Volumetric and spectroscopic characterizations of the native and acid-induced denatured states of staphylococcal nuclease

We have characterized the acid-induced denaturation of staphylococcal nucle ase (SNase) at different urea concentrations by a combination of ultrasonic velocimetry, high precision densimetry, and CD spectroscopy. Our CD spectr oscopic results suggest that, at low salt and acidic pH, the protein is unf olded with disrupted secondary and tertiary structures. Furthermore, as jud ged by far UV CD spectra, the protein is further unfolded at acidic pH upon the addition of urea up to the concentration of 1.5 M. The midpoint of the transition shifts to more neutral pH values and the cooperativity of the t ransition decreases as the acid-induced denaturation of SNase occurs at hig her urea concentrations. We find that the change in volume, Delta v, accomp anying the acid-induced denaturation of SNase increases from -0.013 cm(3) g (-1) (-218 cm(3) mol(-1)) in the absence of urea to 0.011 cm(3) g(-1) (185 cm(3) mol(-1)) at 1.5 M urea. At all urea concentrations, the partial speci fic adiabatic compressibility, k(s)(o), of the protein decreases upon its u nfolding with the values of Delta k(s)(o) equal to -6.3 x 10(-6) (-0.106 cm (3) mol(-1) bar(-1)), -4.5 x 10(-6) (-0.076 cm(3) mol(-1) bar(-1)), -4.6 x 10(-6) (-0.077 cm(3) mol(-1) bar(-1)), and -3.8 x 10(-6) (-0.064 cm(3) mol( -1) bar(-1)) cm(3) g(-1) bar(-1) at urea concentrations of 0, 0.5, 1.0, and 1.5 M, respectively. In general, our volumetric results suggest that the a cid-induced denatured state of SNase is only partially unfolded with the so lvent-exposed surface area Equal to 70-80% of that expected for the fully e xtended conformation. (C) 2000 Academic Press.