Corbett R J, Ahmad F, Roche R S
Biochem Cell Biol. 1986 Oct;64(10):953-61. doi: 10.1139/o86-127.
Equilibrium and kinetic studies of the unfolding and autolysis of the two domain protein thermolysin in guanidine hydrochloride are described. Enzyme activity, circular dichroism, fluorescence, sedimentation, size exclusion chromatography, and viscosity measurements were used to monitor conformational transitions and characterize the native and denatured states. The observation of biphasic transitions for the unfolding of apothermolysin and the spectroscopic changes associated with each phase of the overall unfolding process suggest unfolding of the N-terminal domain at less than 1 M guanidine hydrochloride, followed by the unfolding of the C-terminal domain, with the transition midpoint at 3 M guanidine hydrochloride. The refolding of the C-terminal domain is reversible; however, refolding of the N-terminal domain could not be demonstrated owing to protein aggregation. A quantitative analysis of the two transitions suggest that the unfolding of the two structural domains of thermolysin is not completely independent. Attempts to measure the unfolding of holothermolysin were hampered by autolysis. However, it was possible to show that at least three calcium ions serve to stabilize thermolysin against autolysis or unfolding in guanidine hydrochloride. Similar stabilization was observed for thermolysin with a single terbium ion bound at calcium site S(1). This result is consistent with our earlier findings, which suggest that calcium bound at sites S(1)-S(2) are located at a critical point on the unfolding pathway of thermolysin and serve to act as an interdomain lock.
本文描述了在盐酸胍中双结构域蛋白嗜热菌蛋白酶的去折叠和自溶的平衡及动力学研究。使用酶活性、圆二色性、荧光、沉降、尺寸排阻色谱和粘度测量来监测构象转变，并表征天然态和变性态。脱辅基嗜热菌蛋白酶去折叠的双相转变以及与整体去折叠过程各阶段相关的光谱变化表明，在盐酸胍浓度低于1 M时N端结构域去折叠，随后C端结构域去折叠，转变中点在盐酸胍浓度为3 M时。C端结构域的重折叠是可逆的；然而，由于蛋白质聚集，无法证明N端结构域的重折叠。对这两个转变的定量分析表明，嗜热菌蛋白酶的两个结构域的去折叠并非完全独立。测量全嗜热菌蛋白酶去折叠的尝试因自溶而受阻。然而，有可能表明至少三个钙离子有助于稳定嗜热菌蛋白酶以防止其在盐酸胍中自溶或去折叠。在钙位点S(1)结合单个铽离子的嗜热菌蛋白酶也观察到类似的稳定作用。这一结果与我们早期的发现一致，即结合在S(1)-S(2)位点的钙位于嗜热菌蛋白酶去折叠途径的关键点上，并起到结构域间锁的作用。
