Ultrafast Infrared Spectroscopy of an Isotope-Labeled Photoactivatable Flavoprotein
Ultrafast Infrared Spectroscopy of an Isotope-Labeled Photoactivatable Flavoprotein
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DOI：
10.1021/bi101589a
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发表时间：
2011-03-01
期刊：
影响因子：
2.9
通讯作者：
Tonge, Peter J.
中科院分区：
文献类型：
--
作者：
Haigney, Allison;Lukacs, Andras;Tonge, Peter J.
使用黄素（BLUF）结构域的蓝光光传感器，例如转录抗阻遏物AppA，利用非共价结合的黄素作为光感受的发色团。由于生色团的异咯嗪环在光吸收后不能发生大规模的结构变化，因此人们对了解BLUF蛋白质基质如何感知和响应黄素光激发产生了浓厚的兴趣。光吸收被认为是导致在超快时间尺度上围绕黄素发色团的氢键网络发生变化的原因，而光激发引起的结构变化正在被振动光谱探测。在这里，我们报告超快时间分辨红外光谱的AppA BLUF域（AppA（BLUF））重建与同位素标记的核黄素（RI）和黄素腺嘌呤二核苷酸（FAD），这允许第一明确的地面和激发态模式直接从黄素羰基的分配。模型化合物的研究和DFT计算的基态振动光谱揭示了这些模式对环境的敏感性，表明它们可以用作结构动力学的探针。
The blue light using Flavin (BLUF) domain photosensors, such as the transcriptional antirepressor AppA, utilize a noncovalently bound flavin as the chromophore for photoreception. Since the isoalloxazine ring of the chromophore is unable to undergo large-scale structural change upon light absorption, there is intense interest in understanding how the BLUF protein matrix senses and responds to flavin photoexcitation. Light absorption is proposed to result in alterations in the hydrogen-bonding network that surrounds the Flavin chromophore on an ultrafast time scale, and the structural changes caused by photoexcitation are being probed by vibrational spectroscopy. Here we report ultrafast time-resolved infrared spectra of the AppA BLUF domain (AppA(BLUF)) reconstituted with isotopically labeled riboflavin (RI) and flavin adenine dinucleotide (FAD), which permit the first unambiguous assignment of ground and excited state modes arising directly from the flavin carbonyl groups. Studies of model compounds and DFT calculations of the ground state vibrational spectra reveal the sensitivity of these modes to their environment, indicating that they can be used as probes of structural dynamics.