title: Multiple probes reveal a native-like intermediate during low-temperature refolding of ubiquitin.
abstract: We investigate the refolding of ubiquitin Phe45Trp/Ile61Ala (Ub(*)I61A) in a low-temperature, high-viscosity buffer, where folding is slowed so that apparent two-state and three-state mechanisms are readily distinguishable. Ub(*)I61A forms a compact ensemble rapidly (as judged from stopped-flow, small-angle X-ray scattering) with a secondary structure signature similar to that of the native state (as judged from stopped-flow circular dichroism from 215 nm to 250 nm), but the fluorescence signature still resembles the guanidinium-denatured state. The compact ensemble forms over a range of solvent and temperature conditions. The native fluorescence signature, which requires the tryptophan residue to be packed tightly, is acquired at least 500 times more slowly. Molecular dynamics simulations at 495 K show no contraction of the backbone in ethylene glycol buffer compared to pure aqueous buffer, and no significant effect on the local backbone structure of the unfolded protein. Only at higher simulation temperature does a backbone contraction appear. 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.
authors.affiliation: Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: E
authors.initials: E
authors.lastname: Larios
authors.forename: J S
authors.initials: JS
authors.lastname: Li
authors.forename: K
authors.initials: K
authors.lastname: Schulten
authors.forename: H
authors.initials: H
authors.lastname: Kihara
authors.forename: M
authors.initials: M
authors.lastname: Gruebele
mesh_terms: D002942:Circular Dichroism; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012542:Scattering, Radiation; D013696:Temperature; D025801:Ubiquitin / Q000737:chemistry*
chemical_list: D025801:Ubiquitin