Physical properties of the glycoprotein of vesicular stomatitis virus measured by intrinsic fluorescence and aggregation.

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We have studied the denaturation and renaturation of the purified glycoprotein (G) of vesicular stomatitis virus by using intrinsic fluorescence spectroscopy and an aggregation assay. Our studies were carried out with G containing two complex oligosaccharide chains, with the asialo form of the protein, and for some experiments with G containing altered oligosaccharide structures. Fluorescence quenching using acrylamide showed no differences between the native and denatured states of G due to sialic acid content. Denaturation by guanidinium chloride (GdmCl) at 25 degrees C was reversible for the major transition region. The data analyzed by a two-state denaturation model gave a free energy of unfolding in the absence of denaturant of approximately 1.4 kcal/mol. For renaturation, two types of dialysis protocols were employed. The first (direct dialysis) involved dialysis against standard buffer [140 mM NaCl, 10 mM sodium phosphate, 1 mM disodium ethylenediaminetetraacetate, and 0.2% (w/v) poly(oxyethylene) 10-tridecyl ether, pH 7.4]. Recovery of the native emission maximum did not occur for any of the G proteins by using this procedure. The second (annealing dialysis) involved slow removal of GdmCl against decreasing concentrations of GdmCl in standard buffer over a period of 2-3 days. Only in this case was recovery of the native emission maximum and fluorescence intensity obtained. For those G proteins in which the oligosaccharide chains were decreased in size, this protocol led to extensive aggregation.