What fluorescence correlation spectroscopy can tell us about unfolded proteins.

Abstract

Fluorescence correlation spectroscopy (FCS) measures rates of diffusion, chemical reaction, and other dynamic processes of fluorescent molecules. Studies of unfolded proteins benefit from the fact that FCS can provide information about rates of protein conformational change both by a direct readout from conformation-dependent fluorescence changes and by changes in diffusion coefficient. Although the diffusion coefficient is relatively insensitive to protein conformation changes, it can be measured with high accuracy by FCS and so can be useful for monitoring structural changes that occur as the protein passes among partially unfolded states. As the protein takes on more compact or more extended conformations, the hydrodynamic radius and consequently the diffusion coefficient change correspondingly; if these changes are large enough, they can be observed in FCS measurements. Among kinetics approaches used to study dynamics within the unfolded state, FCS is closely related to relaxation methods in which small free energy perturbations displace the equilibrium point of a reaction system. The kinetic parameters are deduced from the rate of relaxation to the new equilibrium.