Approaching the speed limit for Greek Key beta-barrel formation: transition-state movement tunes folding rate of zinc-substituted azurin.

Azurin is a blue-copper protein with a beta-barrel structure of Greek Key topology. In vitro, copper can be substituted with zinc without change in protein structure. We here analyze the kinetic folding behavior of zinc-substituted Pseudomonas aeruginosa azurin. Our findings can be summarized in three key conclusions: first, zinc remains strongly bound to the polypeptide upon unfolding, suggesting that the cofactor may bind to the protein before polypeptide folding in vivo. Second, the semi-logarithmic plot of folding and unfolding rates for zinc-substituted azurin as a function of denaturant concentration exhibits curvature due to a changing transition-state structure. Third, the extrapolated folding speed in water for zinc-substituted azurin is similar to that of other proteins with the same topology, implying that there is a speed limit that can be modulated by stability-driven transition-state movement for formation of beta-barrel structures with Greek Key topology.