Mutational investigation of protein folding transition states by Φ-value analysis and beyond: lessons from SH3 domain foldingThis paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

Understanding how proteins adopt their unique native structures requires a complete structural characterization of the rate-limiting transition state(s) along the folding pathway. By definition, transition states are not significantly populated and are only accessible via folding kinetics studies. In this respect, interpreting the kinetic effects of amino acid substitutions (especially to Ala) via Φ-value analysis is the most common method to probe the structure of these transient, yet important states. A critical review of the key assumptions required for rigorous interpretation of Φ values reveals that a multiple substitution strategy in which a position of interest is mutated to a variety of amino acids, and not exclusively to Ala, provides the best means to characterize folding transition states. This approach has proven useful in revealing non-native interactions and (or) conformations in folding transition states. Moreover, by simultaneously examining the folding kinetics of multiple substitutions made at a single surface-exposed position using the Brønsted analysis the backbone conformation in a folding transition state can be investigated. For folding equilibria with exchange rates on the order of milliseconds, the kinetic parameters for Φ-value analysis can be obtained from NMR relaxation dispersion experiments, under fully native conditions, along with a wealth of high-resolution structural information about the states in exchange (native, denatured, and intermediate states that populate the pathway). This additional structural information, which is not readily obtained through stopped-flow based methods, can significantly facilitate the interpretation of Φ values because it often reports on the validity of the assumptions required for a rigorous interpretation of Φ values.