Short alanine peptides, containing 16 or 17 residues, appear to form alpha-helices in aqueous solution. But the main spectroscopic analyses used on helical peptides (circular dichroism and nuclear magnetic resonance) cannot distinguish between an alpha-helix (in which the ith residue is hydrogen-bonded to residue i + 4; ref. 9) and the next most common peptide helix, the 3(10)-helix10 (i-->i + 3 hydrogen-bonding). To address this problem we have designed single and doubly spin-labelled analogues of alanine-based peptides in which the nitroxide spin label forms an unbranched side chain extending from the sulphur atom of a cysteine residue. Here we report the circular dichroism, Fourier-transform infrared and electron-spin resonance spectra of these peptides under helix-forming conditions. The infrared absorbance gives an amide I' band with a frequency that is substantially different from that observed for alpha-helices. The electron-spin resonance spectra of doubly labelled helices show that the ranking of distances between side chains, around a single turn (residues 4-8), is inconsistent with an alpha-helical structure. Our experiments suggest that the more likely peptide geometry is a 3(10)-helix.

Ala-based peptides form marginally stable helices at low temperature and are conventionally considered as mixtures of alpha-helix and random coil. However, recent work with doubly spin-labeled peptides suggests that short 16-residue sequences contain a significant fraction of 3(10)-helix near the N-terminus (positions 4-8). Using the same double-label strategy, we report on the helix geometry of the peptides Ac-(AAAAK)nA-NH2 with n = 3 and n = 4. The 16-mer (n = 3) is now examined at a region near the C-terminus, and there is evidence for 3(10)-helix here as well. The 21-mer (n = 4) is examined in three regions of the sequence. In dramatic contrast to the 16-mer, the 21-mer exhibits the signature of alpha-helix at the N-terminus and on through the middle of the peptide. The 21-mer C-terminus, however, adopts the 3(10)-helix geometry as is often found for C-termini in protein alpha-helices. These data indicate that the proportion of alpha-helix and 3(10)-helix in Ala-based peptides depends upon the sequence length.

Alanine-based peptides of defined sequence and length show measurable helix contents, allowing them to be used as a model system both for analyzing the mechanism of helix formation and for investigating the contributions of side-chain interactions to protein stability. Extensive characterization of many peptide sequences with varying amino acid contents indicates that the favorable helicity of alanine-based peptides can be attributed to the large helix-stabilizing propensity of alanine. The resulting model provides a physically plausible resolution of the discrepancies between the two systems and allows the helix contents of both template-nucleated and standard peptide helices to be predicted by using a single set of helix propensities. Helix formation in both standard peptides and template-peptide conjugates can be attributed to the large intrinsic helix-forming tendency of alanine.The energetic cost of ␣-helix nucleation generally is considered to originate from the requirement of constraining the conformation of three consecutive amino acids before the first helical hydrogen bond can form (1). In contrast, helix propagation requires only one additional residue to be constrained for the formation of one additional hydrogen bond. The overall extent of helix formation depends on the energetics of both nucleation and propagation, as well as on chain length. To separate the effects of helix nucleation and propagation experimentally, Kemp and coworkers (2, 3) have designed a template (Ac-Hel 1 ; see Fig. 1A) that overcomes the nucleation penalty for helix formation by providing hydrogen-bond acceptors for the otherwise unsatisfied NH groups at the N terminus of the helix. Short peptides attached to this template show significant helix formation that is nucleated preferentially from the template. Because the template efficiently nucleates helical segments, the macroscopic characteristics of the template-nucleated helix depend primarily on the nucleation properties of the template and the propagation propensities of the attached amino acid residues.Using the Ac-Hel 1 template as both a nucleation site and a reporter of helix content, Kemp and coworkers (2, 4-6) have reported significant helix formation for a number of short alanine-based peptide sequences under a variety of conditions. Quite surprisingly, however, the authors conclude from their data that alanine is helix-indifferent with an equilibrium constant for propagation of approximately one (2, 4). In direct contrast, alanine is observed to be a strong helix former in a variety of standard peptide systems (7-9). To explain this paradox, Kemp and coworkers (2) have suggested that the helix content of standard alanine-based peptides might originate from an anomalously large nucleation propensity of alanine. A large nucleation propensity is inconsistent, however, with the extent of fraying observed in alanine-based peptides (10-12). Furthermore, to model their Ac-Hel 1 -peptide conjugate data, Kemp and coworkers (2, 4-6) assume that the nucleation propen...

AbstractÐThe aromatic diacid residue 4,6-dibenzofuranbispropionic acid (1) was designed to nucleate a parallel b-sheet-like structure in small peptides in aqueous solution via a hydrogen-bonded hydrophobic cluster. Even though a 14-membered ring hydrogen bond necessary for parallel b-sheet formation is favored in simple amides composed of 1, this hydrogen bonding interaction does not appear to be sucient to nucleate parallel b-sheet formation in the absence of hydrophobic clustering between the dibenzofuran portion of 1 and the hydrophobic side chains of the¯anking a-amino acids. The subsequence -hydrophobic residue-1-hydrophobic residue-is required for folding in the context of a nucleated two-stranded parallel b-sheet structure. In all cases where the peptidomimetics can fold into two diastereomeric parallel b-sheet structures having dierent hydrogen bonding networks, these conformations appear to exchange rapidly. The majority of the parallel b-sheet structures evaluated herein undergo linked intramolecular folding and self-assembly, aording a ®brillar b-sheet quaternary structure. To unlink folding and assembly, asymmetric parallel b-sheet structures incorporating N-methylated a-amino acid residues have been synthesized using a new solid phase approach. Residue 1 facilitates the folding of several peptides described within aording a monomeric parallel b-sheet-like structure in aqueous solution, as ascertained by a variety of spectroscopic and biophysical methods, increasing our understanding of parallel b-sheet structure. #

Arginine is a stabilizing element in both thermophilic and low molecular weight proteins. Similarly Lys+-->Arg+ substitutions increase the helix content of designed helical peptides. Here we explore this 'arginine effect' by examining how Lys+-->Arg+ substitutions influence the 3(10)-helix-->alpha-helix equilibrium in the helical peptide Ac-(AAAAK)3A-NH2. The unsubstituted sequence contains a significant amount of 3(10)-helix, however, single Lys+-->Arg+ substitutions shift the peptide conformation toward alpha-helix in a position-dependent fashion. The single substitution closest to the carboxy terminus induces the largest conformational change at the helix amino terminus. These findings suggest that a single strategically-placed arginine can exert long range control on helix structure.

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