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首页> 外文期刊>Biochimica et Biophysica Acta. Protein Structure and Molecular Enzymology >Probing the ATP binding site of tubulin with thiotriphosphate analogues of ATP
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Probing the ATP binding site of tubulin with thiotriphosphate analogues of ATP

机译：用ATP的硫代三磷酸类似物探测微管蛋白的ATP结合位点

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摘要

Tubulin assembly studies with GTPαS diastereoisomers have shown that there is stereoselectivity at the α-phosphate binding region of tubulin. GTPαS(S_p) bound tighter than GTPαS(R_P) and promoted nucleation and assembly better than GTP and GTPαS(R_p) and dATP have been reported to bind weakly to tubulin and to be less effective than GTP and dGTP in promoting tubulin assembly. This study was done to learn if ATPαS(S_p) and dATPαS(S_p) are good promoters of tubulin assembly and to compare these ATP thiotriphosphate analogues to the corresponding GTP analogues in tubulin assembly. Studies were also done with ATPαS(R_p), GTP, ATPβS(S_p) and ATPγS. At least three cycles of tubulin (25 μM) assembly-disassembly were found with 1 mM ATP250L罶(S_p) and dATPαS(S_p) and both nucleotides were incorporated and hydrolyzed in the polymers. Less dATPαS(S_p) (25 μM) than ATPαS(S_p) (100 μM) promoted assembly to 50% of the maximum value. The critical concentrations (Cc) for assembly with 1 mM nucleotide were low for ATPαS(S_p) (3 μM) and dATPαS(S_p) (2 μM) and compared favorably with GTP (5 μM), GTPαS(S_p) (2 μM) and dGTPαS(S_p) (1 μM). Both 1 mM ATP and dATP were poor promoters of tubulin assembly and were not detected in the polymers. The predominant structures induced by 1 mM ATPαS(S_p) and dATPαS(S_p) were bundles of sheets and microtubules, which were more stable to the cold and to Ca(II) than microtubules assembled with GTP, ATP or dATP. ATPαS(R_p) (1 mM) did not promote assembly suggesting that there is stereoselectivity at the ATPαS α-phosphate binding region of tubulin is there is with GTPαS diastereoisomers. ATPαS(S_p) and dATPαS(S_p) mimic GTPαS(S_p) and dGTPαS(S_p) in tubulin assembly since all four nucleotides promote bundles of tubulin in buffer with glycerol, and the deoxy nucleotides have lower Cc, shorter lags and faster rates for tubulin assembly.
机译：用GTPαS非对映异构体进行的微管蛋白组装研究表明，在微管蛋白的α-磷酸结合区域存在立体选择性。 GTPαS（S_p）比GTPαS（R_P）结合更紧密，并且比GTP更好地促进了成核和组装，并且据报道GTPαS（R_p）和dATP与微管蛋白的结合较弱，并且在促进微管蛋白组装方面不如GTP和dGTP有效。进行这项研究是为了了解ATPαS（S_p）和dATPαS（S_p）是否是微管蛋白装配的良好启动子，并将这些ATP硫代三磷酸酯类似物与微管蛋白装配中的相应GTP类似物进行比较。还对ATPαS（R_p），GTP，ATPβS（S_p）和ATPγS进行了研究。用1 mMATP250L罶（S_p）和dATPαS（S_p）发现了至少三个微管蛋白（25μM）组装-拆卸循环，并且两个核苷酸均被掺入并水解了聚合物。少于ATPαS（S_p）（100μM）的dATPαS（S_p）（25μM）促使组装达到最大值的50％。 ATPαS（S_p）（3μM）和dATPαS（S_p）（2μM）的1 mM核苷酸组装的临界浓度（Cc）较低，与GTP（5μM），GTPαS（S_p）（2μM）相比要好和dGTPαS（S_p）（1μM）。 1 mM ATP和dATP都是微管蛋白组装的不良启动子，在聚合物中未检测到。 1 mMATPαS（S_p）和dATPαS（S_p）诱导的主要结构是片状和微管束，比用GTP，ATP或dATP组装的微管对冷和Ca（II）更为稳定。 ATPαS（R_p）（1 mM）不能促进组装，这表明微管蛋白的ATPαSα-磷酸结合区域存在立体选择性，与GTPαS非对映异构体相同。 ATPαS（S_p）和dATPαS（S_p）模仿微管蛋白装配中的GTPαS（S_p）和dGTPαS（S_p），因为所有四个核苷酸都促进甘油在缓冲液中的微管蛋白束，并且脱氧核苷酸的Cc较低，滞后时间较短且微管蛋白的速率更快部件。

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