Testing the correlation between delta A and delta V of protein unfolding using m value mutants of staphylococcal nuclease.
The application of hydrostatic pressure to aqueous protein solutions results in the unfolding of the protein structure because the protein-solvent system volume is smaller for the unfolded state. Contributions to this decrease in volume upon unfolding (delta Vu) derive from altered interactions of the protein with solvent and are presumed to include electrostriction of charged residues, elimination of packing defects, and hydration of hydrophobic surfaces upon unfolding. If the contribution of hydrophobic surface area solvation to the observed volume change of unfolding were large and negative, as is generally assumed, then one would expect to find a correlation between the amount of surface area exposed on unfolding, delta A(u), and the volume change, delta Vu. In order to test this correlation, we have determined delta Vu for two mutants of staphylococcal nuclease, A69T + A90S and H121P, whose unfolding by denaturant is, respectively, either significantly more (28%) or significantly less (28%) cooperative than that observed for wild-type (WT). This cooperativity coefficient or m value has been shown to correlate with delta A(u). If, in turn, delta Vu is correlated with delta A(u), we would expect the m+ mutant, A69T + A90S, to exhibit a delta Vu that is more negative than WT nuclease, while the delta Vu for the m- mutant, H121P, should be smaller in absolute value. To verify the correlation between m value and delta A(u) for these mutants, we determined the xylose concentration dependence of the stability of each mutant at atmospheric pressure and as a function of pressure. The efficiency of xylose stabilization was found to be much greater for the m+ mutant than for WT, consistent with an increase in delta A(u), while that of the m- mutant was found to be only slightly greater than for WT, indicating that other factors may contribute to the denaturant m value in this case. Regardless of the denaturant m value or the effect of xylose on stability, the volume changes upon unfolding for both mutants were found to be within error of that observed for WT. Thus, there does not appear to be a correlation between the volume change and the change in exposed surface area upon unfolding. We have previously shown a lack of pH dependence of the volume change, ruling out electrostriction as a dominant contribution to delta Vu of nuclease. These studies implicate either compensation between polar and nonpolar hydration or excluded volume effects as the major determinant for the value of delta Vu.
Frye
K J
KJ
School of Pharmacy, University of Wisconsin-Madison 53706, USA.
Perman
C S
CS
Royer
C A
CA
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004926:Escherichia coli; D006874:Hydrostatic Pressure; D007700:Kinetics; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008961:Models, Structural; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013816:Thermodynamics; D014994:Xylose
D011994:Recombinant Proteins; D014994:Xylose; D008836:Micrococcal Nuclease