ANALYSIS OF I,I+5 AND I,I+8 I-HYDROPHOBIC INTERACTIONS IN A HELICAL MODEL PEPTIDE BEARING THE HYDROPHOBIC STAPLE MOTIF

In this work we have analyzed by far-UV circular dichroism the contribution to alpha-helix stability of pairwise hydrophobic interactions in the hydrophobic staple motif [Munoz et al. (1995) Nat. Struct. Biol. 2, 380-385]. For this, we have used a new series of alanine-based model peptides having a capping box motif (Ser-X-X-Glu) and no other charged residues to facilitate the determination of the interaction energies with a helix/coil transition algorithm. Our results show that the favorable i,i+5 interaction between a hydrophobic residue (Leu, Met, Ile, Val, Phe) at position N' (before the N-cap) and a Leu at position N+4 (inside the helix) contributes up to -1.48 +/- 0.18 kcal/mol to alpha-helix stability at 278 and pH 7. More interestingly, the same hydrophobic residues at position N' interact favorably with an Ala at position N+4, although the interaction is weaker than that with Leu (up to -0.8 +/- 0.14 kcal/mol at 278 K and pH 7). To our knowledge, this is the first example in which a strong pairwise interaction with Ala is described and suggests that Ala could be less neutral in terms of side chain-side chain interactions than normally assumed. We observe a strong stereospecificity for the position N' which could be explained based on the extreme rigidity imposed by the formation in phase of the hydrophobic staple and capping-box motifs, as is seen in the protein structure database. We have also investigated the contribution to alpha-helix stability of a geometrically feasible i,i+8 hydrophobic interaction between residues N' and N+7. In this regard, we find that most of the i,i+8 hydrophobic pairs have completely negligible interactions, while the Leu-Leu and perhaps the Ile-Leu pairs have a weak favorable i,i+8 interaction.