Abstract
Fluorescence correlation spectroscopy (FCS) is a sensitive analytical tool that allows dynamics and hydrodynamics of biomolecules to be studied under a broad range of experimental conditions. One application of FCS of current interest is the determination of the size of protein molecules in the various states they sample along their folding reaction coordinate, which can be accessed through the measurement of diffusion coefficients. It has been pointed out that the analysis of FCS curves is prone to artifacts that may lead to erroneous size determination. To set the stage for FCS studies of unfolded proteins, we first show that the diffusion coefficients of small molecules as well as proteins can be determined accurately even in the presence of high concentrations of co-solutes that change the solution refractive index significantly. Indeed, it is found that the Stokes-Einstein relation between the measured diffusion coefficient and solution viscosity holds even in highly concentrated glycerol or guanidinium hydrochloride (GuHCl) solutions. These measurements form the basis for an investigation of the structure of the denatured state of two proteins, the small protein L and the larger, three-domain protein adenylate kinase (AK). FCS is found useful for probing expansion in the denatured state beyond the unfolding transition. It is shown that the denatured state of protein L expands as the denaturant concentration increases, in a process akin to the transition from a globule to a coil in polymers. This process continues at least up to 5 M GuHCl. On the other hand, the denatured state of AK does not seem to expand much beyond 2 M GuHCl, a result that is in qualitative accord with single-molecule fluorescence histograms. Because both the unfolding transition and the coil-globule transition of AK occur at a much lower denaturant concentration than those of protein L, a possible correlation between the two phenomena is suggested.
| Original language | English |
|---|---|
| Pages (from-to) | 4819-4827 |
| Number of pages | 9 |
| Journal | Biophysical Journal |
| Volume | 94 |
| Issue number | 12 |
| DOIs | |
| State | Published - 15 Jun 2008 |
| Externally published | Yes |
Bibliographical note
Funding Information:This research was made possible in part by the generosity of the Harold Perlman Family, as well as by partial financial support of the US-Israel Binational Science Foundation (grant 2002371 to G.H. and grant 2005270 to E.H.), the National Institutes of Health (grant 1R01GM080515-01 to GH), and the European Union (grant MTKD-CT-2005-029936 to E.H.).