On the mechanism of activation of the BLUF domain of AppA
On the mechanism of activation of the BLUF domain of AppA
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DOI：
10.1021/bi051367p
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发表时间：
2006-01-10
期刊：
影响因子：
2.9
通讯作者：
Hellingwerf, KJ
中科院分区：
文献类型：
--
作者：
Laan, W;Gauden, M;Hellingwerf, KJ
AppA是一种转录抗阻遏物，在高强度蓝光和氧化还原信号下调节球形红细菌光合作用基因的稳定表达。它的蓝光传感是由一个N-末端BLUF结构域，一个新的黄素折叠的成员介导的。该结构域（AppA（5 - 125））的光循环包括轻微红移的长寿命信号传导状态的形成，其在亚纳秒时间尺度上直接由黄素的单重激发态形成[Gauden等人（2005）Biochemistry）44，3653 - 3662]。这种信号状态的吸收光谱的红移已被归因于其与周围的载脂蛋白的氢键相互作用的重排。在这项研究中，我们的特点是一个AppA突变体与改变芳香族氨基酸：W104F。这种突变体表现出增加的寿命的单重激发态的黄素发色团。然而，最引人注目的是。它显示其信号状态形成的量子产率增加了1.5倍。此外，它还显示出基态恢复率略有增加。除此之外，在该突变蛋白和野生型BLUF结构域中咪唑的存在显著加速基态恢复速率，表明该速率受到氢键重排的限制。总的来说，回收率提高了700倍，这使得AppA的W104F BLUF域适合于未来使用步进扫描FTIR进行分析。AppA的BLUF结构域的基态恢复速率遵循Arrhenius动力学。这表明，该域本身不经历大的结构变化，光照后，在全长AppA的结构转变是由域间重排为主。
AppA, a transcriptional antirepressor, regulates the steady expression of photosynthesis genes in Rhodobacter sphaeroides in response to high-intensity blue light and to redox signals. Its blue-light sensing is mediated by an N-terminal BLUF domain, a member of a novel flavin fold. The photocycle of this domain (AppA(5-125)) includes formation of a slightly red-shifted long-lived signaling state, which is formed directly from the singlet excited state of the flavin on a subnanosecond time scale [Gauden et al. (2005) Biochemistry) 44, 3653-3662]. The red shift of the absorption spectrum of this signaling state has been attributed to a rearrangement of its hydrogen-bonding interactions with the surrounding apoprotein. In this study we have characterized an AppA mutant with an altered aromatic amino acid: W104F. This mutant exhibits an increased lifetime of the singlet excited state of the flavin chromophore. Most strikingly, however. it shows a 1.5-fold increase in its quantum yield of signaling state formation. In addition, it shows a slightly increased rate of ground-state recovery. On top of this, the presence of imidazole, both in this mutant protein and in the wild-type BLUF domain, significantly accelerates the rate of groundstate recovery, suggesting that this rate is limited by rearrangement of (a) hydrogen bond(s). In total, an similar to 700-fold increase in recovery rate has been obtained, which makes the W104F BLUF domain of AppA, for example, suitable for future analyses with step-scan FTIR. The rate of ground-state recovery of the BLUF domain of AppA follows Arrhenius kinetics. This suggests that this domain itself does not undergo large structural changes upon illumination and that the structural transitions in full-length AppA are dominated by interdomain rearrangements.