Exploring one-state downhill protein folding in single molecules

Loading...
Thumbnail Image
Issue Date
2012-01-3
Embargo End Date
Authors
Liu, Jianwei
Campos, Luis A.
Cerminara, Michele
Wang, Xiang
Ramanathan, Ravishankar
English, Douglas S.
Munoz, Victor
Advisor
Citation

Liu, J., Campos, L.A., Cerminara, M., Wang, X., Ramanathan, R., English, D.S., Munoz, V. Exploring one-state downhill protein folding in single molecules (2012) Proceedings of the National Academy of Sciences of the United States of America, 109 (1), pp. 179-184

Abstract

A one-state downhill protein folding process is barrierless at all conditions, resulting in gradual melting of native structure that permits resolving folding mechanisms step-by-step at atomic resolution. Experimental studies of one-state downhill folding have typically focused on the thermal denaturation of proteins that fold near the speed limit (ca. 10 6 s -1) at their unfolding temperature, thus being several orders of magnitude too fast for current single-molecule methods, such as single-molecule FRET. An important open question is whether one-state downhill folding kinetics can be slowed down to make them accessible to single-molecule approaches without turning the protein into a conventional activated folder. Here we address this question on the small helical protein BBL, a paradigm of one-state downhill thermal (un)folding. We decreased 200-fold the BBL folding-unfolding rate by combining chemical denaturation and low temperature, and carried out free-diffusion single-molecule FRET experiments with 50-?s resolution and maximal photoprotection using a recently developed Troloxcysteamine cocktail. These experiments revealed a single conformational ensemble at all denaturing conditions. The chemical unfolding of BBL was then manifested by the gradual change of this unique ensemble, which shifts from high to low FRETefficiency and becomes broader at increasing denaturant. Furthermore, using detailed quantitative analysis, we could rule out the possibility that the BBL single-molecule data are produced by partly overlapping folded and unfolded peaks. Thus, our results demonstrate the one-state downhill folding regime at the single-molecule level and highlight that this folding scenario is not necessarily associated with ultrafast kinetics.

Table of Content
Description
Click on the DOI link below to access the article (may not be free).
publication.page.dc.relation.uri
DOI