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

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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, I1 and I2, during unfolding/refolding at the folding temperature, Tf = 335 K. Equilibrium intermediate I1 demonstrates discrete structure in regions α0-β6 whereas intermediate I2 is a loose ensemble of states with N-terminal structure varying from at least β1-β3 (denoted I2A) to α0-β4 at most (denoted I2B). The structures of I1 and I2 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, I120Q, I200Q, I300Q, and I360Q, during refolding. Kinetic intermediates I120Q, I200Q, and I300Q are highly similar to equilibrium αTS intermediates I2A, I2B, and I1, respectively, consistent with kinetic experiments on αTS from E. coli. A small population (∼10%) of kinetic trajectories are trapped in the I120Q intermediate ensemble and require a slow and complete unfolding step to properly refold. Both the on-pathway and off-pathway I120Q intermediates show structure in β1-β3, which is also strikingly consistent with kinetic folding experiments of αTS. In the off-pathway intermediate I120Q, helix α2 is wrapped in a nonnative chiral arrangement around strand β3, sterically preventing the subsequent folding step between β3 and β4. 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.

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Biophysical Journal





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open access

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