EFFECTS OF AMINO-ACID SUBSTITUTIONS ON THE PRESSURE DENATURATION OF STAPHYLOCOCCAL NUCLEASE AS MONITORED BY FLUORESCENCE AND NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY

In the present study we have used high hydrostatic pressure coupled wi th either time-resolved and steady-state fluorescence or NMR spectrosc opy in order to investigate the effects of amino acid substitutions on the high-pressure denaturation properties of staphylococcal nuclease. This protein has been shown previously to be structurally heterogeneo us in its native state. On the NMR time scale, four distinct interconv erting conformational forms arise from the population of both cis and trans Xaa-Pro peptide bonds (His46-Pro47 and Lys116-Pro117) [Evans et al. (1989) Biochemistry 28, 362; Loh et al. (1991) in Techniques in Pr otein Chemistry II, pp 275-282, Academic Press, New York]. Mutations i n the protein sequence have been shown to change the distribution amon g the various forms [Alexandrescu et al. (1989) Biochemistry 28, 204; Alexandrescu et al. (1990) Biochemistry 29, 4516]. Time-resolved fluor escence on a series of mutants with altered equilibria for cis/trans i somerism about the 116-117 peptide bond did not reveal any simple rela tionship between the position of the cis/trans equilibrium in the fold ed state and the heterogeneity of the fluorescence decay. However, the specific dynamic properties of each mutant, as revealed by time-resol ved fluorescence, do appear to be correlated with their partial molar volume changes of denaturation. A striking finding is that mutation of either (or both) of the prolines that exhibits structural heterogenei ty to glycine greatly alters the stability of the protein to pressure. These mutations also result in decreased chain mobility as assessed b y time-resolved fluorescence. It appears that packing defects, which a llow for peptide bond cis/trans heterogeneity in the wild-type protein , are removed by the Pro --> Gly substitutions.