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

The folding thermodynamics and kinetics of the Pin WW domain, a three-stran ded antiparallel beta -sheet, have been characterized extensively. Folding and activation free energies were determined as a function of temperature f or 16 mutants, which sample all strands and turns of the molecule. The muta tional phi value (Phi (m)) diagram is a smooth function of sequence, indica ting a prevalence of local interactions in the transition state (TS). At 37 degreesC, 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 r esidues for thermodynamic stability are located in the strand hydrophobic c lusters, indicating that factors contributing to protein stability and fold ing kinetics are not correlated. The location of the TS along the entropic reaction coordinate Phi (T), obtained by temperature-tuning the kinetics, r eveals that sufficiently destabilizing mutants in loop 2 or in the Leu7-Trp 11-Tyr24-Pro37 hydrophobic cluster can cause a switch to a late TS. Phi (m) analysis is usually applied "perturbatively" (methyl truncation), but with Phi (T) to quantitatively assess TS shifts along a reaction coordinate, mo re severe mutations can be used to probe regions of the free energy surface beyond the TS. (C) 2001 Academic Press.