|Title||Illuminating Photochemistry of an Excitation Ratiometric Fluorescent Protein Calcium Biosensor.|
|Publication Type||Journal Article|
|Year of Publication||2017|
|Authors||Tang, Longteng, Yanli Wang, Weimin Liu, Yongxin Zhao, Robert E. Campbell, and Chong Fang|
|Journal||J Phys Chem B|
|Date Published||2017 Apr 13|
Fluorescent protein (FP)-based biosensors have become an important and promising tool to track metal ion movement inside living systems. Their working principles after light irradiation, however, remain elusive. To facilitate the rational design of biosensors, we dissect the fluorescence modulation mechanism of a newly developed excitation ratiometric green FP-based Cabiosensor, GEX-GECO1, using femtosecond stimulated Raman spectroscopy (FSRS) in the electronic excited state. Upon 400 nm photoexcitation, characteristic vibrational marker bands at ∼1180 and 1300 cmshow concomitant decay and rise dynamics, probing the progression of an ultrafast excited state proton transfer (ESPT) reaction. The Ca-bound biosensor exhibits two distinct populations that undergo ESPT with ∼6 and 80 ps time constants, in contrast to one dominant population with a 25 ps time constant in the Ca-free biosensor. This result is supported by key structural constraints from molecular dynamics simulations with and without Ca. The blueshift of the ∼1265 cmC-O stretch mode unravels the vibrational cooling dynamics of the protonated chromophore regardless of Cabinding events. This unique line of inquiry reveals the essential structural dynamics basis of fluorescence modulation inside an excitation ratiometric protein biosensor, correlating the uncovered chromophore structural heterogeneity with different H-bonding configurations and intrinsic proton transfer rate in the photoexcited state.
|Alternate Journal||J Phys Chem B|