Conformational Studies on a Glycosylated Bovine Pancreatic Ribonuclease

Abstract

Abstract Various spectral properties (circular dichroism, optical rotatory dispersion, and ultraviolet denaturation difference spectroscopy) of a glycosylated bovine pancreatic ribonuclease (RNase B') have been measured. This glycoprotein contains an average of 1 mole of glucosamine and 2 to 3 moles of mannose per mole of protein. The amino acid composition of RNase B' is identical with that of ribonuclease A (RNase A), and the amino acid sequence of the two proteins is believed to be identical. The CD spectrum of RNase B' was resolved into Gaussian bands, and assignments were made to optically active side chain chromophores and peptide groups. The rotational strength of each transition was determined and found to be essentially the same as the corresponding value for RNase A. The ORD spectrum of RNase B' was also virtually identical with that of RNase A. The denaturation difference spectrum indicated about three buried tyrosyl groups in RNase B', in agreement with the structure and spectrum of RNase A. These spectral results, which reflect different portions of the protein, strongly suggest that the secondary and tertiary structure of the glycoprotein is identical with that of the nonglycosylated protein, RNase A. Consequently, the carbohydrate moiety has no effect on the conformation of the protein. Kinetic studies of unfolding and refolding also indicated no appreciable effect of the oligosaccharide on the rate constants. Evidence is presented which shows that RNase B' unfolds and refolds in guanidine hydrochloride at neutral pH via the two-state mechanism, as does RNase A. Thus, the equilibrium spectral parameters, measured at various denaturant concentrations, could be converted to the free energy for the reaction, native protein → unfolded protein, which does not involve an appreciable concentration of stable intermediates. From these data, the free energy of unfolding in the absence of denaturant, ΔG0, was obtained by means of various extrapolation methods. The average value found for ΔG0 was 15.3 Cal per mole, which is only several hundred calories per mole greater than that of RNase A, i.e. the carbohydrate moiety has only a small stabilizing effect on the protein. The acid-induced unfolding of RNase B, like that of RNase A, was quite complicated and difficult to interpret quantitatively. The data do, however, suggest that several masked carboxyl groups are responsible for the acid transition. Herein, we have emphasized how spectral data, in particular near ultraviolet CD spectroscopy, can be quantitatively analyzed to permit very detailed comparisons on the conformation and stability of similar proteins.