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

What are the selective pressures on protein sequences during evolution? Ami no acid residues may be highly conserved for functional or structural (stab ility) reasons. Theoretical studies have proposed that residues involved in the folding nucleus may also be highly conserved. To test this we are usin g an experimental "fold approach" to the study of protein folding. This com pares 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 f old selected for this study is the immunoglobulin-like beta-sandwich fold, which is a fold that has no specifically conserved function. Four model pro teins are used from two distinct superfamilies that share the immunoglobuli n-like fold, the fibronectin type III and immunoglobulin superfamilies. Her e, 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 beta -sandwich proteins characterised here, the tyrosine is the only residue tha t is absolutely conserved at equivalent sites. By mutating this position to phenylalanine, we show that the tyrosine hydroxyl is not required to nucle ate folding in the immunoglobulin superfamily, whereas it is involved to so me extent in early structure formation in the fibronectin type III superfam ily. The tyrosine corner is important for stability, mutation to phenylalan ine 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 th e loop connecting the beta-sheets, representing an evolutionary "cul-de-sac ". (C) 2000 Academic Press.