We describe a novel N-terminal α-helix local
motif that involves three hydrophobic residues and a Pro
residue (Pro-box motif ). Database analysis shows
that when Pro is the N-cap of an α-helix the distribution
of amino acids in adjacent positions changes dramatically
with respect to the average distribution in an α-helix,
but not when Pro is at position N1. N-cap Pro residues
are usually associated to Ile and Leu, at position N′,
Val at 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 positions N′ and
N4 make favorable interactions. To analyze the role of
this putative 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 interactions
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 formers results in a small overall stabilization
of the α-helix with respect to other peptides having
Gly as the N-cap and Ala at N3 and N4. This suggests that
the Pro-box motif will not specially contribute
to protein stability but to the specificity of its fold.
In fact, 80% of the sequences that contain the fingerprint
sequence in the protein database are adopting the described
structural motif, and in none of them is the helix extended
to place Pro at the more favorable N1 position.