Evidence for strained interactions between side-chains and the polypeptide backbone.

Abstract

In most proteins, a small but significant fraction of residues adopt phi,psi angles that generate unfavorable steric interactions between side-chain atoms and the peptide backbone. For the small protein staphylococcal nuclease, the X-ray structure reveals that 18 of 133 residues occupy unusual and, presumably, energetically unfavorable backbone conformations. To quantify the amount of strain energy generated by these local interactions, we have analyzed the changes in stability that accompany replacement of the wild-type side-chain with glycine, a residue that can access a much larger set of phi,psi angles without energy penalty. To correct for the many other sources of stability loss that might accompany this mutation, the glycine mutant was compared to an alanine mutant at the same position and the resulting free energy difference delta delta GG-->A was then compared to the average delta delta GG-->A at all other, unstrained positions in the nuclease occupied by similar amino acid types. In addition, potential steric clashes were introduced by substituting alanine at each of six positions occupied in the wild-type by glycine with phi,psi angles that are unfavorable for all other residue types. The data suggest that residues with phi,psi angles outside the preferred alpha-helical and beta-sheet regions represent sites of local strain energy that lower the stability of the native state by 1 to 2 kcal/mol and, in some cases, as much as 3 to 4 kcal/mol. Given that 10 to 20% of residues in globular proteins adopt phi,psi angles outside the preferred alpha-helical and beta-sheet regions, this implies that there is on the order of 20 kcal/mol of strain energy in a protein of 100 residues that may be relieved by appropriate mutations.