Dramatic acceleration of protein folding by stabilization of a nonnative backbone conformation

Abstract

Through a mutagenic investigation of Gly-48, a highly conserved position in the Src homology 3 domain, we have discovered a series of amino acid substitutions that are highly destabilizing, yet dramatically accelerate protein folding, some up to 10-fold compared with the wild-type rate. The unique folding properties of these mutants allowed for accurate measurement of their folding and unfolding rates in water with no denaturant by using an NMR spin relaxation dispersion technique. A strong correlation was found between ␤-sheet propensity and the folding rates of the Gly-48 mutants, even though Gly-48 lies in an unusual non-␤-strand backbone conformation in the native state. This finding indicates that the accelerated folding rates of the Gly-48 mutants are the result of stabilization of a nonnative ␤-strand conformation in the transition-state structure at this position, thus providing the first, to our knowledge, experimentally elucidated example of a mechanism by which folding can occur fastest through a nonnative conformation. We also demonstrate that residues that are most stabilizing in the transition-state structure are most destabilizing in the native state, and also cause the greatest reductions in in vitro functional activity. These data indicate that the unusual native conformation of the Gly-48 position is important for function, and that evolutionary selection for function can result in a domain that folds at a rate far below the maximum possible. T he energetics involved as proteins progress from a broad ensemble of unfolded conformations to a single native structure are still not thoroughly understood. A widely used approach to study folding is the protein engineering method, which measures the thermodynamic and kinetic effects of single amino acid substitutions (usually with Ala) at many different sites in a protein as a means to define the most structured regions of the folding transition state (1, 2). Mapping the transition state identifies regions that fold more quickly than others, and such knowledge has led to important theoretical advances in our understanding of the folding process (3, 4). Although this method provides a general picture of folding pathways, it does not define the mechanisms by which individual residues can facilitate or retard the folding process. Our approach to study these mechanisms is to investigate the effects of multiple amino acid substitutions at single positions (5-7). In the present study, this approach is used to investigate the role in folding and function of position 48 of the Src homology 3 (SH3) domain, which is one of most conserved positions in this domain (8).The SH3 domain is well suited for protein-folding studies due to its small size and amenability to biophysical analysis (9-11). Composed of two three-stranded ␤-sheets packed orthogonally against one another (Fig. 1A), SH3 domains function as proteinprotein interaction modules in a wide variety of eukaryotic proteins. The structure of the SH3 domain transition state has been extensively characterized...

β‐strand interactions at the domain interface critical for the stability of human lens γD‐crystallin

“…1 Such a favorable role of non-native interaction in the formation of protein folding nucleus is in line with previous experimental and theoretical studies, suggesting that specific non-native interactions may accelerate folding by reducing conformational search to the native state. [53][54][55][56][57][58][59][60][61][62][63] The identification of a critical salt-bridge in a crystallin folding/unfolding intermediate is another example of non-native interactions crucial for protein folding. Similarly, Presta and Rose 59 described helix stop signals that were not necessarily retained in the native state of the folded proteins.…”

Reconstruction of the src-SH3 Protein Domain Transition State Ensemble using Multiscale Molecular Dynamics Simulations

“…Interestingly, earlier work in which the TSE for SH3 was extracted directly from discrete molecular dynamics simulations showed more structure in this region, in particular between residues Leu16 and Gly46, 20 the latter being a kinetically important residue for folding. 45 The method of identification of the transition state in this previous work was based on the P fold analysis presented here. Hence, structures with the Leu16-Gly46 contact formed and P fold values of 0.5 are true TSE conformations (based on the P fold definition used here).…”

Protein folding: Then and now

“…Moreover, they also predicted that the two most kinetically important residues in folding are L24 and G64. Both L24 and G64 are experimentally-verified to be important kinetically [148].…”