目的 TePixD(T110078)是一种来自细长嗜热聚球藻(Thermosynechococcus elongatus BP-1)的 blue light-using flavin(BLUF)感光蛋白。TePixD蛋白在FAD 口袋周围有一个保守的Tyr8-Gln50-Met93三联体，以介导质子偶联电子转移(proton-coupled electron transfer，PCET)过程。但具体的光响应机制有待进一步研究。本文的目的是解析TePixD在光响应关键位点突变体的结构以及生化性质，以此分析TePixD的光响应过程。方法 利用X射线衍射法解析TePixDY8F突变体的晶体结构，Tyr8侧链在PCET过程中有重要作用，而突变体Phe8的侧链失去了羟基从而使分子无法介导PCET。本文比较了TePixD野生型和Y8F突变体之间的结构，并比较了同蛋白家族的SyPixDY8F与TePixDY8F的结构。此外，使用多角度光散射法分析几种TePixD在光响应关键位点突变体(Y8F、Q50L、W91F、Y8F/W91F和Q50L/W91F)在溶液中的生化特性。结果 本文以2。54 Å分辨率解析了TePixD Y8F突变体的晶体结构，发现其与TePixD野生型的整体结构相似，而与SyPixD Y8F存在显著差异。生化分析表明，对比黑暗和光照条件时，TePixD突变体与野生型的分子质量变化以及洗脱峰表现出差异，表明突变体对光诱导蛋白质构象变化产生干扰。结论 本文的结构测定和生化分析为揭示BLUF蛋白的光响应机制提供了新信息。
Structure of The BLUF Protein TePixD Y8F Mutant
Objective TePixD(T110078)is a blue light-using flavin(BLUF)photoreceptor protein from Thermosynechococcus elongatus BP-1.TePixD protein has a conserved Tyr8-Gln50-Met93 triad around the FAD pocket to mediate the proton-coupled electron transfer(PCET)process.But the detailed light response mechanism needs further study.We aimed to elucidate the structure and biochemical properties of TePixD mutants at key light response sites to analyze the light response process of TePixD.Methods We employed X-ray crystallography to resolve the crystal structure of the TePixD Y8F mutant.The side chain of Tyr8 is involved in PCET while Phe8 in mutation loses the function due to the loss of its hydroxyl group.We compared the structure of TePixD Y8F mutation to TePixD wild type(WT)and its homology protein SyPixD Y8F.Using multi-angle light scattering(MALS),we analyzed the oligomerization of multiple TePixD mutations(Y8F,Q50L,W91F,Y8F/W91F,and Q50L/W91F),focusing specifically on mutational sites that are critical residues for the protein's photo response to dark and light conditions.Results We resolved the crystal structure of TePixD Y8F mutant at a resolution of 2.54 Å and found that it shares a similar overall structure with the TePixD WT but exhibits significant differences from the SyPixD Y8F structure.Biochemical analysis revealed differences in molecular mass and elution profiles between the TePixD mutants and the WT under dark and light conditions,indicating the perturbation on the light-induced conformational change by the mutants.Conclusion Our structure determination and biochemical analyses will add information to reveal the light response mechanism of BLUF proteins.
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