Conservation of folding and stability within a protein family: the tyrosine corner as an evolutionary cul-de-sac

Abstract

What are the selective pressures on protein sequences during evolution? Amino acid residues may be highly conserved for functional or structural (stability) reasons. Theoretical studies have proposed that residues involved in the folding nucleus may also be highly conserved. To test this we are using an experimental “fold approach” to the study of protein folding. This compares the folding and stability of a number of proteins that share the same fold, but have no common amino acid sequence or biological activity. The fold selected for this study is the immunoglobulin-like β-sandwich fold, which is a fold that has no specifically conserved function. Four model proteins are used from two distinct superfamilies that share the immunoglobulin-like fold, the fibronectin type III and immunoglobulin superfamilies. Here, the fold approach and protein engineering are used to question the role of a highly conserved tyrosine in the “tyrosine corner” motif that is found ubiquitously and exclusively in Greek key proteins. In the four model β-sandwich proteins characterised here, the tyrosine is the only residue that is absolutely conserved at equivalent sites. By mutating this position to phenylalanine, we show that the tyrosine hydroxyl is not required to nucleate folding in the immunoglobulin superfamily, whereas it is involved to some extent in early structure formation in the fibronectin type III superfamily. The tyrosine corner is important for stability, mutation to phenylalanine costs between 1.5 and 3 kcal mol−1. We propose that the high level of conservation of the tyrosine is related to the structural restraints of the loop connecting the β-sheets, representing an evolutionary “cul-de-sac”.