Phytochromes are photosensitive proteins with a covalently bound open‐chain chromophore that can switch between two principal states: red light absorbing Pr and far‐red light absorbing Pfr. Our group has previously shown that the bacteriophytochrome from Xanthomonas campestris pv. campestris (XccBphP) is a bathy‐like phytochrome that uses biliverdin IXα as a co‐factor and is involved in bacterial virulence. To date, the XccBphP crystal structure could only be solved in the Pr state, while the structure of its Pfr state remains elusive. The aims of this work were to develop an efficient screening methodology for the rapid characterization and to identify XccBphP variants that favor the Pfr form. The screening approach developed here consists in analyzing the UV‐Vis absorption behavior of clarified crude extracts containing recombinant phytochromes. This strategy has allowed us to quickly explore over a hundred XccBphP variants, characterize multiple variants and identify Pfr‐favored candidates. The high‐quality data obtained enabled not only a qualitative, but also a quantitative characterization of their photochemistry. This method could be easily adapted to other phytochromes or other photoreceptor families.

中文翻译：

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一种基于光谱学的快速筛选和表征植物色素光化学以寻找 Pfr 偏爱变体的方法

光敏色素是具有共价结合的开链发色团的光敏蛋白，可以在两种主要状态之间切换：吸收红光的 Pr 和吸收远红光的 Pfr。我们小组之前已经表明，来自油菜黄单胞菌 pv. 的细菌植物色素。campestris (XccBphP) 是一种类似深海植物的光敏色素，它使用胆绿素 IXα 作为辅助因子并参与细菌毒力。迄今为止，XccBphP 晶体结构只能在 Pr 状态下解决，而其 Pfr 状态的结构仍然难以捉摸。这项工作的目的是为快速表征开发一种有效的筛选方法，并确定有利于 Pfr 形式的 XccBphP 变体。这里开发的筛选方法包括分析含有重组光敏色素的澄清粗提物的 UV-Vis 吸收行为。这种策略使我们能够快速探索一百多个 XccBphP 变体，表征多个变体并确定 Pfr 偏好的候选者。获得的高质量数据不仅可以对它们的光化学进行定性和定量表征。这种方法可以很容易地适应其他光敏色素或其他光感受器家族。