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首页> 外文期刊>Biochemistry >A Q63E Rhodobacter sphaeroides AppA BLUF Domain Mutant Is Locked in a Pseudo-Light-Excited Signaling State
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A Q63E Rhodobacter sphaeroides AppA BLUF Domain Mutant Is Locked in a Pseudo-Light-Excited Signaling State

机译：Q63E球形红细菌AppA BLUF域突变体被锁定在伪光激发信号状态

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摘要

The AppA BLUF photoreceptor from Rhodobacter sphaeroides contains a conserved key residue,nGln63, that is thought to undergo a shift in hydrogen-bonding interactions when a bound flavin is lightnexcited. In this study we have characterized two substitutionmutants ofGln63 (Q63E, Q63L) in the context ofntwo constructs of the BLUF domain that have differing lengths,AppA1-126 andAppA17-133.Q63Lmuta-ntions in both constructs exhibit a blue-shifted flavin absorption spectrum as well as a loss of the photocycle.nAltered fluorescence emission and fluorescence quenching of the Q63L mutant indicate significantnperturbations of hydrogen bonding to the flavin and surrounding amino acids which is confirmed byn1nH-15nN HSQC NMR spectroscopy. The Q63E substitution mutant is constitutively locked in a lit signalingnstate as evidenced by a permanent 3 nm red shift of the flavin absorption, quenching of flavin fluorescencenemission, analysis ofn1nH-15nN HSQC spectra, and the inability of full-length AppA Q63E to bind to the PpsRnrepressor. The significance of these findings on the mechanism of light-induced output signaling is discussed.
机译：球形球形红细菌的AppA BLUF感光体包含一个保守的关键残基nGln63，当轻键结合的黄素时，该残基被认为会发生氢键相互作用的变化。在这项研究中，我们在BLUF结构域的两个长度不同的构建体（AppA1-126和AppA17-133）中表征了Gln63的两个取代突变体（Q63E，Q63L）。两个构建体中的Q63L突变体均显示出蓝移的黄素吸收光谱，因为n63n突变的荧光发射和荧光猝灭表明Q63L突变体的氢键与黄素和周围氨基酸的结合显着扰动，这已通过n1nH-15nN HSQC NMR光谱学证实。 Q63E取代突变体被组成性地锁定在点亮的信号状态中，黄素吸收发生永久性3 nm红移，黄素荧光发射淬灭，n1nH-15nN HSQC光谱分析，以及全长AppA Q63E无法与Q63E结合PpsRnrepressor。讨论了这些发现对光诱导输出信号传导机制的意义。

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