Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt Am Main, Max-von-Laue-Straße 7, 60438, Frankfurt, Germany.
Institute for Analytical Chemistry, University of Leipzig, Linnéstraße 3, 04103, Leipzig, Germany.
Photochem Photobiol Sci. 2022 Sep;21(9):1627-1636. doi: 10.1007/s43630-022-00245-9. Epub 2022 Jun 10.
The ability of some knotless phytochromes to photoconvert without the PHY domain allows evaluation of the distinct effect of the PHY domain on their photodynamics. Here, we compare the ms dynamics of the single GAF domain (g1) and the GAF-PHY (g1g2) construct of the knotless phytochrome All2699 from cyanobacterium Nostoc punctiforme. While the spectral signatures and occurrence of the intermediates are mostly unchanged by the domain composition, the presence of the PHY domain slows down the early forward and reverse dynamics involving chromophore and protein binding pocket relaxation. We assign this effect to a more restricted binding pocket imprinted by the PHY domain. The photoproduct formation is also slowed down by the presence of the PHY domain but to a lesser extent than the early dynamics. This indicates a rate limiting step within the GAF and not the PHY domain. We further identify a pH dependence of the biphasic photoproduct formation hinting towards a pKa dependent tuning mechanism. Our findings add to the understanding of the role of the individual domains in the photocycle dynamics and provide a basis for engineering of phytochromes towards biotechnological applications.
一些无结藻胆体的光转化能力无需 PHY 结构域即可实现，这使得我们能够评估 PHY 结构域对其光动力学的独特影响。在这里，我们比较了来自蓝细菌鱼腥藻的无结藻胆体 All2699 的单个 GAF 结构域（g1）和 GAF-PHY（g1g2）结构的毫秒动力学。虽然域组成对光谱特征和中间体的出现影响不大，但 PHY 结构域的存在会减缓涉及发色团和蛋白质结合口袋松弛的早期正向和反向动力学。我们将这种效应归因于 PHY 结构域所产生的更受限的结合口袋印记。光产物的形成也会因 PHY 结构域的存在而减缓，但程度不如早期动力学那么大。这表明在 GAF 结构域内存在限速步骤，而不是 PHY 结构域。我们进一步确定了光产物形成的双相 pH 依赖性，暗示存在 pKa 依赖性调节机制。我们的研究结果增加了对单个结构域在光循环动力学中作用的理解，并为针对生物技术应用的藻胆体工程提供了基础。
