Φ value versus ψ analysis
- Cambridge University Chemical Laboratory and Medical Research Council Centre for Protein Engineering, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, United Kingdom
Knowing the structures of transition states is crucial to the understanding of any chemical mechanism. As the transition state is an ephemeral species, its properties can be studied experimentally only indirectly from kinetics, with fine structural details inferred from the analysis of reaction kinetics of reagents whose structures have been subtly altered. A battery of protein engineering techniques has been developed to apply to study protein folding, of which the application of the latest method, ψ analysis, to ubiquitin is described by Sosnick et al. (1) in this issue of PNAS. Their results suggest that more structure is formed in the transition state than indicated by Φ-value analysis, which has been the standard method for nearly two decades, and the transition state is heterogeneous.
Transition States and Φ-Value Analysis
The key procedure in transition-state analysis is to mutate the structures of amino acids and measure the changes in kinetics and equilibria of protein folding. In Φ analysis, mutations are designed to delete or alter existing weak interactions. The parameter Φ (= ΔΔG ‡/ΔΔG 0, where ΔΔG ‡ is the change of free energy of activation and ΔΔG 0 the change in free energy of folding on mutation) is a measure of the average extent of structure formation at the mutated site on a scale of 0 to 1, relative to denatured and native states, respectively (2). Φ Analysis gives the degree of formation of elements of secondary structure and tertiary interactions (3) and even backbone-backbone hydrogen-bonding interactions (4). Formation of pairwise interactions may be identified unambiguously by the application of double-mutant cycles in which both partners are mutated. Movement of the transition state around the energy landscape also can be detected (5). Importantly, Φ values are the raw stuff of simulation: computer modeling reconstructs the 3D structure of the …
