Maity Haripada, Eftink Maurice R
Department of Chemistry and Biochemistry, University of Mississippi, University, MS 38677, USA.
Arch Biochem Biophys. 2004 Nov 1;431(1):119-23. doi: 10.1016/j.abb.2004.07.023.
The sodium perchlorate-induced conformational transition of Staphylococcal nuclease has been monitored by both circular dichroism (CD) and fluorescence spectroscopy. The perchlorate-induced transition is cooperative as observed by both spectroscopic signals. However, the protein loses only about one-third of its native far-UV CD signal at high perchlorate concentrations, indicating that a significant amount of secondary structure remains in the post-transition state. The remaining CD signal can be further diminished in a cooperative manner by the addition of the strong denaturant, urea. Near-UV CD spectra clearly show that the protein loses its tertiary structure in the perchlorate-induced denatured state. The perchlorate-induced transition curves were fit to the standard two-state model and the standard free energy change and m value of the transition are 2.3kcal/mol and 1.8kcal/(molM), respectively. By comparison, the urea-induced unfolding of Staphylococcal nuclease (in the absence of perchlorate) yields an unfolding free energy change, DeltaG(0,un), of 5.6kcal/mol and an m value of 2.3kcal/(molM). Thus, the thermodynamic state obtained in the post-transition region of perchlorate-induced conformation transition has a significantly lower free energy change, a high content of secondary structure, and diminished tertiary structure. These results suggest that the perchlorate-induced denatured state is a partially folded equilibrium state. Whether this intermediate is relevant to the folding/unfolding path under standard conditions is unknown at this time.
高氯酸钠诱导的葡萄球菌核酸酶构象转变已通过圆二色性（CD）和荧光光谱进行监测。如两种光谱信号所观察到的，高氯酸盐诱导的转变是协同的。然而，在高氯酸盐浓度较高时，该蛋白质仅失去其天然远紫外CD信号的约三分之一，这表明在转变后的状态中仍保留大量二级结构。通过添加强变性剂尿素，剩余的CD信号可以以协同方式进一步减弱。近紫外CD光谱清楚地表明，该蛋白质在高氯酸盐诱导的变性状态下失去其三级结构。高氯酸盐诱导的转变曲线符合标准的两态模型，转变的标准自由能变化和m值分别为2.3kcal/mol和1.8kcal/(mol·M)。相比之下，尿素诱导的葡萄球菌核酸酶（在不存在高氯酸盐的情况下）展开产生的展开自由能变化ΔG(0,un)为5.6kcal/mol，m值为2.3kcal/(mol·M)。因此，在高氯酸盐诱导的构象转变的转变后区域中获得的热力学状态具有明显更低的自由能变化、高含量的二级结构和减弱的三级结构。这些结果表明，高氯酸盐诱导的变性状态是一种部分折叠的平衡状态。目前尚不清楚这种中间体是否与标准条件下的折叠/展开路径相关。
