Tuning the Attempt Frequency of Protein Folding Dynamics via Transition-State Rigidification: Application to Trp-Cage

[Image: see text] The attempt frequency or prefactor (k(0)) of the transition-state rate equation of protein folding kinetics has been estimated to be on the order of 10(6) s(–1), which is many orders of magnitude smaller than that of chemical reactions. Herein we use the mini-protein Trp-cage to show that it is possible to significantly increase the value of k(0) for a protein folding reaction by rigidifying the transition state. This is achieved by reducing the conformational flexibility of a key structural element (i.e., an α-helix) formed in the transition state via photoisomerization of an azobenzene cross-linker. We find that this strategy not only decreases the folding time of the Trp-cage peptide by more than an order of magnitude (to ∼100 ns at 25 °C) but also exposes parallel folding pathways, allowing us to provide, to the best of our knowledge, the first quantitative assessment of the curvature of the transition-state free-energy surface of a protein.