Photocycle dynamics of the E149A mutant of cryptochrome 3 from Arabidopsis thaliana.
Photocycle dynamics of the E149A mutant of cryptochrome 3 from Arabidopsis thaliana.
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DOI：
10.1016/j.jphotobiol.2009.08.005
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发表时间：
2009-11
期刊：
影响因子：
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通讯作者：
P. Zirak;A. Penzkofer;Julia Moldt;R. Pokorný;A. Batschauer;L. Essen
中科院分区：
文献类型：
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作者：
P. Zirak;A. Penzkofer;Julia Moldt;R. Pokorný;A. Batschauer;L. Essen
通过体外光学吸收和发射光谱研究，对拟南芥cryDASH成员隐花色素3（cry 3）的E149 A突变体进行了表征。突变蛋白非共价结合发色团黄素腺嘌呤二核苷酸（FAD）。与野生型蛋白相反，它不结合N5，N10-亚甲基-5，6，7，8-四氢叶酸（MTHF）。因此，由FAD引起的光动力学是可获得的，而无需与MTHF的介入偶联。在暗适应cry 3-E149 A中，FAD以氧化形式（FADox）、半醌形式（FADH）和阴离子氢醌形式（FADredH−）存在。先前未曝光的cry 3-E149 A的蓝光光激发将FADox转移为阴离子半醌形式（FAD-），量子效率约为2%，反向恢复时间约为10秒（光循环I）。长时间的光激发导致不可逆的蛋白质重新构象，具有U形FAD的结构修饰并使质子转移成为可能。因此，光循环动力学的变化伴随着FADox到FADH、FADH到FADredH-的光转换以及黑暗中的热反平衡（光循环II）而发生。cry 3-E149 A的光循环动力学与野生型cry 3和其他光敏隐花色素的光循环行为进行了比较。
The E149A mutant of the cryDASH member cryptochrome 3 (cry3) from Arabidopsis thaliana was characterized in vitro by optical absorption and emission spectroscopic studies. The mutant protein non-covalently binds the chromophore flavin adenine dinucleotide (FAD). In contrast to the wild-type protein it does not bind N5,N10-methenyl-5,6,7,8-tetrahydrofolate (MTHF). Thus, the photo-dynamics caused by FAD is accessible without the intervening coupling with MTHF. In dark adapted cry3-E149A, FAD is present in the oxidized form (FADox), semiquinone form (FADH), and anionic hydroquinone form (FADredH−). Blue-light photo-excitation of previously unexposed cry3-E149A transfers FADoxto the anionic semiquinone form (FAD−) with a quantum efficiency of about 2% and a back recovery time of about 10s (photocycle I). Prolonged photo-excitation leads to an irreversible protein re-conformation with structure modification of the U-shaped FAD and enabling proton transfer. Thus, a change in the photocycle dynamics occurs with photo-conversion of FADoxto FADH, FADH to FADredH−, and thermal back equilibration in the dark (photocycle II). The photocycle dynamics of cry3-E149A is compared with the photocycle behaviour of wild-type cry3 and other photo-sensory cryptochromes.