φ-Value Analysis of Apo-Azurin Folding: Comparison between Experiment and Theory†

Pseudomonas aeruginosa azurin is a 128-residue β-sandwich metalloprotein; in vitro kinetic experiments have shown that it folds in a two-state reaction. Here, we used a variational free energy functional to calculate the characteristics of the transition state ensemble (TSE) for folding of the apo-form of P. aeruginosa azurin and investigate how it responds to thermal and mutational changes. The variational method directly yields predicted chevron plots for wild-type and mutant apo-forms of azurin. In parallel, we performed in vitro kinetic-folding experiments on the same set of azurin variants using chemical perturbation. Like the wild-type protein, all apo-variants fold in apparent two-state reactions both in calculations and in stopped-flow mixing experiments. Comparisons of phi (φ) values determined from the experimental and theoretical chevron parameters reveal an excellent agreement for most positions, indicating a polarized, highly structured TSE for folding of P. aeruginosa apo-azurin. We also dem...

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