Watching an Engineered Calcium Biosensor Glow: Altered Reaction Pathways before Emission

• Published in: The journal of physical chemistry. B Vol. 122; no. 50; pp. 11986 - 11995
• Main Authors: Tachibana, Sean R, Tang, Longteng, Zhu, Liangdong, Liu, Weimin, Wang, Yanli, Fang, Chong
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 20-12-2018
• ISSN: 1520-6106; 1520-5207
• DOI: 10.1021/acs.jpcb.8b10587

Biosensors have become an indispensable tool set in life sciences. Among them, fluorescent protein-based biosensors have great biocompatibility and tunable emission properties but their development is largely on trial and error. To facilitate a rational design, we implement tunable femtosecond stimulated Raman spectroscopy, aided by transient absorption and quantum calculations, to elucidate the working mechanisms of a single-site Pro377Arg mutant of an emission ratiometric Ca2+ biosensor based on a green fluorescent protein–calmodulin complex. Comparisons with the parent protein and the Ca2+-free/bound states unveil more structural inhomogeneity yet an overall faster excited-state proton-transfer (ESPT) reaction inside the Ca2+-bound biosensor. The correlated photoreactant and photoproduct vibrational modes in the excited state reveal more chromophore twisting and trapping in the Ca2+-bound state during ESPT and the largely conserved chromophore dynamics in the Ca2+-free state from parent protein. The uncovered structural dynamics insights throughout an ESPT reaction inside a calcium biosensor provide important design principles in maintaining a hydrophilic, less compact, and more homogeneous environment with directional H-bonding (from the chromophore to surrounding protein residues) via bioengineering methods to improve the ESPT efficiency and quantum yield while maintaining photostability.