Multiple time scale backbone dynamics of homologous thermophilic and mesophilic ribonuclease HI enzymes
Multiple time scale backbone dynamics of homologous thermophilic and mesophilic ribonuclease HI enzymes
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DOI：
10.1016/j.jmb.2004.03.055
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发表时间：
2004-06-11
影响因子：
5.6
通讯作者：
Palmer, AG III
中科院分区：
文献类型：
--
作者：
Butterwick, JA;Loria, JP;Palmer, AG III
使用N-15核磁自旋弛豫对无半胱氨酸嗜热栖热菌核糖核酸酶HI突变体（ttRNH*）中多个时间尺度上的骨架构象波动进行定量。在310 K和静态磁场强度为11.7，14.1和18.8 T下获得的解析框架弛豫数据进行了分析，使用减少的谱密度映射和无模型的方法。在290-320 K温度范围内和14.1 T静磁场强度下，利用Hahn回波横向和多重量子弛豫数据表征了化学交换谱线展宽。ttRNH* 的结果进行了比较，以前发表的数据嗜温同系物，大肠杆菌核糖核酸酶HI（ecRNH）。对于ttRNH* 和ecRNH，皮秒至纳秒时间尺度上的分子内构象波动通常是相似的。β-链3和5以及富含甘氨酸的区域更刚性，而底物结合柄区域和C-末端尾部在ttRNH* 中比在ecRNH中更柔性。两条β链和位于中心β折叠周围沿着的富含甘氨酸的区域的刚性可能与嗜热酶的热力学稳定性增加有关。化学交换线加宽，反映微秒到毫秒的时间尺度构象变化，是更明显的ttRNH* 比ecRNH，特别是在手柄和周围的催化位点的残基。化学交换的温度依赖性表明，与ecRNH相比，手柄中ttRNH* 残基的表观活化能增加了约15 kJ/mol。增加的活化屏障，加上不存在于ecRNH中的α-螺旋B和C之间的运动，可能与嗜热酶在310 K下的催化活性降低有关。(C)2004爱思唯尔有限公司保留所有权利。
Backbone conformational fluctuations on multiple time scales in a cysteine-free Thermus thermophilus ribonuclease HI mutant (ttRNH*) are quantified using N-15 nuclear magnetic spin relaxation. Laboratory-frame relaxation data acquired at 310 K and at static magnetic field strengths of 11.7, 14.1 and 18.8 T are analysed using reduced spectral density mapping and model-free approaches. Chemical exchange line broadening is characterized using Hahn-echo transverse and multiple quantum relaxation data acquired over a temperature range of 290-320 K and at a static magnetic field strength of 14.1 T. Results for ttRNH* are compared to previously published data for a mesophilic homologue, Escherichia coli ribonuclease HI (ecRNH). Intramolecular conformational fluctuations on the pico-second-to-nanosecond time scale generally are similar for ttRNH* and ecRNH. beta-Strands 3 and 5 and the glycine-rich region are more rigid while the substrate-binding handle region and C-terminal tail are more flexible in ttRNH* than in ecRNH. Rigidity in the two beta-strands and the glycine-rich region, located along the periphery of the central beta-sheet, may be associated with the increased thermodynamic stability of the thermophilic enzyme. Chemical exchange line broadening, reflecting microsecond-to-millisecond time scale conformational changes, is more pronounced in ttRNH* than in ecRNH, particularly for residues in the handle and surrounding the catalytic site. The temperature dependence of chemical exchange show an increase of similar to15 kJ/mol in the apparent activation energies for ttRNH* residues in the handle compared to ecRNH. Increased activation barriers, coupled with motion between alpha-helices B and C not present in ecRNH, may be associated with the reduced catalytic activity of the thermophilic enzyme at 310 K. (C) 2004 Elsevier Ltd. All rights reserved.