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首页> 外文期刊>Journal of Molecular Biology >Conformation changes in E. coli Rho monitored by hydrogen/deuterium exchange and mass spectrometry: response to ligand binding.
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Conformation changes in E. coli Rho monitored by hydrogen/deuterium exchange and mass spectrometry: response to ligand binding.

机译：通过氢/氘交换和质谱监测的大肠杆菌Rho中的构型变化：对配体结合的反应。

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Escherichia coli Rho is a doughnut-shaped homohexameric ATP-dependent RNA-DNA helicase that releases newly synthesized RNA molecules from transcription complexes. Rho binds 60-80 bases of RNA among six primary RNA binding sites around the inside of its N-terminal crown; the RNA then passes through the central hole of the hexamer. Here it triggers ATP hydrolysis and is moved with respect to the protein. We study protein conformation changes upon ligand binding using amide proton hydrogen/deuterium exchange and mass spectrometry. Global-exchange studies indicate net mass differences of about 15 Da after 1 h of exchange in the presence--versus in the absence--of the ligand MgATP or the RNA poly(C). Sites of ligand-dependent exchange differences were localized by mass determination of the peptic peptides of Rho. A peptide of the N-terminal domain near the known primary RNA sites (aa 56-63) was protected from amide proton exchange in the presence of poly(C), as was a novel N-terminal domain peptide that is not near RNA in the crystal structures or in NMR structures with RNA oligomers (aa 37-46). This result may further define the primary interaction site of RNA with Rho. The Q-loop-containing peptide in the central hole of the protein that interacts with RNA was also protected by RNA (aa 271-286). The exchange rate of one peptide near the ATPase active site (aa 206-218) slowed in the presence of MgATP and increased in the presence of RNA. Overall, the results show changes in a few protein segments rather than a different overall conformation.
机译：大肠杆菌Rho是一种甜甜圈形的同六聚体ATP依赖性RNA-DNA解旋酶，可从转录复合物中释放出新合成的RNA分子。 Rho在其N末端冠的内部周围的六个主要RNA结合位点中结合60-80个RNA碱基。然后，RNA通过六聚体的中心孔。在这里，它触发ATP水解并相对于蛋白质移动。我们使用酰胺质子氢/氘交换和质谱研究配体结合后的蛋白质构象变化。全球交换研究表明，在存在配体MgATP或RNA poly（C）的情况下（相对于不存在的情况下）交换1小时后，净质量差异约为15 Da。通过质量测定Rho的消化肽来定位依赖配体的交换差异的位点。在poly（C）的存在下，已知的主要RNA位点附近的N末端结构域的肽段（aa 56-63）可以防止酰胺质子交换，而在RNA中不靠近RNA的新型N末端结构域的肽段RNA低聚物（aa 37-46）的晶体结构或NMR结构。该结果可以进一步定义RNA与Rho的主要相互作用位点。与RNA相互作用的蛋白质中心孔中含有Q环的肽也受到RNA的保护（aa 271-286）。在存在MgATP的情况下，靠近ATPase活性位点（aa 206-218）的一种肽的交换速率变慢，而在RNA的存在下增加。总体而言，结果显示了一些蛋白质片段的变化，而不是不同的整体构象。

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