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首页> 外文期刊>Biochemistry >Mutational and Structural Studies of the PixD BLUF Output Signal That Affects Light-Regulated Interactions with PixE
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Mutational and Structural Studies of the PixD BLUF Output Signal That Affects Light-Regulated Interactions with PixE

机译：影响与PixE进行光调节相互作用的PixD BLUF输出信号的突变和结构研究

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摘要

PixD (Slr1694) is a BLUF (blue-light-using FAD) photoreceptor used by the cyanobacterium Synechocystis sp. PCC6803 to control phototaxis toward blue light. In this study, we probe the involvement of a conserved Tyr8-Gln50-Met93 triad in promoting an output signal upon blue light excitation of the bound flavin. Analysis of acrylamide quenching of Trp91 fluorescence shows that the side chain of this residue remains partially solvent exposed in both the lit and dark states. Mutational analysis demonstrates that substitution mutations at Tyr8 and Gln50 result in the loss of the photocycle while a mutation of Met93 does not appreciably disturb the formation of the light-excited state and only minimally accelerates its decay from 5.7 to 4.5 s. However, mutations of Tyr8, Gln50, and Met93 disrupt the ability of PixD dimers to interact with PixE to form a higher-order PixD_(10)-PixE_5 complex, which is indicative of a lit conformational state. Solution nuclear magnetic resonance spectroscopy and X-ray crystallographic analyses confirm that a Tyr8 to Phe mutation is locked in a pseudo-light-excited state revealing flexible areas in PixD that likely constitute part of an output signal upon light excitation of wild-type PixD.
机译：PixD（Slr1694）是蓝细菌蓝藻（Synechocystcystis sp。）使用的BLUF（蓝光FAD）感光体。 PCC6803可以控制趋向于蓝光。在这项研究中，我们探讨了保守的Tyr8-Gln50-Met93三联体在结合黄素的蓝光激发下促进输出信号的参与。 Trp91荧光的丙烯酰胺淬灭分析表明，该残基的侧链在亮和暗状态下均部分暴露于溶剂中。突变分析表明，Tyr8和Gln50处的取代突变会导致光循环丧失，而Met93的突变不会明显干扰光激发态的形成，只会将其衰减从5.7最小加速到4.5 s。但是，Tyr8，Gln50和Met93的突变破坏了PixD二聚体与PixE相互作用以形成更高阶的PixD_（10）-PixE_5复合物的能力，这表明该构象状态是亮的。溶液核磁共振波谱和X射线晶体学分析证实，Tyr8到Phe突变被锁定在伪光激发状态，揭示了PixD中的柔性区域，该区域在野生型PixD的光激发下很可能构成输出信号的一部分。

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