Selective neoglycosylation increases the structural stability of vicilin, the 7S storage globulin from pea seeds

The effects of glycosylation on the stability and subunit interactions of v icilin, the major storage protein in pea seeds, were investigated. Glycosyl ated vicilin derivatives were prepared by alkylation of lysine epsilon -ami no groups with various carbohydrates. Average modification levels of 13.4 /- 3.0, 11.1 +/- 3.6, 7.5 +/- 4.2, and 4.7 +/- 0.3 moles of carbohydrate/mo l of vicilin were obtained with glucose, galactose, galacturonic acid, and lactose, respectively. Nondenaturing polyacrylamide gel electrophoresis and size-exclusion chromatography indicated that the quaternary structure and hydrodynamic radius of vicilin were not affected by glycosylation at the le vels used. We have previously shown that application of hydrostatic pressur e causes dissociation of vicilin subunits [C. Pedrosa and S. T. Ferreira (1 994) Biochemistry 33, 4046-4055]. Analysis of pressure dissociation data al lowed determination of the Gibbs free energy change (DeltaG(diss)) and mola r volume change (DeltaV(diss)) of dissociation of vicilin subunits. For unm odified vicilin, DeltaG(diss) = 18.2 kcal/mol and DeltaV(diss) = -102 ml/mo l. Glycosylated vicilin derivatives were significantly stabilized against s ubunit dissociation, with DeltaG(diss) of 19.4, 19.2, 20.6, and 22.1 kcal/m ol for glucose, galactose, lactose, and galacturonic acid derivatives, resp ectively. No changes in DeltaV(diss) were found for the glucose and galacto se derivatives, whereas DeltaV(diss) of -128 and -135 ml/mol, respectively, were found for the lactose and galacturonic acid derivatives. The glycosyl ated derivatives also appeared more resistant to unfolding by guanidine hyd rochloride than unmodified vicilin. Intrinsic fluorescence lifetime measure ments showed that glycosylation caused a significant increase in heterogene ity of the fluorescence decay, possibly reflecting increased conformational heterogeneity of glycosylated derivatives relative to unmodified vicilin. These results indicate that the stability and subunit interactions of vicil in may be modulated by mild, selective glycosylation at low modification le vels, an effect that may be of interest in the study of other oligomeric pr oteins. (C) 2000 Academic Press.