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 with either time-resolved and steady-state fluorescence or NMR spectroscopy 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 heterogeneous in its native state. On the NMR time scale, four distinct interconverting 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 Protein Chemistry II, pp 275-282, Academic Press, New York]. Mutations in the protein sequence have been shown to change the distribution among the various forms [Alexandrescu et al. (1989) Biochemistry 28, 204; Alexandrescu et al. (1990) Biochemistry 29, 4516]. Time-resolved fluorescence on a series of mutants with altered equilibria for cis/trans isomerism about the 116-117 peptide bond did not reveal any simple relationship between the position of the cis/trans equilibrium in the folded state and the heterogeneity of the fluorescence decay. However, the specific dynamic properties of each mutant, as revealed by time-resolved 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 heterogeneity to glycine greatly alters the stability of the protein to pressure. These mutations also result in decreased chain mobility as assessed by time-resolved fluorescence. It appears that packing defects, which allow for peptide bond cis/trans heterogeneity in the wild-type protein, are removed by the Pro-->Gly substitutions.
Royer
C A
CA
School of Pharmacy, University of Wisconsin-Madison 53706.
Hinck
A P
AP
Loh
S N
SN
Prehoda
K E
KE
Peng
X
X
Jonas
J
J
Markley
J L
JL
D000595:Amino Acid Sequence; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008836:Micrococcal Nuclease / Q000737:chemistry*; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011312:Pressure; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D008836:Micrococcal Nuclease