.alpha.-Helical versus 310-Helical Conformation of Alanine-Based Peptides in Aqueous Solution: An Electron Spin Resonance Investigation

Abstract

Due to the difficulties in experimentally differentiating between the a-and 3io-helical conformations in solution, isolated helical peptides have been assumed to be in the -helical conformation. However, recent electron spin resonance (ESR) studies have suggested that such peptides, in particular short alanine-based peptides, are 310helical (Miick, S. M.; et al. Nature 1992, 359, 653-5). This result prompted us to further investigate the helical conformations of alanine-based peptides in solution using electron spin resonance spectroscopy. Unlike previous investigations with a flexible link connecting the spin-label to the peptide backbone, we used a conformationally constrained spin-label (4-amino-4-carboxy-2,2,6,6-tetramethylpiperidine-1 -oxyl, Toac) that is rigidly attached to the peptide backbone. From a combination of molecular modeling and ESR spectroscopy investigations, it was concluded that these alanine-based peptides exist primarily in the -helical conformation, and not the 3io-form as previously suggested for an analogous set of peptides in aqueous environments. This discrepancy is thought to be due to the differences in flexibility of the spin-labels employed. The conformationally constrained spin-label Toac used in this study should accurately reflect the backbone conformation. Free energy surfaces, or potentials of mean force, for the conformational transition of the spin-label used in previous studies (Miick S. M.; et al. Nature 1992, 359, 653-5) suggest that this spin-label is too flexible to accurately distinguish between the a-and 3io-helical conformations.

Back to the future: Ribonuclease A

“…These plots indicate why proline, or any other N-methyl amino acid, has difficulties being accommodated within the helical segments of proteins. The most dramatic change was seen, however, when the a-hydrogen of the alanine was replaced by a methyl group, acetyl-L-aminoisobutyric N-methylamide ( Figure 7D), simultaneously restricting both u and w. This prediction that a-methyl amino acids would restrict the flexibility of the peptide backbone to only those regions of u,w space common to both acetyl-L-alanine N-methylamide ( Figure 7A) and acetyl-D-alanine N-methylamide ( Figure 7E) has been confirmed subsequently by numerous experimental/theoretical studies by the Marshall, [65][66][67][68][69] Karle, 70,71 Toniolo,72,73 Balaram, 70 and Millhauser 74 groups. By focusing the minimal energy states of the peptide backbone to u,w space associated with right-or left-handed helices, substitutions with a-methyl amino acids preorganize the peptide…”

Determination of the Solution Structures of Conantokin-G and Conantokin-T by CD and NMR Spectroscopy

“…Although theoretical studies on the relative energies of 3 10 versus ␣-helix suggest that the latter conformation is preferred in helices of greater than 10 -12 residues due to a lower degree of steric hindrance (35,36), this is offset in con-T by a large number of putative electrostatic i,iϩ3 interactions which would favor 3 10 helix formation. For example, helix stabilizing salt-bridges may be formed between Gla 4 -Lys could promote the alignment of Gla residues so that the above mentioned interactions become favorable.…”

Hydrophobic Aided Replica Exchange: an Efficient Algorithm for Protein Folding in Explicit Solvent

“…The representative protein system we study here is the R-helix 3K(I) 23 with a sequence of AAAAKAAAAKAAAAKA. The N and C terminals are capped with Ace and Nme groups, respectively.…”