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.
