Tachibana Sean R, Tang Longteng, Chen Cheng, Zhu Liangdong, Takeda Yuka, Fushimi Keiji, Seevers Travis K, Narikawa Rei, Sato Moritoshi, Fang Chong
Department of Chemistry, Oregon State University, 153 Gilbert Hall, Corvallis, OR 97331-4003, United States.
Graduate School of Integrated Science and Technology, Shizuoka University, 422-8529 Shizuoka, Japan.
Spectrochim Acta A Mol Biomol Spectrosc. 2021 Apr 5;250:119379. doi: 10.1016/j.saa.2020.119379. Epub 2020 Dec 23.
Cyanobacteriochromes (CBCRs) are an emerging class of photoreceptors that are distant relatives of the phytochromes family. Unlike phytochromes, CBCRs have gained popularity in optogenetics due to their highly diverse spectral properties spanning the UV to near-IR region and only needing a single compact binding domain. AnPixJg2 is a CBCR that can reversibly photoswitch between its red-absorbing (P) and green-absorbing (P) forms of the phycocyanobilin (PCB) cofactor. To reveal primary events of photoconversion, we implemented femtosecond transient absorption spectroscopy with a homemade LED box and a miniature peristaltic pump flow cell to track transient electronic responses of the photoexcited AnPixJg2 on molecular time scales. The 525 nm laser-induced P-to-P reverse conversion exhibits a ~3 ps excited-state lifetime before reaching the conical intersection (CI) and undergoing further relaxation on the 30 ps time scale to generate a long-lived Lumi-G ground state intermediate en route to P. The 650 nm laser-induced P-to-P forward conversion is less efficient than reverse conversion, showing a longer-lived excited state which requires two steps with ~13 and 217 ps time constants to enter the CI region. Furthermore, using a tunable ps Raman pump with broadband Raman probe on both the Stokes and anti-Stokes sides, we collected the pre-resonance ground-state femtosecond stimulated Raman spectroscopy (GS-FSRS) data with mode assignments aided by quantum calculations. Key vibrational marker bands at ~850, 1050, 1615, and 1649 cm of the P conformer exhibit a notable blueshift to those of the P conformer inside AnPixJg2, reflecting the PCB chromophore terminal D (major) and A (minor) ring twist along the primary photoswitching reaction coordinate. This integrated ultrafast spectroscopy and computational platform has the potential to elucidate photochemistry and photophysics of more CBCRs and photoactive proteins in general, providing the highly desirable mechanistic insights to facilitate the rational design of functional molecular sensors and devices.
蓝细菌色素（CBCRs）是一类新兴的光感受器，是光敏色素家族的远亲。与光敏色素不同，CBCRs因其跨越紫外到近红外区域的高度多样的光谱特性以及仅需一个紧凑的结合结构域而在光遗传学中受到欢迎。AnPixJg2是一种CBCR，其藻蓝胆素（PCB）辅因子的吸收红光（P）和吸收绿光（P）形式之间可进行可逆的光开关转换。为了揭示光转换的主要事件，我们使用自制的LED盒和微型蠕动泵流通池实施了飞秒瞬态吸收光谱，以在分子时间尺度上跟踪光激发的AnPixJg2的瞬态电子响应。525 nm激光诱导的P到P的反向转换在到达锥形交叉点（CI）之前表现出约3 ps的激发态寿命，并在30 ps时间尺度上进一步弛豫，以在通往P的途中产生长寿命的Lumi-G基态中间体。650 nm激光诱导的P到P的正向转换效率低于反向转换，显示出寿命更长的激发态，这需要两个步骤，时间常数约为13和217 ps才能进入CI区域。此外，我们使用可调谐的皮秒拉曼泵浦以及在斯托克斯和反斯托克斯两侧的宽带拉曼探针，通过量子计算辅助的模式分配收集了预共振基态飞秒受激拉曼光谱（GS-FSRS）数据。P构象体在~850、1050、1615和1649 cm处的关键振动标记带相对于AnPixJg2内部的P构象体表现出明显的蓝移，反映了PCB发色团末端D（主要）和A（次要）环沿主要光开关反应坐标的扭曲。这种集成的超快光谱和计算平台有潜力阐明更多CBCRs以及一般光活性蛋白的光化学和光物理过程，提供非常需要的机理见解，以促进功能性分子传感器和器件的合理设计。
