The folding mechanism of a β-sheet: The WW domain

The folding thermodynamics and kinetics of the Pin WW domain, a three-stranded antiparallel β-sheet, have been characterized extensively. Folding and activation free energies were determined as a function of temperature for 16 mutants, which sample all strands and turns of the molecule. The mutational phi value (Φ_m) diagram is a smooth function of sequence, indicating a prevalence of local interactions in the transition state (TS). At 37°C, the diagram has a single pronounced maximum at turn 1: the rate-limiting step during folding is the formation of loop 1. In contrast, key residues for thermodynamic stability are located in the strand hydrophobic clusters, indicating that factors contributing to protein stability and folding kinetics are not correlated. The location of the TS along the entropic reaction coordinate Φ_T, obtained by temperature-tuning the kinetics, reveals that sufficiently destabilizing mutants in loop 2 or in the Leu7-Trp11-Tyr24-Pro37 hydrophobic cluster can cause a switch to a late TS. Φ_m analysis is usually applied "perturbatively" (methyl truncation), but with Φ_T to quantitatively assess TS shifts along a reaction coordinate, more severe mutations can be used to probe regions of the free energy surface beyond the TS.