Volume, expansivity and isothermal compressibility changes associated withtemperature and pressure unfolding of staphylococcal nuclease

We have characterized the temperature- and pressure-induced unfolding of st aphylococcal nuclease (Snase) using high precision densitometric measuremen ts. The changes in the apparent specific volume, expansion coefficient and isothermal compressibility were determined by these measurements. To our kn owledge, these are the first measurements of the volume and isothermal comp ressibility changes of a protein undergoing pressure-induced unfolding. In order to aid in interpreting the temperature and pressure dependence of the apparent specific volume of Snase, we have also carried out differential s canning calorimetry under the solution conditions which are used for the vo lumetric studies. We have seen that large compensating volume and compressi bility effects accompany the temperature and pressure-induced protein unfol ding. Measurements of the apparent specific volume and thermal expansion co efficient of Snase at ambient pressure indicate the formation of a pretrans itional, molten globule type of intermediate structure about 10 degreesC be low the actual unfolding temperature of the protein. Compared to the folded state, the apparent specific volume of the unfolded protein is about 0.3-0 .5 % smaller. In addition, we investigated the pressure dependence of the a pparent specific volume of Snase at a number of different temperatures. At 45 degreesC we calculate a decrease in apparent specific volume due to pres sure-induced unfolding of -3.3 10(-3) cm(3) g(-1) or -55 cm(3) mol(-1). The threefold increase in compressibility between 40 and 70 MPa reflects a tra nsition to a partially unfolded state, which is consistent with our results obtained for the radius of gyration of the pressure-denatured state of Sna se. At the lower temperature of 35 degreesC, a significant increase in comp ressibility around 30 MPa is indicative of the formation of a pressure-indu ced molten globule-like intermediate. Changes in the apparent volume, expan sion coefficient and isothermal compressibility are discussed in terms of i nstrinsic, hydrational and thermal contributions accompanying the unfolding transition. (C) 2001 Academic Press.