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

The Center for Theoretical Biological Physics, University of California at San Diego, La Jolla, California 92093-0374

Correspondence: Address reprint requests to José N. Onuchic, Center of Theoretical Biological Physics, University of California at San Diego, 9500 Gilman Dr., La Jolla, CA 92093. Tel.: 858-534-7067; E-mail: jonuchic{at}ucsd.edu.

The role of native contact topology in the folding of a TIM barrel model based on the -subunit of tryptophan synthase (TS) from Salmonella typhimurium (Protein Data Bank structure 1BKS) was studied using both equilibrium and kinetic simulations. Equilibrium simulations of TS reveal the population of two intermediate ensembles, I₁ and I₂, during unfolding/refolding at the folding temperature, T_f = 335 K. Equilibrium intermediate I₁ demonstrates discrete structure in regions ₀-ß₆ whereas intermediate I₂ is a loose ensemble of states with N-terminal structure varying from at least ß₁-ß₃ (denoted I_2A) to ₀-ß₄ at most (denoted I_2B). The structures of I₁ and I₂ match well with the two intermediate states detected in equilibrium folding experiments of Escherichia coli TS. Kinetic folding simulations of TS 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 TS intermediates I_2A, I_2B, and I₁, respectively, consistent with kinetic experiments on TS 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 ß₁-ß₃, which is also strikingly consistent with kinetic folding experiments of TS. In the off-pathway intermediate I_120Q, helix ₂ is wrapped in a nonnative chiral arrangement around strand ß₃, sterically preventing the subsequent folding step between ß₃ and ß₄. 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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