A novel method for measuring the effect of volume excluding polymers on the kinetics of refolding of an enzymatically active protein has been developed. Carbonic anhydrase, a small single-subunit protein containing no sulfhydryl crosslinks is denatured in 5 M guanidine chloride. Refolding is initiated by dilution into 0.5 M urea containing a small molecule substrate that develops color upon being proteolyzed by enzymatically active protein. Using a spectrophotometric plate reader, parallel measurements are made of the time course of the development of absorbance in solutions containing no enzyme, native enzyme (incubated in 0.5 M GdnCl rather than 5 M GdnCl) and refolding enzyme. Global analysis of the three absorbance versus time curves yields information about the fraction of enzymatic activity recovered as a function of time in the initially denatured protein. These experiments were conducted in buffer and in the presence of various concentrations of Ficoll 70, a crosslinked polymer of sucrose. Initial analysis of the data indicates that increasing concentrations of Ficoll significantly inhibit recovery of enzymatic activity, and we are attempting to discern the mechanistic basis of this effect (Monterroso, Minton).? ? Additional applications are being developed for the light scattering apparatus developed in our laboratory and described in previous annual reports. A hybrid system that can perform both size-exclusion chromatography and composition gradient experiments has been constructed and is being currently tested (Fernandez). The option of using a differential refractometer in place of an absorbance monitor as an indicator of solution composition has been added and measurements aimed at validating changes in both hardware and data processing software have been completed (Liang, Minton). Application of the new instrumentation to the measurement of protein-protein interactions is in progress.? ? A novel laser light scattering apparatus has been constructed specifically for the measurement of very small changes in Rayleigh scattering. This apparatus is intended for use in characterization of initial stages of the formation of amyloid or other pathological protein aggregates (Minton, McPhie, Fernandez).? ? A number of previously published Brownian dynamics simulations and a set of elegant experiments carried out by M. Hayer-Hartl and colleagues at the Max Planck Institute for Biochemistry have indicated that confinement of a protein within a cavity substantially affects the rate of refolding in a bimodal fashion: when the confining cavity is large relative to the size of the native protein, refolding is accelerated, but when the confining cavity is only slighly larger than the native protein, refolding is decelerated. A simple model based upon the differential effect of confinement upon the entropy of the native and unfolded configurations of the protein has been proposed (Hayer-Hartl, Minton).
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