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首页> 外文期刊>Journal of Molecular Biology >Conformational Preferences Underlying Reduced Activity of a Thermophilic Ribonuclease H
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Conformational Preferences Underlying Reduced Activity of a Thermophilic Ribonuclease H

机译：嗜热核糖核酸酶H的潜在活性的构象偏好

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摘要

The conformational basis for reduced activity of the thermophilic ribonuclease HI enzyme from Thermus thermophilus, compared to its mesophilic homolog from Escherichia coli, is elucidated using a combination of NMR spectroscopy and molecular dynamics (MD) simulations. Explicit-solvent all-atom MD simulations of the two wild-type proteins and an E. coil mutant in which a glycine residue is inserted after position 80 to mimic the T. thermophilus protein reproduce the differences in conformational dynamics determined from N-15 spinrelaxation NMR spectroscopy of three loop regions that surround the active site and contain functionally important residues: the glycine-rich region, the handle region, and the beta(5)/alpha(E) loop. Examination of the MD trajectories indicates that the thermophilic protein samples conformations productive for substrate binding and activity less frequently than the mesophilic enzyme, although these differences may manifest as either increased or decreased relative flexibility of the different regions. Additional MD simulations indicate that mutations increasing activity of the T. thermophilus enzyme at mesophilic temperatures do so by reconfiguring the local environments of the mutated sites to more closely resemble active conformations. Taken together, the results show that both locally increased and decreased flexibility contribute to an overall reduction in activity of T. thermophilus ribonuclease H compared to its mesophilic E. coil homolog. (C) 2014 Elsevier Ltd. All rights reserved.
机译：使用NMR光谱和分子动力学（MD）模拟的组合，阐明了与热嗜热素从热嗜热嗜热素中减少热药核糖核酸酶Hi酶活性的构象基础。两种野生型蛋白质和E.的明确溶剂全原子MD模拟和E.线圈突变体，其中将甘氨酸残基插入甘氨酸残基以模仿T.嗜热蛋白质再现由N-15旋转的构象动态的差异围绕活性部位的三个环区域的NMR光谱谱系，并含有功能重要的残基：富含甘氨酸的区域，手柄区域和β（5）/α（E）环。对MD轨迹的检查表明，嗜热蛋白样品的样品兼容底物结合和常见的脂肪酶的活性，尽管这些差异可能表现为增加或降低不同区域的相对柔韧性。额外的MD模拟表明，通过重新配置突变位点的局部环境以更紧密地类似于主动构象的局部环境，突变增加了嗜热温度的活性。结果表明，与其嗜培素的E.线圈同源物相比，局部增加和灵活性降低有助于T.Mircophilus Ribonuclease H的总体活性。 （c）2014年elestvier有限公司保留所有权利。

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