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

Abstract

We have characterized the acid-induced denaturation of staphylococcal nuclease (SNase) at different urea concentrations by a combination of ultrasonic velocimetry, high precision densimetry, and CD spectroscopy. Our CD spectroscopic results suggest that, at low salt and acidic pH, the protein is unfolded with disrupted secondary and tertiary structures. Furthermore, as judged 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 transition decreases as the acid-induced denaturation of SNase occurs at higher urea concentrations. We find that the change in volume, Δν, accompanying the acid-induced denaturation of SNase increases from −0.013 cm3 g−1 (−218 cm3 mol−1) in the absence of urea to 0.011 cm3 g−1 (185 cm3 mol−1) at 1.5 M urea. At all urea concentrations, the partial specific adiabatic compressibility, k°s, of the protein decreases upon its unfolding with the values of Δk°s equal to −6.3×10−6 (−0.106 cm3 mol−1 bar−1), −4.5×10−6 (−0.076 cm3 mol−1 bar−1), −4.6×10−6 (−0.077 cm3 mol−1 bar−1), and −3.8×10−6 (−0.064 cm3 mol−1 bar−1) cm3 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 acid-induced denatured state of SNase is only partially unfolded with the solvent-exposed surface area equal to 70–80 % of that expected for the fully extended conformation.