Dissecting the energetics of protein α-helix C-cap termination through chemical protein synthesis

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Abstract
The α-helix is a fundamental protein structural motif and is frequently terminated by a glycine residue1,2,3,4,5. Explanations for the predominance of glycine at the C-cap terminal portions of α-helices have invoked uniquely favorable energetics of this residue in a left-handed conformation4 or enhanced solvation of the peptide backbone because of the absence of a side chain6. Attempts to quantify the contributions of these two effects have been made previously, but the issue remains unresolved. Here we have used chemical protein synthesis to dissect the energetic basis of α-helix termination by comparing a series of ubiquitin variants containing an L-amino acid or the corresponding D-amino acid at the C-cap Gly35 position. D-Amino acids can adopt a left-handed conformation without energetic penalty, so the contributions of conformational strain and backbone solvation can thus be separated. Analysis of the thermodynamic data revealed that the preference for glycine at the C′ position of a helix is predominantly a conformational effect.