Equilibrium and Kinetic Folding Pathways of a TIM Barrel with a Funneled Energy Landscape

Abstract

The role of native contact topology in the folding of a TIM barrel model based on the α-subunit of tryptophan synthase ( αTS) from Salmonella typhimurium (Protein Data Bank structure 1BKS) was studied using both equilibrium and kinetic simulations. Equilibrium simulations of αTS reveal the population of two intermediate ensembles, I 1 and I 2, during unfolding/refolding at the folding temperature, T f = 335 K. Equilibrium intermediate I 1 demonstrates discrete structure in regions α 0- β 6 whereas intermediate I 2 is a loose ensemble of states with N-terminal structure varying from at least β 1- β 3 (denoted I 2A) to α 0- β 4 at most (denoted I 2B). The structures of I 1 and I 2 match well with the two intermediate states detected in equilibrium folding experiments of Escherichia coli αTS. Kinetic folding simulations of αTS reveal the sequential population of four intermediate ensembles, I 120Q, I 200Q, I 300Q, and I 360Q, during refolding. Kinetic intermediates I 120Q, I 200Q, and I 300Q are highly similar to equilibrium αTS intermediates I 2A, I 2B, and I 1, respectively, consistent with kinetic experiments on αTS from E. coli. A small population (∼10%) of kinetic trajectories are trapped in the I 120Q intermediate ensemble and require a slow and complete unfolding step to properly refold. Both the on-pathway and off-pathway I 120Q intermediates show structure in β 1- β 3, which is also strikingly consistent with kinetic folding experiments of αTS. In the off-pathway intermediate I 120Q, helix α 2 is wrapped in a nonnative chiral arrangement around strand β 3, sterically preventing the subsequent folding step between β 3 and β 4. These results demonstrate the success of combining kinetic and equilibrium simulations of minimalist protein models to explore TIM barrel folding and the folding of other large proteins.