Engineered Metal-Binding Sites to Probe Protein Folding Transition States: Psi Analysis

The formation of the transition state ensemble (TSE) represents the rate-limiting step in protein folding. The TSE is the least populated state on the pathway, and its characterization remains a challenge. Properties of the TSE can be inferred from the effects on folding and unfolding rates for various perturbations. A difficulty remains on how to translate these kinetic effects to structural properties of the TSE. Several factors can obscure the translation of point mutations in the frequently used method, "mutational Phi analysis." We take a complementary approach in "Psi analysis," employing rationally inserted metal binding sites designed to probe pairwise contacts in the TSE. These contacts can be confidently identified and used to construct structural models of the TSE. The method has been applied to multiple proteins and consistently produces a considerably more structured and native-like TSE than Phi analysis. This difference has significant implications to our understanding of protein folding mechanisms. Here we describe the application of the method and discuss how it can be used to study other conformational transitions such as binding.