Biophysics Group, Department of Physics and Astronomy, Faculty of Sciences, VU University, De Boelelaan 1081, 1081HV Amsterdam, The Netherlands.
Proc Natl Acad Sci U S A. 2010 May 18;107(20):9170-5. doi: 10.1073/pnas.0911535107. Epub 2010 Apr 30.
Phytochromes are red-light photoreceptor proteins that regulate a variety of responses and cellular processes in plants, bacteria, and fungi. The phytochrome light activation mechanism involves isomerization around the C15 horizontal lineC16 double bond of an open-chain tetrapyrrole chromophore, resulting in a flip of its D-ring. In an important new development, bacteriophytochrome (Bph) has been engineered for use as a fluorescent marker in mammalian tissues. Here we report that an unusual Bph, RpBphP3 from Rhodopseudomonas palustris, denoted P3, is fluorescent. This Bph modulates synthesis of light-harvesting complex in combination with a second Bph exhibiting classical photochemistry, RpBphP2, denoted P2. We identify the factors that determine the fluorescence and isomerization quantum yields through the application of ultrafast spectroscopy to wild-type and mutants of P2 and P3. The excited-state lifetime of the biliverdin chromophore in P3 was significantly longer at 330-500 ps than in P2 and other classical phytochromes and accompanied by a significantly reduced isomerization quantum yield. H/D exchange reduces the rate of decay from the excited state of biliverdin by a factor of 1.4 and increases the isomerization quantum yield. Comparison of the properties of the P2 and P3 variants shows that the quantum yields of fluorescence and isomerization are determined by excited-state deprotonation of biliverdin at the pyrrole rings, in competition with hydrogen-bond rupture between the D-ring and the apoprotein. This work provides a basis for structure-based conversion of Bph into an efficient near-IR fluorescent marker.
光敏色素是一种红光光受体蛋白，可调节植物、细菌和真菌中的多种反应和细胞过程。光敏色素的光激活机制涉及到开链四吡咯发色团的 C15 横线 C16 双键周围的异构化，导致其 D 环翻转。在一个重要的新进展中，细菌光敏色素（Bph）已被设计用于哺乳动物组织中的荧光标记。在这里，我们报告一种来自沼泽红假单胞菌的不寻常的 Bph，RpBphP3，称为 P3，是荧光的。这种 Bph 与另一种表现出经典光化学的 Bph（称为 P2）一起调节光捕获复合物的合成。我们通过超快光谱学应用于 P2 和 P3 的野生型和突变体，确定了决定荧光和异构化量子产率的因素。在 P3 中，胆绿素发色团的激发态寿命在 330-500 ps 之间明显长于 P2 和其他经典光敏色素，并伴随着显著降低的异构化量子产率。H/D 交换将胆绿素从激发态的衰减速率降低了 1.4 倍，并增加了异构化量子产率。比较 P2 和 P3 变体的性质表明，荧光和异构化的量子产率是由吡咯环上胆绿素的激发态去质子化决定的，与 D 环和脱辅基蛋白之间氢键的断裂竞争。这项工作为基于结构的将 Bph 转化为高效近红外荧光标记物提供了基础。
