Seemann H, Winter R, Royer C A
Department of Chemistry, Physical Chemistry I, University of Dortmund, Otto-Hahn-Str. 6, Dortmund, D-44227, Germany.
J Mol Biol. 2001 Apr 6;307(4):1091-102. doi: 10.1006/jmbi.2001.4517.
We have characterized the temperature- and pressure-induced unfolding of staphylococcal nuclease (Snase) using high precision densitometric measurements. The changes in the apparent specific volume, expansion coefficient and isothermal compressibility were determined by these measurements. To our knowledge, these are the first measurements of the volume and isothermal compressibility 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 scanning calorimetry under the solution conditions which are used for the volumetric studies. We have seen that large compensating volume and compressibility effects accompany the temperature and pressure-induced protein unfolding. Measurements of the apparent specific volume and thermal expansion coefficient of Snase at ambient pressure indicate the formation of a pre-transitional, molten globule type of intermediate structure about 10 degrees C below 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 apparent specific volume of Snase at a number of different temperatures. At 45 degrees C we calculate a decrease in apparent specific volume due to pressure-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 transition to a partially unfolded state, which is consistent with our results obtained for the radius of gyration of the pressure-denatured state of Snase. At the lower temperature of 35 degrees C, a significant increase in compressibility around 30 MPa is indicative of the formation of a pressure-induced molten globule-like intermediate. Changes in the apparent volume, expansion coefficient and isothermal compressibility are discussed in terms of instrinsic, hydrational and thermal contributions accompanying the unfolding transition.
我们利用高精度密度测量法对葡萄球菌核酸酶（Snase）的温度和压力诱导去折叠过程进行了表征。通过这些测量确定了表观比容、膨胀系数和等温压缩率的变化。据我们所知，这些是对蛋白质压力诱导去折叠过程中体积和等温压缩率变化的首次测量。为了有助于解释Snase表观比容的温度和压力依赖性，我们还在用于体积研究的溶液条件下进行了差示扫描量热法。我们发现，温度和压力诱导的蛋白质去折叠伴随着巨大的补偿体积和压缩率效应。在常压下对Snase表观比容和热膨胀系数的测量表明，在比蛋白质实际去折叠温度低约10摄氏度时形成了一种预转变的、熔融球状的中间结构。与折叠态相比，去折叠蛋白质的表观比容小约0.3 - 0.5%。此外，我们研究了Snase在多个不同温度下表观比容的压力依赖性。在45摄氏度时，我们计算出由于压力诱导去折叠导致表观比容下降了 -3.3×10⁻³ cm³ g⁻¹ 或 -55 cm³ mol⁻¹。在40至70兆帕之间压缩率增加了三倍，这反映了向部分去折叠状态的转变，这与我们对Snase压力变性态回转半径的研究结果一致。在35摄氏度的较低温度下，在约30兆帕时压缩率显著增加，这表明形成了一种压力诱导的类似熔融球状的中间体。从去折叠转变伴随的内在、水化和热贡献方面讨论了表观体积、膨胀系数和等温压缩率的变化。
