Sasahara K, Demura M, Nitta K
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan.
Biochemistry. 2000 May 30;39(21):6475-82. doi: 10.1021/bi992560k.
Equilibrium unfolding of hen egg white lysozyme as a function of GdnCl concentration at pH 0.9 was studied over a temperature range 268.2-303.2 K by means of CD spectroscopy. As monitored by far- and near-UV CD at 222 and 289 nm, the lack of coincidence between two unfolding transition curves was observed, which suggests the existence of a third conformational species in addition to native and unfolded states. The three-state model, in which a stable intermediate is populated, was employed to estimate the thermodynamic parameters for the GdnCl-induced unfolding. It was found that the transition from the native to intermediate states proceeds with significant changes in enthalpy and entropy due to an extremely cooperative process, while the transition from the intermediate to unfolded states shows a low cooperativity with small enthalpy and entropy changes. These results indicate that the highest energy barrier for the GdnCl-induced unfolding of hen lysozyme is located in the process from the native state to the intermediate state, and this process is largely responsible for the cooperativity of protein unfolding.
通过圆二色光谱法，在268.2 - 303.2 K的温度范围内研究了pH值为0.9时，蛋清溶菌酶在不同浓度盐酸胍（GdnCl）作用下的平衡去折叠过程。通过在222和289 nm处的远紫外和近紫外圆二色光谱监测发现，两条去折叠转变曲线不重合，这表明除了天然态和去折叠态之外，还存在第三种构象状态。采用包含稳定中间体的三态模型来估算GdnCl诱导去折叠的热力学参数。研究发现，从天然态到中间体状态的转变过程中，由于一个极其协同的过程，焓和熵发生了显著变化；而从中间体状态到去折叠态的转变协同性较低，焓和熵的变化较小。这些结果表明，GdnCl诱导蛋清溶菌酶去折叠的最高能量屏障位于从天然态到中间体状态的过程中，并且这一过程在很大程度上决定了蛋白质去折叠的协同性。
