Modulation of a protein free-energy landscape by circular permutation

Circular permutations usually retain the native structure and function of a protein while inevitably perturb its folding dynamics. By using simulations with a structure-based model and a rigorous methodology to determine free-energy surfaces from trajectories we evaluate the effect of a circular permutation on the free-energy landscape of the protein T4 lysozyme. We observe changes which, while subtle, largely affect the cooperativity between the two subdomains. Such a change in cooperativity has been previously experimentally observed and recently also characterized using single molecule optical tweezers and the Crooks relation. The free-energy landscapes show that both the wild type and circular permutant have an on-pathway intermediate, previously experimentally characterized, where one of the subdomains is completely formed. The landscapes, however, differ in the position of the rate-limiting step for folding, which occurs before the intermediate in the wild-type and after in the circular permutant. This shift of transition state explains the observed change in the cooperativity. The underlying free-energy landscape thus provides a microscopic description of the folding dynamics and the connection between circular permutation and the loss of cooperativity experimentally observed.