Panick G, Malessa R, Winter R, Rapp G, Frye K J, Royer C A
Department of Chemistry, University of Dortmund, Germany.
J Mol Biol. 1998 Jan 16;275(2):389-402. doi: 10.1006/jmbi.1997.1454.
The pressure-induced unfolding of wild-type staphylococcal nuclease (Snase WT) was studied using synchrotron X-ray small-angle scattering (SAXS) and Fourier-transform infrared (FT-IR) spectroscopy, which monitor changes in the tertiary and secondary structural properties of the protein upon pressurization. The experimental results reveal that application of high-pressure up to 3 kbar leads to an approximate twofold increase of the radius of gyration Rg of the native protein (Rg approximately 17 A) and a large broadening of the pair-distance-distribution function, indicating a transition from a globular to an ellipsoidal or extended chain structure. Analysis of the FT-IR amide I' spectral components reveals that the pressure-induced denaturation process sets in at 1.5 kbar at 25 degrees C and is accompanied by an increase in disordered and turn structures while the content of beta-sheets and alpha-helices drastically decreases. The pressure-induced denatured state above 3 kbar retains nonetheless some degree of beta-like secondary structure and the molecule cannot be described as a fully extended random coil. Temperature-induced denaturation involves a further unfolding of the protein molecule which is indicated by a larger Rg value and significantly lower fractional intensities of IR-bands associated with secondary-structure elements. In addition, we have carried out pressure-jump kinetics studies of the secondary-structural evolution and the degree of compactness in the folding/unfolding reactions of Snase. The effect of pressure on the kinetics arises from a larger positive activation volume for folding than for unfolding, and leads to a significant slowing down of the folding rate with increasing pressure. Moreover, the system becomes two-state under pressure. These properties make it ideal for probing multiple order parameters in order to compare the kinetics of changes in secondary structure by pressure-jump FT-IR and chain collapse by pressure-jump SAXS. After a pressure jump from 1 bar to 2.4 kbar at 20 degrees C, the radius of gyration increases in a first-order manner from 17 A to 22.4 A over a timescale of approximately 30 minutes. The increase in Rg value is caused by the formation of an extended (ellipsoidal) structure as indicated by the corresponding pair-distance-distribution function. Pressure-jump FT-IR studies reveal that the reversible first order changes in beta-sheet, alpha-helical and random structure occur on the same slow timescale as that observed for the scattering curves and for fluorescence. These studies indicate that the changes in secondary structure and chain compactness in the folding/unfolding reactions of Snase are probably dependent upon the same rate-limiting step as changes in tertiary structure.
利用同步辐射X射线小角散射（SAXS）和傅里叶变换红外（FT-IR）光谱研究了压力诱导的野生型葡萄球菌核酸酶（Snase WT）的去折叠过程，这两种技术可监测加压时蛋白质三级和二级结构性质的变化。实验结果表明，施加高达3千巴的高压会导致天然蛋白质的回转半径Rg（Rg约为17 Å）增加约两倍，并且对距分布函数大幅展宽，这表明从球状结构转变为椭球状或伸展链结构。对FT-IR酰胺I'光谱成分的分析表明，在25℃下，压力诱导的变性过程在1.5千巴时开始，同时无序结构和转角结构增加，而β-折叠和α-螺旋的含量急剧减少。然而，高于3千巴的压力诱导变性状态仍保留一定程度的β样二级结构，分子不能被描述为完全伸展的无规卷曲。温度诱导的变性涉及蛋白质分子的进一步去折叠，这由更大的Rg值和与二级结构元件相关的红外波段的显著更低的分数强度表明。此外，我们对Snase折叠/去折叠反应中的二级结构演化和紧密程度进行了压力跳跃动力学研究。压力对动力学的影响源于折叠的正活化体积大于去折叠的正活化体积，并且导致折叠速率随压力增加而显著减慢。此外，该系统在压力下变为两态。这些特性使其成为探测多个序参的理想选择，以便通过压力跳跃FT-IR比较二级结构变化的动力学和通过压力跳跃SAXS比较链塌陷的动力学。在20℃下从1巴压力跳跃到2.
