Department of Chemistry, Oregon State University, Corvallis, Oregon, USA.
School of Molecular Sciences, Center for Bioenergy and Photosynthesis, Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, Arizona, USA.
Protein Sci. 2023 Jan;32(1):e4517. doi: 10.1002/pro.4517.
Green-to-red photoconvertible fluorescent proteins (FPs) are vital biomimetic tools for powerful techniques such as super-resolution imaging. A unique Kaede-type FP named the least evolved ancestor (LEA) enables delineation of the evolutionary step to acquire photoconversion capability from the ancestral green fluorescent protein (GFP). A key residue, Ala69, was identified through several steady-state and time-resolved spectroscopic techniques that allows LEA to effectively photoswitch and enhance the green-to-red photoconversion. However, the inner workings of this functional protein have remained elusive due to practical challenges of capturing the photoexcited chromophore motions in real time. Here, we implemented femtosecond stimulated Raman spectroscopy and transient absorption on LEA-A69T, aided by relevant crystal structures and control FPs, revealing that Thr69 promotes a stronger π-π stacking interaction between the chromophore phenolate (P-)ring and His193 in FP mutants that cannot photoconvert or photoswitch. Characteristic time constants of ~60-67 ps are attributed to P-ring twist as the onset for photoswitching in LEA (major) and LEA-A69T (minor) with photoconversion capability, different from ~16/29 ps in correlation with the Gln62/His62 side-chain twist in ALL-GFP/ALL-Q62H, indicative of the light-induced conformational relaxation preferences in various local environments. A minor subpopulation of LEA-A69T capable of positive photoswitching was revealed by time-resolved electronic spectroscopies with targeted light irradiation wavelengths. The unveiled chromophore structure and dynamics inside engineered FPs in an aqueous buffer solution can be generalized to improve other green-to-red photoconvertible FPs from the bottom up for deeper biophysics with molecular biology insights and powerful bioimaging advances.
绿到红光可转换荧光蛋白（FPs）是强大技术（如超分辨率成像）的重要仿生工具。一种名为最原始祖先（LEA）的独特的 Kaede 型 FP 可以描绘从原始绿色荧光蛋白（GFP）获得光转换能力的进化步骤。通过几种稳态和时间分辨光谱技术鉴定出一个关键残基，丙氨酸 69（Ala69），该残基允许 LEA 有效地光开关并增强绿到红的光转换。然而，由于实时捕获光激发发色团运动的实际挑战，这种功能蛋白的内部工作机制仍然难以捉摸。在这里，我们通过相关晶体结构和对照 FP 实施了飞秒受激拉曼光谱和瞬态吸收在 LEA-A69T 上的应用，揭示了 Thr69 促进了发色团酚盐（P-）环和 FP 突变体中不能光转化或光开关的 His193 之间更强的π-π堆积相互作用。60-67 ps 的特征时间常数归因于 P-环扭曲，这是 LEA（主要）和具有光转化能力的 LEA-A69T 光开关的起始，与 Gln62/His62 侧链扭曲相关的16/29 ps 不同，表明在各种局部环境中光诱导构象松弛的偏好。通过靶向光辐照波长的时间分辨电子光谱揭示了具有正光开关能力的 LEA-A69T 的次要亚群。在水缓冲溶液中构建的 FP 内部揭示的发色团结构和动力学可以推广到其他绿到红光可转换 FP，从底层改进，以提高具有分子生物学见解和强大生物成像进展的更深层次的生物物理学。
