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首页> 外文期刊>Biochemistry >Equilibrium and kinetic analysis of nucleotide binding to the DEAD-box RNA helicase DbpA.
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Equilibrium and kinetic analysis of nucleotide binding to the DEAD-box RNA helicase DbpA.

机译：核苷酸与DEAD-box RNA解旋酶DbpA结合的平衡和动力学分析。

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The Escherichia coli DEAD-box protein A (DbpA) is an RNA helicase that utilizes the energy from ATP binding and hydrolysis to facilitate structural rearrangements of rRNA. We have used the fluorescent nucleotide analogues, mantADP and mantATP, to measure the equilibrium binding affinity and kinetic mechanism of nucleotide binding to DbpA in the absence of RNA. Binding generates an enhancement in mant-nucleotide fluorescence and a corresponding reduction in intrinsic DbpA fluorescence, consistent with fluorescence resonance energy transfer (FRET) from DbpA tryptophan(s) to bound nucleotides. Fluorescent modification does not significantly interfere with the affinities and kinetics of nucleotide binding. Different energy transfer efficiencies between DbpA-mantATP and DbpA-mantADP complexes suggest that DbpA adopts nucleotide-dependent conformations. ADP binds (K(d) approximately 50 microM at 22 degrees C) 4-7 times more tightly than ATP (K(d) approximately 400 microM at 22 degrees C). Both nucleotides bind with relatively temperature-independent association rate constants (approximately 1-3 microM(-1) s(-1)) that are much lower than predicted for a diffusion-limited reaction. Differences in the binding affinities are dictated primarily by the dissociation rate constants. ADP binding occurs with a positive change in the heat capacity, presumably reflecting a nucleotide-induced conformational rearrangement of DbpA. At low temperatures (<22 degrees C), the binding free energies are dominated by favorable enthalpic and unfavorable entropic contributions. At physiological temperatures (>22 degrees C), ADP binding occurs with positive entropy changes. We favor a mechanism in which ADP binding increases the conformational flexibility and dynamics of DbpA.
机译：大肠杆菌DEAD-box蛋白A（DbpA）是一种RNA解旋酶，利用来自ATP结合和水解的能量促进rRNA的结构重排。我们已经使用荧光核苷酸类似物mantADP和mantATP来测量在不存在RNA的情况下核苷酸与DbpA的平衡结合亲和力和动力学机制。结合产生了mant-核苷酸荧光增强，固有DbpA荧光相应降低，这与从DbpA色氨酸到结合核苷酸的荧光共振能量转移（FRET）相一致。荧光修饰不会显着干扰核苷酸结合的亲和力和动力学。 DbpA-mantATP和DbpA-mantADP复合物之间的不同能量转移效率表明DbpA采用核苷酸依赖性构象。 ADP的结合力（22摄氏度下的K（d）约为50 microM）比ATP的结合力（22摄氏度下的K（d）约为400 microM）更紧密地结合4至7倍。这两个核苷酸与相对温度无关的缔合速率常数（大约1-3 microM（-1）s（-1））结合，该常数比对扩散受限反应的预测要低得多。结合亲和力的差异主要由解离速率常数决定。 ADP结合发生时热容量发生正向变化，大概反映了核苷酸诱导的DbpA构象重排。在低温（<22摄氏度）下，结合自由能以有利的焓和不利的熵贡献为主导。在生理温度（> 22摄氏度）下，ADP结合会发生正熵变化。我们支持其中ADP结合增加DbpA的构象灵活性和动力学的机制。

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