Some recommendations for the practitioner to improve the precision of experimentally determined protein folding rates and phi values.

The mechanism by which proteins fold from an initially random conformation into a functional, native structure remains a major unsolved question in molecular biology. Of particular interest to the protein folding community is the structure that the protein adopts in the folding transition state (the highest free energy state on the pathway from unfolded to folded), as that state forms the barrier that defines the folding pathway. Unfortunately, however, unlike those of the initial, unfolded state and the final, folded state of the protein, the structure in the transition state cannot be directly assessed via experiment. Instead, experimentalists infer the structure of the transition state, often by estimating changes in its free energy by measuring the effects of amino acid substitutions on folding and unfolding rates (Phi-value analysis). In this article we show how to obtain more efficient estimates of these important quantities via improved experimental designs, and how to avoid common pitfalls in the analysis of kinetic data during the extraction of these parameters. Copyright 2008 Wiley-Liss, Inc.