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首页> 外文期刊>The journal of physical chemistry, A. Molecules, spectroscopy, kinetics, environment, & general theory >Primary reactions of bacteriophytochrome observed with ultrafast mid-infrared spectroscopy
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Primary reactions of bacteriophytochrome observed with ultrafast mid-infrared spectroscopy

机译：超快中红外光谱法观察到的细菌性植物色素的主要反应

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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 C _(15)? -C_(16) double bond of an open-chain tetrapyrrole chromophore, resulting in a flip of its D-ring. In an important recent development, bacteriophytochrome (Bph) has been engineered for use as a fluorescent marker in mammalian tissues. Bphs covalently bind a biliverdin (BV) chromophore, naturally abundant in mammalian cells. Here, we report an ultrafast time-resolved mid-infrared spectroscopic study on the Pr state of two highly related Bphs from Rps. palustris, RpBphP2 (P2) and RpBphP3 (P3) with distinct photoconversion and fluorescence properties. We observed that the BV excited state of P2 decays in 58 ps, while the BV excited state of P3 decays in 362 ps. By combining ultrafast mid-IR spectroscopy with FTIR spectroscopy on P2 and P3 wild type and mutant proteins, we demonstrate that the hydrogen bond strength at the ring D carbonyl of the BV chromophore is significantly stronger in P3 as compared to P2. This result is consistent with the X-ray structures of Bph, which indicate one hydrogen bond from a conserved histidine to the BV ring D carbonyl for classical bacteriophytochromes such as P2, and one or two additional hydrogen bonds from a serine and a lysine side chain to the BV ring D carbonyl for P3. We conclude that the hydrogen-bond strength at BV ring D is a key determinant of excited-state lifetime and fluorescence quantum yield. Excited-state decay is followed by the formation of a primary intermediate that does not decay on the nanosecond time scale of the experiment, which shows a narrow absorption band at ?1540 cm~(- 1). Possible origins of this product band are discussed. This work may aid in rational structure- and mechanism-based conversion of BPh into an efficient near-IR fluorescent marker.
机译：植物色素是红光感光蛋白，可调节植物，细菌和真菌中的各种响应和细胞过程。植物色素的光激活机制涉及C_（15）？附近的异构化。开链四吡咯发色团的-C_（16）双键，导致其D环翻转。在最近的重要发展中，细菌植物色素（Bph）已被工程化以用作哺乳动物组织中的荧光标记。 Bph共价结合在肝脏细胞中自然丰富的biliverdin（BV）发色团。在这里，我们报告了来自Rps的两个高度相关的Bph的Pr状态的超快时间分辨中红外光谱研究。 palustris，RpBphP2（P2）和RpBphP3（P3）具有明显的光转换和荧光特性。我们观察到，P2的BV激发态衰减为58 ps，而P3的BV激发态衰减为362 ps。通过将超快中红外光谱与FTIR光谱结合在P2和P3野生型和突变蛋白上，我们证明，与P2相比，P3中BV生色团的环D羰基处的氢键强度明显更强。此结果与Bph的X射线结构一致，表明X射线结构是从保守的组氨酸到BV环D羰基的一个氢键，对于经典的细菌性植物色素（如P2），以及一个或两个从丝氨酸和赖氨酸侧链的另外的氢键P3的BV环D羰基。我们得出的结论是，BV环D处的氢键强度是激发态寿命和荧光量子产率的关键决定因素。激发态衰变之后，形成初级中间产物，该中间产物在实验的纳秒时间尺度上不衰变，这表明在1540 cm〜（-1）处有一个窄吸收带。讨论了该产品系列的可能来源。这项工作可能有助于将BPh合理地基于结构和机制转换为有效的近红外荧光标记。

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