (1976) Biochemistry 15, 146; Gabbay, E. J., Adawadkar, P. D., Kapicak, L., Pearce, S., & Wilson, W. D. (1976) Biochemistry 15, 152]. The results also indicate a great dependence on the length of the side chain for intercalation of the aromatic ring. Specifically, if the side chain is long enough, and flexible enough, the aromatic ring can fully or partially intercalate, regardless of the chirality of the N-terminal amino acid alpha-carbon. However, if the side chain is too short, only partial intercalation is observed for peptides of the L-D designation, and no intercalation is observed for peptides of the L-D designation.     

Disruption of the beta-sheet structure of a protected pentapeptide, related to the beta-amyloid sequence 17-21, induced by a single, helicogenic C(alpha)-tetrasubstituted alpha-amino acid.

Fifteen years ago it was shown that an alpha-aminoisobutyric acid (Aib) residue is significantly more effective than an L-Pro or a D-amino acid residue in inducing beta-sheet disruption in short model peptides. As this secondary structure element is known to play a crucial role in the neuropathology of Alzheimer's disease, it was decided to check the effect of Aib (and other selected, helix inducer, C(alpha)-tetrasubstituted alpha-amino acids) on the beta-sheet conformation adopted by a protected pentapeptide related to the sequence 17-21 of the beta-amyloid peptide. By use of FT-IR absorption and 1H NMR techniques it was found that the strong self-association characterizing the pentapeptide molecules in weakly polar organic solvents is completely abolished by replacing a single residue with Aib or one of its congeners.     

Terminal effect of chiral residue on helical screw sense in achiral peptides

To understand the terminal effect of chiral residue for determining a helical screw sense, we adopted five kinds of peptides I – V containing N‐ and/or C‐terminal chiral Leu residue(s): Boc–L ‐Leu–(Aib–ΔPhe)₂–Aib–OMe ( I ), Boc–(Aib–ΔPhe)₂–L ‐Leu–OMe ( II ), Boc–L ‐Leu–(Aib–ΔPhe)₂–L ‐Leu–OMe ( III ), Boc–D ‐Leu–(Aib–ΔPhe)₂–L ‐Leu–OMe ( IV ), and Boc–D ‐Leu–(Aib–ΔPhe)₂–Aib–OMe ( V ). The segment –(Aib–ΔPhe)₂– was used for a backbone composed of two “enantiomeric” (left‐/right‐handed) helices. Actually, this could be confirmed by ¹H‐nmr [nuclear Overhauser effect (NOE) and solvent accessibility of NH resonances] and CD spectroscopy on Boc–(Aib–ΔPhe)₂–Aib–OMe, which took a left‐/right‐handed 3₁₀‐helix. Peptides I – V were also found to take 3₁₀‐type helical conformations in CDCl₃, from difference NOE measurement and solvent accessibility of NH resonances. Chloroform, acetonitrile, methanol, and tetrahydrofuran were used for CD measurement. The CD spectra of peptides I – III in all solvents showed marked exciton couplets with a positive peak at longer wavelengths, indicating that their main chains prefer a left‐handed screw sense over a right‐handed one. Peptide V in all solvents showed exciton couplets with a negative peak at longer wavelengths, indicating it prefers a right‐handed screw sense. Peptide IV in chloroform showed a nonsplit type CD pattern having only a small negative signal around 280 nm, meaning that left‐ and right‐handed helices should exist with almost the same content. In the other solvents, peptide IV showed exciton couplets with a negative peak at longer wavelengths, corresponding to a right‐handed screw sense. From conformational energy calculation and the above ¹H‐nmr studies, an N‐ or C‐terminal L ‐Leu residue in the lowest energy left‐handed 3₁₀‐helical conformation was found to take an irregular conformation that deviates from a left‐handed helix. The positional effect of the L ‐residue on helical screw sense was discussed based on CD data of peptides I – V and of Boc–(L ‐Leu–ΔPhe)_n–L ‐Leu–OMe (n = 2 and 3). © 1999 John Wiley & Sons, Inc. Biopoly 49: 551–564, 1999     

New tools for the control of peptide conformation and supramolecular chemistry: crown-carrier, C(alpha)-methyl L-DOPA amino acids

The preferred conformation of five, terminally protected, model peptide series to the hexamer level, based on three novel crowned, C(alpha)-methyl L-DOPA amino acids combined with either L-Ala/Aib or Gly/Aib, were assessed in structure supporting solvents using FT-IR absorption, (1)H NMR, and CD techniques. The FT-IR absorption spectra strongly suggest that the contribution of the crowned C(alpha)-tetrasubstituted residue to intramolecular H-bonding is equivalent to that of Aib and is much more significant than that of either L-Ala or Gly. In addition, the (1)H NMR titrations and the CD patterns resemble those typically exhibited by (right-handed) 3(10)-helical structures.     

Helix-helix interconversion rates of short 13C-labeled helical peptides as measured by dynamic NMR spectroscopy

The rates at which a peptide hexamer and a peptide octamer interconvert between left- and right-handed helical forms in CD2Cl2 solution have been characterized by 13C dynamic NMR (DNMR) spectroscopy. The peptide esters studied are Fmoc-(Aib)n-OtBu (n = 6 and 8), where Fmoc is 9-fluorenylmethyoxycarbonyl and Aib is the strongly helix-forming residue alpha-aminoisobutyric acid. Because the Aib residue is itself achiral, homooligomers of this residue form a 50/50 mixture of enantiomeric 3(10)-helices in solution. It has been demonstrated (R.-P. Hummel, C. Toniolo, and G. Jung, Angewandte Chemie International Edition, 1987, Vol. 26, pp. 1150-1152) that oligomers of Aib interconvert on the millisecond timescale. We have performed lineshape analysis of 13C-NMR spectra collected for our peptides enriched with 13C at a single residue. Rate constants for the octamer range from 6 s(-1) at 196 K to about 56,500 s(-1) at 320 K. At all temperatures, the hexamer interconverts about three times faster than the octamer. Eyring plots of the data reveal experimentally indistinguishable DeltaH++ values for the hexamer and octamer of 37.8 +/- 0.6 and 37.6 +/- 0.4 kJ mol(-1) respectively. The difference in the rates of interconversion is dictated by entropic factors. The hexamer and octamer exhibit negative DeltaS++ values of -29.0(-1) +/- 2.5 and -37.3 +/- 1.7 J K(-1) mol(-1), respectively. A mechanism for the helix-helix interconversion is proposed. and calculated DeltaG++ values are compared to the estimate for a decamer undergoing a helix-helix interconversion.     

Long, chiral polypeptide 3(10)-helices at atomic resolution

The crystal-state preferred conformation of the terminally blocked hepta- and octapeptides with the general formula -(Aib)n L-Leu-(Aib)2- (n = 4 and 5, respectively), determined by X-ray diffraction, was found to be a right-handed 3(10)-helix stabilized by five and six consecutive intramolecular NH...O = C H-bonds of the C(10)-III type, respectively. The octapeptide structure represents the first observation at atomic resolution of a regular, chiral 3(10)-helix larger than two complete turns. In both cases the right handed screw sense of the helix is dictated by the presence of the single, internal L-residue. This study confirms the propensity of short peptides rich in Aib, the prototype of the amino acid residues dialkylated at the alpha carbon, to adopt a 3(10)-helical structure and is expected to help our understanding of the conformational preferences of the membrane-active, channel-forming, ion-transporting peptaibol antibiotics.     

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.