Conformation changes in E. coli Rho monitored by hydrogen/deuterium exchange and mass spectrometry: response to ligand binding.
Conformation changes in E. coli Rho monitored by hydrogen/deuterium exchange and mass spectrometry: response to ligand binding.
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通过氢/氘交换和质谱法监测大肠杆菌 Rho 的构象变化：对配体结合的响应。
DOI：
10.1016/j.jmb.2010.08.004
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发表时间：
2010
影响因子：
5.6
通讯作者：
Xiao,Hui
中科院分区：
文献类型：
--
作者：
Stitt,BarbaraL;Xiao,Hui
大肠杆菌Rho是一种甜甜圈形状的依赖于ATP的同源六聚体RNA-DNA解旋酶，它能从转录复合体中释放新合成的RNA分子。Rho在其N-末端冠状内侧的六个主要RNA结合位点中结合60-80个碱基的RNA；然后RNA通过六聚体的中心孔。在这里，它触发三磷酸腺苷的水解，并相对于蛋白质移动。我们利用酰胺、质子、氢/氢交换和质谱学研究了蛋白质在配体结合时的构象变化。全球交换研究表明，在存在配体镁ATP或RNA聚(C)的情况下，交换1小时后的净质量差异约为15Da。通过对Rho的多肽进行质量测定，确定了配体依赖的交换差异的位置。在聚(C)的存在下，N-末端结构域的已知初级RNA位点附近的多肽(AA-56-63)被保护而不被酰胺质子交换，因为它是一种新的N-末端结构域的多肽，它在晶体结构中或在与RNA低聚体(AA-37-46)的核磁共振结构中不靠近RNA。这一结果可能进一步确定了RNA与Rho的主要相互作用部位。与RNA相互作用的蛋白质中央孔洞中含有Q-环的多肽也受到RNA的保护(AA-271-286)。靠近ATPase活性部位的一个肽(AA-206-218)的交换速率在MgATP存在下减慢，而在RNA存在下增加。总体而言，结果显示了几个蛋白质片段的变化，而不是不同的整体构象。
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.