Fluorescence Modulation by Ultrafast Chromophore Twisting Events: Developing a Powerful Toolset for Fluorescent-Protein-Based Imaging.

The advancement of modern life sciences has benefited tremendously from the discovery and development of fluorescent proteins (FPs), widely expressed in live cells to track a myriad of cellular events. The chromophores of various FPs can undergo many ultrafast photophysical and/or photochemical processes in the electronic excited state and emit fluorescence with different colors. However, the chromophore becomes essentially nonfluorescent in solution environment due to its intrinsic twisting capability upon photoexcitation. To study "microscopic" torsional events and their effects on "macroscopic" fluorescence, we have developed an integrated ultrafast characterization platform involving femtosecond transient absorption (fs-TA) and wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS). A wide range of naturally occurring, circularly permuted, non-canonical amino-acid-decorated FPs and FP-based optical highlighters with photochromicity, photoconversion, and/or photoswitching capabilities have been recently investigated in great detail. Twisting conformational motions were elucidated to exist in all of these systems but to various extents. The associated different ultrafast pathways can be monitored via frequency changes of characteristic Raman bands during primary events and functional processes. The mapped electronic and structural dynamics information is crucial and has shown great potential and initial success for the rational design of proteins and other photoreceptors with novel functions and fluorescence properties.