Photoreaction of photoactivated adenylate cyclase from cyanobacterium Microcoleus chthonoplastes
Photoreaction of photoactivated adenylate cyclase from cyanobacterium Microcoleus chthonoplastes
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DOI：
10.1016/j.jphotobiol.2021.112252
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发表时间：
2021-07-12
影响因子：
5.4
通讯作者：
Terazima，Masahide
中科院分区：
文献类型：
--
作者：
Ikoma，Misato;Nakasone，Yusuke;Terazima，Masahide
采用时间分辨瞬态光栅法研究了蓝细菌microcoleus chthonoplastesPCC 7420 （mPAC）的光激活腺苷酸环化酶（per - art - sim (PAS)）、光氧电压（LOV）和腺苷酸环化酶（AC）结构域的光化学反应。在其发色团（黄素单核苷酸）和半胱氨酸残基之间发生加合物形成的吸收光谱变化，时间常数为0.66 μs。反应后，扩散系数(D)-发生了显著变化，时间常数为38 ms。测定的d值与浓度有关，表明二聚体和四聚体之间存在快速平衡。结合尺寸排除色谱和CD光谱分析结果，我们认为光致d变化主要是由于光照射后平衡态从富二聚体转变为富四聚体。由于反应速率不依赖于浓度，四聚体形成的速率决定步骤不是蛋白质通过扩散碰撞，而是构象变化。利用不同的截断突变体研究了PAS和AC结构域以及LOV结构域的N端和c端侧翼螺旋（A′α-和j α-螺旋）的作用。PAS结构域被发现是一个强二聚化位点，与有效的信号转导有关。发现mPAC中A′α-和j′α-螺旋的同时存在是导致光诱导构象变化的重要因素，从而诱导四聚体的形成。结果表明，A′α和j α-螺旋螺旋结构的角度变化是导致这种构象变化的关键。提出了mPAC的反应方案。
The photochemical reaction of photoactivated adenylate cyclase from cyanobacteriumMicrocoleus chthonoplastesPCC 7420 (mPAC), which consists of a Per-Arnt-Sim (PAS), a light‑oxygene-voltage (LOV), and an adenylate cyclase (AC) domain, was investigated mainly using the time-resolved transient grating method. An absorption spectral change associated with an adduct formation between its chromophore (flavin mononucleotide) and a cysteine residue was observed with a time constant of 0.66 μs. After this reaction, a significant diffusion coefficient (D)-change was observed with a time constant of 38 ms. The determinedD-value was concentration-dependent indicating a rapid equilibrium between the dimer and tetramer. Combining the results of size exclusion chromatography and CD spectroscopy, we concluded that the photoinducedD-change was mainly attributed to the equilibrium shift from the dimer rich to the tetramer rich states upon light exposure. Since the reaction rate does not depend on concentration, the rate determining step of the tetramer formation is not the collision of proteins by diffusion, but a conformation change. The roles of the PAS and AC domains as well as the N- and C-terminal flanking helices of the LOV domain (A'α- and Jα-helices) were investigated using various truncated mutants. The PAS domain was found to be a strong dimerization site and is related to efficient signal transduction. It was found that simultaneous existence of the A'α- and Jα-helices in mPAC is important for the light-induced conformation change to lead the conformation change which induces the tetramer formation. The results suggest that the angle changes of the coiled-coil structures in the A'α and Jα-helices are essential for this conformation change. The reaction scheme of mPAC is proposed.