TCB Publications - Abstract

E. Larios, J. S. Li, K. Schulten, H. Kihara, and M. Gruebele. Multiple probes reveal a native-like intermediate during the low-temperature refolding of ubiquitin. Journal of Molecular Biology, 340:115-125, 2004.

LARI2004 We investigate the refolding of ubiquitin Phe45Trp/Ile61Ala (Ub*I61A) in a low temperature high viscosity buffer, where folding is slowed down so that apparent two- and three-state mechanisms are readily distinguishable. Ub*I61A rapidly forms a compact ensemble (as judged from stopped-flow small angle X-ray scattering) with a secondary structure signature similar to the native state (as judged from stopped- flow circular dichroism from 215-250 nm), but the fluorescence signature still resembles the guanidinium-denatured state. The native fluorescence signature, which requires the tryptophan residue to be tightly packed, is acquired at least 400 times more slowly. Molecular dynamics simulations yield an unfolded radius of gyration smaller in aqueous ethylene glycol buffer compared to pure aqueous buffer, but without a significant effect on the local backbone structure of the unfolded protein. Thus it appears unlikely that the aqueous ethylene glycol buffer fundamentally changes the folding mechanism of ubiquitin. We suggest that ubiquitin forms a compact ensemble with native-like secondary structure, but without tight packing, long before the native state.

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