Talk at the 40th annual meeting of the Biopysical Society . Abstract: Biophys. J. 70, A262 The technique of fluorescence correlation spectroscopy (FCS, originally proposed in ref.1) has been utilized to determine the hydrodynamic radii of protein molecules as a function of denaturant concentration. Measuring the size of proteins by FCS has the advantage that a very small amount of protein (<1 picomole ) in a dilute solution (a few nM) is sufficient for the experiment and that the measurements are insensitive to the presence of non-fluorescent impurities. FCS supplies a wealth of information on diffusion and chemical kinetics, but the experimental requiremnts of having a small and open probe-volume, low background, minimal system fluctuations and the ability to efficiently compute and repeatedly average mathematical transforms are rather stringent. Exploiting the advantages offered by two-photon induced fluorescence, we have developed a completely automated FCS apparatus that easily meets these requirements. With this apparatus, the diffusion constants of a few fluorescent and fluorescently labeled proteins (e.g. R-Phycoerythrin and Adipocyte Lipid Binding Protein labeled with acrylodan) have been measured as a function of urea concentration. The characteristic decrease of the diffusion constant (i.e. increase of the hydrodynamic radius) associated with protein unfolding at higher denaturant concentrations has been observed. The possibility of also obtaining the kinetic constants of protein folding/unfolding from the FCS data is currently under investigation. We are deeply indebted to E. Kurian and F. G. Prendergast for their kind gift of Acrylodan labeled proteins.
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