Stable proline box motif at the N-terminal end of alpha-helices

We describe a novel N-terminal alpha-helix local motif that involves three hydrophobic residues and a Pro residue (Pro-box motif). Database analysis s hows that when Pro is the N-cap of an alpha-helix the distribution of amino acids in adjacent positions changes dramatically with respect to the avera ge distribution in an alpha-helix, but not when Pro is at position N1. N-ca p Pro residues are usually associated to Ile and Leu, at position N', Val a t position N3 and a hydrophobic residue (h) at position N4. The side chain of the N-cap Pro packs against Val, while the hydrophobic residues at posit ions N' and N4 make favorable interactions. To analyze the role of this put ative motif (sequence fingerprint hPXXhh), we have synthesized a series of peptides and analyzed them by circular dichroism (CD) and NMR. We find that this motif is formed in peptides, and that the accompanying hydrophobic in teractions contribute up to 1.2 kcal/mol to helix stability. The fact that some of the residues in this fingerprint are not good N-cap and helix forme rs results in a small overall stabilization of the alpha-helix with respect to other peptides having Gly as the N-cap and Ala at N3 and N4. This sugge sts that the Pro-box motif will not specially contribute to protein stabili ty but to the specificity of its fold. In fact, 80% of the sequences that c ontain the fingerprint sequence in the protein database are adopting the de scribed structural motif, and in none of them is the helix extended to plac e Pro at the more favorable N1 position.