Commitment and Nucleation in the Protein G Transition State

An accurate characterization of the transition state ensemble (TSE) is central to furthering our understanding of the protein folding reaction. We have extensively tested a recently reported method for studying a proteinʹs TSE, utilizing φ-value data from protein engineering experiments and computational studies as restraints in all-atom Monte Carlo (MC) simulations. The validity of interpreting experimental φ-values as the fraction of native contacts made by a residue in the TSE was explored, revealing that this definition is unable to uniquely specify a TSE. The identification of protein Gʹs second hairpin, in both pre and post-transition conformations demonstrates that high experimental φ-values do not guarantee a residueʹs importance in the TSE. An analysis of simulations based on structures restrained by experimental φ-values is necessary to yield this result, which is not obvious from a simplistic interpretation of individual φ-values. The TSE that we obtain corresponds to a single, specific nucleation event, characterized by six residues common to all three observed, convergent folding pathways. The same specific nucleus was independently identified from computational and experimental data, and “Conservation of Conservation” analysis in the protein G fold. When associated strictly with complete nucleus formation and concomitant chain collapse, folding is a well-defined two state event. Once the nucleus has formed, the folding reaction enters a slow relaxation process associated with side-chain packing and small, local backbone rearrangements. A detailed analysis of φ-values and their relationship to the transition state ensemble allows us to construct a unified theoretical model of protein G folding.