Temperature and guanidine hydrochloride dependence of the structural stability of ribonuclease T1.
Temperature and guanidine hydrochloride dependence of the structural stability of ribonuclease T1.
复制标题
核糖核酸酶 T1 结构稳定性的温度和盐酸胍依赖性。
DOI：
10.1021/bi00160a033
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Freire,E
中科院分区：
文献类型：
--
作者：
PlazadelPino,IM;Pace,CN;Freire,E
摘要：采用高灵敏度差示扫描量热法研究了核糖核酸酶ti的热展开与温度、[GuHCl]和扫描速率的关系。GuHCl的失稳效应表明，随着转变温度的降低，展开转变的动力学变得非常缓慢。在pH为5.3和GuHCl为0时，展开跃迁的中心在59.1℃；随着GuHCl浓度的增加，转变发生在较低的温度下，并表现出逐渐变慢的动力学；因此，例如，在3 M GuHCl下，转变温度为40.6℃，其特征是时间常数接近10 min。在所有研究条件下（pH 5.3, pH 7.0,[GuHCl]< 3 M），转变是热力学可逆的。转变的缓慢动力学导致转变轮廓形状的显著扭曲，这可能被错误地解释为偏离两态机制。从量热数据中确定热力学参数需要开发一种明确包括热力学和转变动力学的分析形式。利用这一形式，表明两态慢动力学模型能够准确地描述核糖核酸酶的结构稳定性作为温度、GuHCl浓度和扫描速率的函数。利用多维量热数据估计了蛋白质稳定性的内在热力学参数、与GuHCl的相互作用参数、展开转变的时间常数及其温度依赖性。
Revised Manuscript Received September 4, 1992 abstract: The thermal unfolding of ribonuclease T i has been studiedby high-sensitivity differential scanning calorimetry as a function of temperature,[GuHCl], and scanning rate. The destabilizing effect of GuHCl has revealed that the kinetics of the unfolding transition become extremely slow as the transition temperature decreases. At pH 5.3 and zero GuHCl, the unfolding transition is centered at 59.1 C; upon increasing the GuHCl concentration, the transition occurs at lower temperatures and exhibits progressively slower kinetics; so, for example, at 3 M GuHCl, the transition temperature is 40.6 C and is characterized by a time constant close to 10 min. Under all conditions studied (pH 5.3, pH 7.0,[GuHCl]< 3 M), the transition is thermodynamically reversible. Theslow kinetics of the transition induce significant distortions in the shape of the transition profiles that can be mistakenly interpreted as deviations from a two-state mechanism.Determination of the thermodynamic parameters from the calorimetric data has required the development of an analytical formalism that explicitly includes the thermodynamics as well as the kinetics of the transition. Using this formalism, it is shown that a two-state slow-kinetics model is capable of accurately describing the structural stability of ribonuclease T] as a function of temperature, GuHCl concentration, and scanning rate. Multidimensional analysis of the calorimetric data has been used to estimate the intrinsic thermodynamic parameters for protein stability, the interaction parameters with GuHCl, and the time constant for the unfolding transition and its temperature dependence.