Mandel A M, Akke M, Palmer A G
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032, USA.
Biochemistry. 1996 Dec 17;35(50):16009-23. doi: 10.1021/bi962089k.
The temperature dependence of the backbone motions in Escherichia coli ribonuclease HI was studied on multiple time scales by 15N nuclear magnetic spin relaxation. Laboratory frame relaxation data at 285, 300, and 310 K were analyzed using the model-free and reduced spectral density approaches. The temperature dependence of the order parameters was used to define a characteristic temperature for the motions of the backbone N-H bond vectors on picosecond to nanosecond time scales. The characteristic temperatures for secondary structure elements, loops, and the C-terminus are approximately 1000, approximately 300, and approximately 170 K, respectively. The observed variation in the characteristic temperature indicates that the energy landscape, and thus the configurational heat capacity, is markedly structure dependent in the folded protein. The effective correlation times for internal motions do not show significant temperature dependence. Conformational exchange was observed for a large number of residues forming a contiguous region of the protein that includes the coiled coil formed by helices alpha A and alpha D. Exchange broadening in the CPMG experiments decreased with increased temperature, directly demonstrating that the microscopic exchange rate is faster than the pulse repetition rate of 1.2 ms. The temperature dependence of the exchange contributions to the transverse relaxation rate constant shows approximately Arrhenius behavior over the studied temperature range with apparent activation enthalpies of approximately 20-50 kJ/mol. Numerical calculations suggest that these values underestimate the activation barriers by at most a factor of 2. The present results obtained at 300 K are compared to those reported previously [Mandel, A. M., Akke, M., & Palmer, A. G., III (1995) J. Mol. Biol. 246, 144-163] to establish the reproducibility of the experimental techniques.
通过15N核磁共振自旋弛豫在多个时间尺度上研究了大肠杆菌核糖核酸酶HI主链运动的温度依赖性。使用无模型和简化谱密度方法分析了285、300和310 K下实验室坐标系的弛豫数据。序参数的温度依赖性用于定义皮秒到纳秒时间尺度上主链N-H键向量运动的特征温度。二级结构元件、环和C端的特征温度分别约为1000 K、约300 K和约170 K。观察到的特征温度变化表明，在折叠蛋白中，能量景观以及构型热容量明显依赖于结构。内部运动的有效相关时间没有显示出明显的温度依赖性。在形成蛋白质连续区域的大量残基中观察到构象交换，该区域包括由αA和αD螺旋形成的卷曲螺旋。CPMG实验中的交换展宽随温度升高而降低，直接证明微观交换速率快于1.2 ms的脉冲重复速率。交换对横向弛豫速率常数贡献的温度依赖性在研究的温度范围内显示出近似阿仑尼乌斯行为，表观活化焓约为20 - 50 kJ/mol。数值计算表明，这些值最多低估活化势垒2倍。将300 K下获得的当前结果与先前报道的结果[Mandel, A. M., Akke, M., & Palmer, A. G., III (1995) J. Mol. Biol. 246, 144 - 163]进行比较，以确定实验技术的可重复性。
