Do Proteins with Similar Folds Have Similar Transition State Structures? A Diffuse Transition State of the 169 Residue Apoflavodoxin

Apoflavodoxin from Anabaena PCC 7119 is a 169 residue globular protein of known structure and energetics. Here, we present a comprehensive Φ-value analysis to characterize the structure of its transition state. A total of 34 non-disruptive mutations are made throughout the structure and a range of Φ-values from zero to one are observed. In addition, a small set of eight aliphatic small-to-large mutations have been introduced in the hydrophobic core of the protein and they have been analyzed to investigate the feasibility of stabilizing the unfolding transition state by creating new non-native interactions. We find that the transition state of apoflavodoxin (so far the largest protein subjected to Φ-analysis) is diffuse and that it can be stabilized by unspecific hydrophobic interactions that can speed up the folding reaction. The data gathered on the apoflavodoxin transition state are compared with results from experimental studies in other proteins to revisit the relationship between the native state topology and transition state structure.