期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2006
卷号：103
期号：31
页码：11539-11543
DOI：10.1073/pnas.0601395103
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Upon transfer from strongly denaturing to native conditions, proteins undergo a collapse that either precedes folding or occurs simultaneously with it. This collapse is similar to the well known coil-globule transition of polymers. Here we employ single-molecule fluorescence methods to fully characterize the equilibrium coil-globule transition in the denatured state of the IgG-binding domain of protein L. By using FRET measurements on freely diffusing individual molecules, we determine the radius of gyration of the protein, which shows a gradual expansion as the concentration of the denaturant, guanidinium hydrochloride, is increased all the way up to 7 M. This expansion is observed also in fluorescence correlation spectroscopy measurements of the hydrodynamic radius of the protein. We analyze the radius of gyration measurements using the theory of the coil-globule transition of Sanchez [Sanchez, I. C. (1979) Macromolecules 12, 980-988], which balances the excluded volume entropy of the chain with the average interresidue interaction energy. In particular, we calculate the solvation energy of the denatured protein, a property that is not readily accessible in other experiments. The dependence of this energy on denaturant concentration is nonlinear, contrasting with the common linear extrapolation method used to describe denaturation energy. Interestingly, a fit to the binding model of chemical denaturation suggests a single denaturant binding site per protein residue. The size of the denatured protein under native conditions can be extrapolated from the data as well, showing that the fully collapsed state of protein is only {approx}10% larger than the folded state.
关键词：fluorescence correlation spectroscopy ; protein folding ; single-molecule fluorescence
