Hydration of the peptide backbone largely defines the thermodynamic propensity scale of residues at the C′ position of the C-capping box of α-helices

The C′ position of the C-capping box is the second residue outside of the helix. Statistical analysis of residue distribution at the C′ position in the α-helices' C-capping box showed that different amino acid residues occur with different probabilities, with the strongest preference being for glycine. To understand the physico-chemical basis for this preference, we studied the effects that 17 amino acid substitutions at the C′ position in an α-helix of ubiquitin have on the stability of this protein. We determined the following rank order of amino acid residues at the C′ position with respect to their effect on the stability: Gly>His>Asn>Arg>Lys>Gln>Ala>Phe>Met>Ser>Asp>Glu>Trp>Thr>Pro>Ile>Val. The effect of the amino acid substitutions on the structure also was evaluated by comparing the (1)H-(15)N heteronuclear sequential quantum correlation spectra and showed no significant changes in the structures of the most stable (Gly) and the least stable (Val) variants. The obtained changes in stability highly correlate (r = 0.85) with the statistical distribution of the residues at the C′ position indicating that the measured thermodynamic propensities are unbiased by secondary interactions. We also found that the measured thermodynamic propensities correlate well with the amide hydrogen exchange data on short model peptides (r = 0.85) and the calculated hydration of the peptide backbone (r = 0.88). These results combined with the changes in enthalpy and entropy of unfolding of ubiquitin variants suggest that dehydration of the peptide backbone plays a significant role in defining the thermodynamic propensity scale at the C′ position of the C-capping box in α-helices. This propensity scale is useful for protein secondary structure predictions and protein design.