Protein Folding Kinetics Provides a Context-independent Assessment of β-Strand Propensity in the Fyn SH3 Domain

Abstract

Structural database-derived propensities for amino acids to adopt particular local protein structures, such as α-helix and β-strand, have long been recognized and effectively exploited for the prediction of protein secondary structure. However, the experimental verification of database-derived propensities using mutagenesis studies has been problematic, especially for β-strand propensities, because local structural preferences are often confounded by non-local interactions arising from formation of the native tertiary structure. Thus, the overall thermodynamic stability of a protein is not always altered in a predictable manner by changes in local structural propensity at a single position. In this study, we have undertaken an investigation of the relationship between β-strand propensity and protein folding kinetics. By characterizing the effects of a wide variety of amino acid substitutions at two different β-strand positions in an SH3 domain, we have found that the observed changes in protein folding rates are very well correlated to β-strand propensities for almost all of the substitutions examined. In contrast, there is little correlation between propensities and unfolding rates. These data indicate that β-strand conformation is well formed in the structured portion of the SH3 domain transition state, and that local structure propensity strongly influences the stability of the transition state. Since the transition state is known to be packed more loosely than the native state and likely lacks many of the non-local stabilizing interactions seen in the native state, we suggest that folding kinetics studies may generally provide an effective means for the experimental validation of database-derived local structural propensities.