Structural examination of Φ-value analysis in protein folding

Protein folding intermediates and transition states are commonly characterized using a protein engineering procedure (Phi-value analysis) based on several assumptions, including (1) intermediates and transition states have native-like conformations and (2) single mutations from larger hydrophobic residues to smaller ones do not perturb their structures. Although Phi-value analysis has been widely used, these assumptions have not been tested to date because of the lack of high-resolution structures of intermediates and transition states. We recently have determined the structure of a folding intermediate for a four-helix bundle protein (Rd-apocytochrome b(562)) using NMR. The intermediate has the N-terminal helix unfolded. The other three helices fold in a native-like topology with extensive non-native hydrophobic interactions. Here, we have determined the Phi values for 14 hydrophobic core residues, including those with significant non-native interactions. All of the Phi values are in the normal range from 0 to 1, indicating that these non-native interactions cannot be identified by the common Phi-value analysis, and therefore, the first assumption is not valid for this intermediate. We also determined the structure of a mutant (F65A) of the intermediate and found that the structure of the intermediate is not perturbed by the mutation, supporting the second assumption. Together, these results suggest that Phi-value analysis may be valid for characterizing the energetics of the interactions between the mutated residue and others, but not for determining the detailed structures of intermediates and transition states because non-native interactions may exist and may not be identifiable by the common Phi-value analysis.