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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Physical evidence for a phosphorylation-dependent conformational change in the enhancer-binding protein NtrC
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Physical evidence for a phosphorylation-dependent conformational change in the enhancer-binding protein NtrC

机译：增强子结合蛋白NtrC中磷酸化依赖性构象变化的物理证据

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摘要

The bacterial enhancer-binding protein nitro- gen regulatory protein C (NteC) activates transcription by #sigma#~(54)-containing RNA polymerase in a reaction that depends on ATP hydrolysis. Phosphorylation of an aspartate residue in the N-terminal receiver domain of NtrC induces oligomerization of the protein and activates the ATPase activity, which is a function of its central output domain. To study the role of the receiver domain of NtrC, which is known to act positively, we isolated mutant forms ofthe protein carrring single cysteine residues and derivatized them with a sulfhydryl-specific nitroxide reagent for electron paramagnetic resonance stridies. Single cysteines were placed at four positions at which we had obtained constitutive amino acid substitutions, those that yield activity without phos- phorylation. In only one case, derivatized C86 in #alpha#-helix 4 of the receiver domain, did the motion of the side chain become dramatically slower upon phosphorylation. Importantly, deri- vatized NtrC~(N86C) [NtrC~(D86C*)] activated transcription normally. Additional experiments indicated that the spectral change ob- served upon phosphorylation of NtrC~(D86C*) was due to interdo- main interactions rather than a conformational change within the N-terminal domain itself. These interactions did not appear to occur within a monomer. Although it is not clear whether the spectral change seen upon phosphorylation of NtrC~(D86C*) is due to an interaction that occurs within a dimer of NtrC or requires the formation of higher-order oligomers, the change indicated that #alpha#-heli
机译：细菌增强剂结合蛋白氮调节蛋白C（NteC）在依赖ATP水解的反应中通过含#sigma（〜54）的RNA聚合酶激活转录。 NtrC N末端受体结构域中的天冬氨酸残基的磷酸化诱导蛋白质的寡聚化并激活ATPase活性，这是其中央输出域的功能。为了研究已知发挥积极作用的NtrC受体域的作用，我们分离了携带单个半胱氨酸残基的蛋白的突变形式，并用巯基特异性一氧化氮试剂将其衍生化，用于电子顺磁共振谱。将单个半胱氨酸放置在我们获得组成性氨基酸取代的四个位置上，这些位置可产生活性而无需磷酸化。在仅一种情况下，在受体域的＃alpha＃-螺旋4中衍生的C86确实不会使侧链的运动在磷酸化后显着变慢。重要的是，去离子化的NtrC〜（N86C）[NtrC〜（D86C *）]正常激活了转录。额外的实验表明，NtrC〜（D86C *）磷酸化后观察到的光谱变化是由于内部相互作用而不是N末端结构域本身的构象变化引起的。这些相互作用似乎未在单体内发生。尽管尚不清楚NtrC〜（D86C *）磷酸化后看到的光谱变化是由于NtrC二聚体中发生的相互作用还是需要形成更高阶的低聚物，但这种变化表明＃alpha＃-heli

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