Conformational preferences of oligopeptides rich in alpha-aminoisobutyric acid. I. Observation of a 3(10)/alpha-helical transition upon sequence permutation.

The solution conformation of peptides rich in the alpha, alpha-dialkylated amino acid Aib has proven to be a subtle problem, not because of helix/coil transitions, but rather because of alpha-helical/3(10)-helical competition. A special series of peptides containing 75% Aib has been synthesized that feature identical amino acid composition but differing sequences; they are sequence permutation isomers. Nuclear magnetic resonance hydrogen-bonding studies reveal that there is a sequence permutation induced transition between the two alternative helical forms within this set. The implications for the design and conformational prediction of helical Aib-rich peptides are discussed.     

Variants of 3(10)-helices in proteins

An analysis of the shortest 3(10)-helices, containing three helical residues and two flanking capping residues that participate in two consecutive i + 3 --> i hydrogen bonds, shows that not all helices belong to the classic 3(10)-helix, where the three central residues adopt the right-handed helical conformation (alpha(R)). Three variants identified are: 3L10-helix with all residues in the left-handed helical region (alpha(L)), 3EL10-helix where the first residue is in the extended region followed by two residues in the alpha(L) conformation, and its mirror-image, the 3E'R10-helix. In the context of these helices, as well as the equivalent variants of alpha-helices, the length dependence of the handedness of secondary structures in protein structure is discussed. There are considerable differences in the amino acid preferences at different positions in the various types of 3(10)-helices. Each type of 3(10)-helix can be thought to be made up of an extension of a particular type of beta-turn (made up of residues i to i + 3) such that the (i + 3)th residue assumes the same conformation as the preceding residue. Distinct residue preferences at i and i + 3 positions seem to decide whether a particular stretch of four residues will be a beta-turn or a 3(10)-helix in the folded structure.     

A monomeric 3(10)-helix is formed in water by a 13-residue peptide representing the neutralizing determinant of HIV-1 on gp41

The peptide gp41(659-671) (ELLELDKWASLWN) comprises the entire epitope for one of the three known antibodies capable of neutralizing a broad spectrum of primary HIV-1 isolates and is the only such epitope that is sequential. Here we present the NMR structure of gp41(659-671) in water. This peptide forms a monomeric 3(10)-helix stabilized by i,i+3 side chain-side chain interactions favored by its primary sequence. In this conformation the peptide presents an exposed surface, which is mostly hydrophobic and consists of conserved HIV-1 residues. The presence of the 3(10)-helix is confirmed by its characteristic CD pattern. Studies of the 3(10)-helix have been hampered by the absence of a model peptide adopting this conformation. gp41(659-671) can serve as such a model to investigate the spectral characteristics of the 3(10)-helix, the factors that influence its stability, and the propensity of different amino acids to form a 3(10)-helix. The observation that the 3(10)-helical conformation is highly populated in the peptide gp41(659-671) indicates that the corresponding segment in the cognate protein is an autonomous folding unit. As such, it is very likely that the helical conformation is maintained in gp41 throughout the different tertiary structures of the envelope protein that form during the process of viral fusion. However, the exposure of the gp41(659-671) segment may vary, leading to changes in the reactivity of anti-gp41 antibodies in the different stages of viral fusion. Since gp41(659-671) is an autonomous folding unit, peptide immunogens consisting of the complete gp41(659-671) sequence are likely to induce antibodies highly cross-reactive with HIV-1.     

Conformational studies of Aib-rich peptides containing lactam-bridged side chains: evidence of 3(10)-helix formation

Aib-rich side-chain lactam-bridged oligomers Ac-(Glu-Aib-Aib-Lys)n-Ala-OH with n = 1,2,3 were designed and synthesized as putative models of the 3(10)-helix. The lactam bridge between the side chains of L-Glu and L-Lys in (i)--(i + 3) positions was introduced in order to enhance the structural preference toward the right-handed 3(10)-helix. The conformational properties of the three peptides were studied in trifluoroethanol (TFE) solution by CD, NMR, and computer simulations. The structural information was derived mainly from the analysis of nuclear Overhauser effect spectroscopy spectra. The presence of alpha H(i)-HN(i + 2) and of alpha H(i)-HN(i + 3) connectivities and the absence of alpha H(i)-HN(i + 4) connectivities indicate that these peptides fold into a 3(10)-helix rather than into an alpha-helix. Based on these conformational features, stereospecific assignment of the Aib methyl groups was possible. The results of such experiments and of the subsequent distance geometry and restrained molecular dynamics simulations reveal a marked preference of these peptides for 3(10)-helix. The CD spectra of these peptides indicate that the helix content increases upon chain elongation. The CD spectrum of the trimer is characterized by a negative band at 200 nm and by a weak positive band around 220 nm. The CD spectrum in TFE is different from that observed in aqueous solution in the presence of SDS micelles, reported in our previous work, and from those reported by a different research group for 3(10)-helical peptides. A possible reason for these differences could rest in the presence of different equilibria of the conformer populations of the various peptides in different solvent systems.     

Models for the 3(10)-helix/coil,pi-helix/coil,and alpha-helix/3(10)-helix/coil transitions in isolated peptides.

Models for the 3(10)-helix/coil and pi-helix/coil equilibria have been derived. The theory is based on classifying residues into helical or nonhelical (coil) conformations. Statistical weights are assigned to residues in a helical conformation with an associated helical hydrogen bond, a helical conformation with no hydrogen bond, an N-cap position, a C-cap position, or the reference coil conformation. The models for alpha-helix formation and 3(10)-helix formation have also been combined to describe a three-state equilibrium in which alpha-helical, 3(10)-helical, and coil conformations are populated. The results are compared with the modified Lifson-Roig theory for the alpha-helix/coil equilibrium. The comparison accounts for the experimental observations that 3(10)-helices tend to be short and pi-helices are not favored for any length. This work may provide a framework for quantitatively rationalizing experimental work on isolated 3(10)-helices and mixed 3(10)-/alpha-helices.     

Addition of side-chain interactions to 3(10)-helix/coil and alpha-helix/3(10)-helix/coil theory.

An increasing number of experimental and theoretical studies have demonstrated the importance of the 3(10)-helix/ alpha-helix/coil equilibrium for the structure and folding of peptides and proteins. One way to perturb this equilibrium is to introduce side-chain interactions that stabilize or destabilize one helix. For example, an attractive i, i + 4 interaction, present only in the alpha-helix, will favor the alpha-helix over 3(10), while an i, i + 4 repulsion will favor the 3(10)-helix over alpha. To quantify the 3(10)/alpha/coil equilibrium, it is essential to use a helix/coil theory that considers the stability of every possible conformation of a peptide. We have previously developed models for the 3(10)-helix/coil and 3(10)-helix/alpha-helix/ coil equilibria. Here we extend this work by adding i, i + 3 and i, i + 4 side-chain interaction energies to the models. The theory is based on classifying residues into alpha-helical, 3(10)-helical, or nonhelical (coil) conformations. Statistical weights are assigned to residues in a helical conformation with an associated helical hydrogen bond, a helical conformation with no hydrogen bond, an N-cap position, a C-cap position, or the reference coil conformation plus i, i + 3 and i, i + 4 side-chain interactions. This work may provide a framework for quantitatively rationalizing experimental work on isolated 3(10)-helices and mixed 3(10)-/alpha-helices and for predicting the locations and stabilities of these structures in peptides and proteins. We conclude that strong i, i + 4 side-chain interactions favor alpha-helix formation, while the 3(10)-helix population is maximized when weaker i, i + 4 side-chain interactions are present.     

Factors governing 3(10)-helix vs alpha-helix formation in peptides: percentage of C(alpha)-tetrasubstituted alpha-amino acid residues and sequence dependence

As an additional step toward the dissection of the factors responsible for the onset of 3(10)-helix vs alpha-helix in peptides, in this paper we describe the results of a three-dimensional (3D) structural analysis by x-ray diffraction of the N(alpha)-acylated heptapeptide alkylamide mBrBz-L-Iva-L-(alphaMe)Val-L-Abu-L-(alphaMe)Val-L-(alphaMe)Phe-L-(alphaMe)Val-L-Iva-NHMe characterized by a single (L-Abu3) C(alpha)-trisubstituted and six C(alpha)-tetrasubstituted alpha-amino acids. We find that in the crystal state this peptide is folded in a mixed helical structure with short elements of 3(10)-helix at either terminus and a central region of alpha-helix. This finding, taken together with the published NMR and x-ray diffraction data on the all C(alpha)-methylated parent sequence and its L-Val2 analog (also the latter heptapeptide has a single C(alpha)-trisubstituted alpha-amino acid) strongly supports the view that one C(alpha)-trisubstituted alpha-amino acid inserted near the N-terminus of an N(alpha)-acylated heptapeptide alkylamide sequence may be enough to switch a regular 3(10)-helix into an essentially alpha-helical conformation. As a corollary of this work, the x-ray diffraction structure of the N(alpha)-protected, C-terminal tetrapeptide alkylamide Z-L-(alphaMe)Val-L-(alphaMe)Phe-L-(alphaMe)Val-L-Iva-NHMe, also reported here, is clearly indicative of the preference of this fully C(alpha)-methylated, short peptide for the 3(10)-helix. As the same terminally blocked sequence is mixed 3(10)/alpha-helical in the L-Abu3 heptapeptide amide but regular 3(10)-helical in the tetrapeptide amide and in the parent heptapeptide amide, these results point to an evident plasticity even of a fully C(alpha)-methylated short peptide.     

Crystal structure of achiral nonapeptide Boc-(Aib-DeltaZPhe)4-Aib-OMe at atomic resolution: evidence for a 3(10)-helix

An x-ray crystallographic analysis was carried out for Boc-(Aib-DeltaZPhe)4-Aib-OMe (1: Boc = t-butoxycarbonyl; Aib = alpha-aminoisobutyric acid; DeltaZPhe = Z-alpha,beta-didehydrophenylalanine) to provide the precise conformational parameters of the octapeptide segment -(Aib-DeltaZPhe)4-. Peptide 1 adopted a typical 3(10)-helical conformation characterized by = +/-55.8 degrees (50 degrees -65 degrees), = +/-26.7 degrees (15 degrees -45 degrees), and = +/-179.5 degrees (168 degrees -188 degrees) for the average values of the -(Aib-DeltaZPhe)4- segment (the range of the eight values). The 3(10)-helix contains 3.1 residues per turn, being close to the "perfect 3(10)-helix" characterized by 3.0 residues per turn. NMR and Fourier transform infrared (FTIR) spectroscopy revealed that the 3(10)-helical conformation at the atomic resolution is essentially maintained in solution. Energy minimization of peptide 1 by semiempirical molecular orbital calculation converged to a 3(10)-helical conformation similar to the x-ray crystallographic 3(10)-helix. The preference for a 3(10)-helix in the -(Aib-DeltaZPhe)4- segment is ascribed to strong inducers of the 3(10)-helix inherent in Aib and DeltaZPhe residues-in particular, the Aib residues tend to stabilize a 3(10)-helix more effectively. Therefore, the -(Aib-DeltaZPhe)4- segment is useful to rationally design an optically inactive 3(10)-helical backbone, which will be of great importance to provide novel insights into noncovalent and covalent chiral interactions of a helical peptide with a chiral molecule.     

Critical Main-Chain Length for Conformational Conversion From 3(10)-Helix to α-Helix in Polypeptides

To assess the minimal peptide length required for the stabilization of the alpha-helix relative to the 3(10)-helix in Aib-rich peptides, we have solved the X-ray diffraction structures of the terminally blocked sequential hexa- and octapeptides with the general formula-(Aib-L-Ala)n-(n = 3 and 4, respectively). The hexapeptide molecules are completely 3(10)-helical with four 1----4 intramolecular N-H . . . O = C H-bonds. On the other hand, the octapeptide molecules are essentially alpha-helical with four 1----5 H-bonds; however, the helix is elongated at the N-terminus, with two 1----4 H-bonds, giving these molecules a mixed alpha/3(10)-helical character. In both compounds the right-handed screw sense of the helix is dictated by the presence of the Ala residues of L-configuration. This study represents the first experimental proof for a 3(10)----alpha-helix conversion in the crystal state induced by peptide backbone lengthening only.     

Exploring the peptide 310-helix ⇆ α–helix equilibrium with double label electron spin resonance

Over the last several years we have used spin labeling as a means for exploring the structure of helical peptides. Two nitroxide labels are engineered into a peptide sequence and distances are ranked with electron spin resonance (ESR). We have found that there is a significant amount of 3₁₀–helix in 16–residue model peptides containing only L –amino acids. This review covers several facets of the methodology including spin labeling strategy, interpretation of ESR spectra and the influence of molecular dynamics on the spectral line shapes. Also covered are recent findings of a length–dependent 3_l0-helix → α-helix transition and the role of Arg⁺ in the stabilization of specific helix structures. © 1994 John Wiley & Sons, Inc.     

The crystal state conformation of Aib-rich segments of peptaibol antibiotics

Ac-(Aib-Ala)₃-OH (a protected segment of the peptaibols gliodeliquescin and paracelsin), Z-Leu-Aib-Val-Aib-Gly-OtBu (a segment of [Leu]⁷-gliodeliquescin), Z-Val-Aib-Aib-Gln-OtBu (a common segment of alamethicin, paracelsin, and hypelcin), and Ac-Aib-Pro-(Aib-Ala)₂-OMe and Z-Aib-Pro-(Aib-Ala)₂-OMe, which represent differently N^α-protected 1–6 segments of alamethicin and hypelcin, have been synthesized by solution methods. The crystal-state conformations of these five Aib-containing peptides have been determined by X-ray diffraction analysis. We have confirmed that the 3₁₀-helical structure is preferentially adopted by Aib-rich short peptides. An experimentally unambiguous proof for the 3₁₀→α-helix conversion has been provided by the two differently N-blocked -Aib-Pro-(Aib-Ala)₂-OMe hexapeptides. The β-bend ribbon conformation, commonly observed in the (Aib-Pro)_n sequential oligopeptides, is not found in the -Aib-Pro-Aib-Ala-Aib-Ala- sequence. As expected on the basis of the l -configuration of the C^α-monoalkylated residues, a right-handed helix screw sense was found in all peptides investigated. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Development of the multiple sequence approximation within the AGADIR model of α-helix formation: Comparison with Zimm-Bragg and Lifson-Roig formalisms

In this work we present the development of the multiple sequence approximation (AGADIRms) and the standard one-sequence approximation (AGADIRIs) within the framework of AGADIR's α-helix formation model. The extensive comparison between these new formulations and the original one [AGADIR; v. Muñoz and L. Serrano (1994),Nat. Struct. Biol., Vol. 1, pp. 399–409] indicates that the standard one-sequence approximation is virtually identical to the multiple sequence approximation, while the previously used residue partition function approximation [Muñoz and Serrano (1994); (1995), J. Mol. Biol., Vol. 245, pp. 275–296] is less precise. The calculations of the average helical content performed with AGADIR are precise for peptides of less than 30 residues and progressively diverge from the multiple sequence formulation for longer peptides. The helicity distribution of heteropolypeptides with less than 50% average helical content is also well described, while those of quasi-homopolymers with high helical content tend to be flattened. These inaccuracies lead to an underestimation of 0.017 kcal/mol for the mean-residue enthalpic contribution in AGADIR, as compared to AGADIRms and AGADIRIs. The other energy contributions to α-helix stability are not affected by the original statistical approximation. We also discuss the particularities of the model for α-helix formation utilized in AGADIR and compare it with the classical Zimm-Bragg and Lifson-Roig theories. Moreover, we develop the mathematical relationships between the basic AGADIR energy contributions and helix nucleation and elongation, which permit the quantitative comparison between formalisms. Remarkably, the comparison between AGADIRms and the Lifson-Roig formalism shows that, despite the differences on treating helix/coil cooperativity, both theories give virtually identical results when an equivalent set of parameters is used. This indicates that the helix/coil transition is a solid theory independent of the particularities of the model for α-helix formation. © 1997 John Wiley & Sons, Inc. Biopoly 41: 495–509, 1997.     

Constrained phenylalanine analogues. Preferred conformation of the 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) residue.

Three Tic-containing (Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) model peptides were synthesized to assess the tendency of this constrained Phe analogue to fold into a beta-bend and a helical structure, and to adopt a preferred side-chain disposition. The results of the solution conformational analysis, performed by using Fourier transform infrared absorption and 1H nuclear magnetic resonance, indicate that in chloroform the -Aib-D-Tic-Aib-, -(Aib)2-D-Tic-(Aib)2-, and -L-Pro-D-Tic- sequences fold into intramolecularly H-bonded forms to a great extent. An X-ray diffraction analysis on p-BrBz-(Aib)2-DL-Tic-(Aib)2-OMe monohydrate and p-BrBz-L-Pro-D-Tic-NHMe allows us to conclude that, while the pentapeptide methylester forms an incipient (distorted) 3(10)-helix, the dipeptide methylamide adopts a type-II beta-bend conformation. In both cases, the D-Tic side-chain conformation is D, gauche(-). The implications for the use of the Tic residue in designing conformationally restricted analogues of bioactive peptides are briefly discussed.     

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.     

Characterization at atomic resolution of peptide helical structures.

A survey of literature for the various types of helices experimentally observed in high-resolution single crystal x-ray diffraction analyses of peptides has allowed to determine accurate conformational and helical parameters for the various secondary structures such as the alpha-helix, the 3(10)-helix, the fully extended conformation (2(5)-helix) and the beta-bend ribbon spiral. For each of these structures the characteristic phi, psi conformational parameters, n, the number of residues per turn, h, the height per residues and p, the pitch of the helix are described.     

Critical Main-Chain Length for Conformational Conversion From 310-Helix to α-Helix in Polypeptides

Abstract

To assess the minimal peptide length required for the stabilization of the a-helix relative to the 3₁₀-helix in Aib-rich peptides, we have solved the X-ray diffraction structures of the terminally blocked sequential hexa- and octapeptides with the general formula -(Aib-L-Ala)_n-(n = 3 and 4, respectively). The hexapeptide molecules are completely 3₁₀-helical with four 1 ← 4 intramolecular N-H … O=C H-bonds. On the other hand, the octapeptide molecules are essentially α-helical with four 1 ← 5 H-bonds; however, the helix is elongated at the N-terminus, with two 1 ← 4 H-bonds, giving these molecules a mixed α/3₁₀-helical character. In both compounds the right-handed screw sense of the helix is dictated by the presence of the Ala residues of L-configuration. This study represents the first experimental proof for a 3₁₀ →α-helix conversion in the crystal state induced by peptide backbone lengthening only.     

The solvation interface is a determining factor in peptide conformational preferences

The 21 residue polyalanine-based F(s) peptide was studied using thousands of long, explicit solvent, atomistic molecular dynamics simulations that reached equilibrium at the ensemble level. Peptide conformational preference as a function of hydrophobicity was examined using a spectrum of explicit solvent models, and the peptide length-dependence of the hydrophilic and hydrophobic components of solvent-accessible surface area for several ideal conformational types was considered. Our results demonstrate how the character of the solvation interface induces several conformational preferences, including a decrease in mean helical content with increased hydrophilicity, which occurs predominantly through reduced nucleation tendency and, to a lesser extent, destabilization of helical propagation. Interestingly, an opposing effect occurs through increased propensity for 3(10)-helix conformations, as well as increased polyproline structure. Our observations provide a framework for understanding previous reports of conformational preferences in polyalanine-based peptides including (i) terminal 3(10)-helix prominence, (ii) low pi-helix propensity, (iii) increased polyproline conformations in short and unfolded peptides, and (iv) membrane helix stability in the presence and absence of water. These observations provide physical insight into the role of water in peptide conformational equilibria at the atomic level, and expand our view of the complexity of even the most "simple" of biopolymers. Whereas previous studies have focused predominantly on hydrophobic effects with respect to tertiary structure, this work highlights the need for consideration of such effects at the secondary structural level.     

Structural insight into an ankyrin-sensitive lipid-binding site of erythroid beta-spectrin

It was recently shown that the region within beta-spectrin responsible for interactions with ankyrin includes a lipid-binding site which displayed sensitivity to inhibition by ankyrin. We studied its structure by constructing a series of single and double spin-labeled beta-spectrin-derived peptides and analyzing their spin-spin distances via electron paramagnetic resonance spectroscopy and the Fourier deconvolution method. The results indicate that the whole ankyrin-sensitive lipid-binding site of beta-spectrin exhibits a helical conformation revealing a distinct 3(10)-helix contribution at its N-terminus. The start of the helix was located five residues upstream along the sequence compared to the theoretical predictions. A model based on the obtained data provides direct evidence that the examined lipid-binding site is a highly amphipathic helix, which is correlated with the specific conformation of its N-terminal fragment.     

Differences in the amino acid distributions of 3(10)-helices and alpha-helices.

Local determinants of 3(10)-helix stabilization have been ascertained from the analysis of the crystal structure data base. We have clustered all 5-length substructures from 51 nonhomologous proteins into classes based on the conformational similarity of their backbone dihedral angles. Several clusters, derived from 3(10)-helices and multiple-turn conformations, had strong amino acid sequence patterns not evident among alpha-helices. Aspartate occurred over twice as frequently in the N-cap position of 3(10)-helices as in the N-cap position of alpha-helices. Unlike alpha-helices, 3(10)-helices had few C-termini ending in a left-handed alpha conformation; most 3(10) C-caps adopted an extended conformation. Differences in the distribution of hydrophobic residues among 3(10)- and alpha-helices were also apparent, producing amphipathic 3(10)-helices. Local interactions that stabilize 3(10)-helices can be inferred both from the strong amino acid preferences found for these short helices, as well as from the existence of substructures in which tertiary interactions replace consensus local interactions. Because the folding and unfolding of alpha-helices have been postulated to proceed through reverse-turn and 3(10)-helix intermediates, sequence differences between 3(10)- and alpha-helices can also lend insight into factors influencing alpha-helix initiation and propagation.