Structure and dynamics of complex systems in condensed phase govern their functions which broadly impact biological and materials applications. However, a complete understanding of those processes is hindered by the availability of suitable tools which can resolve atomic motions on molecular timescales. We have developed tunable femtosecond stimulated Raman spectroscopy (FSRS) in the mixed time-frequency domain1,2 to investigate the photochemical events of organic chromophores in solution and proteins, correlating the photoacidity and local environment on the crucial femtosecond (fs) to picosecond (ps) timescales. Resonance enhancement conditions for transient molecular species along the multidimensional reaction coordinate are achieved with the guidance from fs transient absorption.3,4 The study of model systems including a photoacid pyranine and a green fluorescent protein (GFP) chromophore in solution reveals how excited state proton transfer (ESPT) competes with ring twisting motions while different energy relaxation pathways are involved.1,3 Upon incorporation into a protein matrix, the chromophore undergoes ESPT in a more complex environment that consists of nearby residues and solvent molecules.5,6 Results from our investigations of a series of FP-based calcium biosensors will be discussed that substantiate the resolving power of FSRS in probing primary structural events in the electronic excited state. This new endeavor enables us to bridge the gap between structure and function and also generate targeted design principles for improved functionalities, which has been recently shown for an FP-biosensor with a single-site mutation.

References:
(1) Liu, W.; Han, F.; Smith, C.; Fang, C. Ultrafast Conformational Dynamics of Pyranine during Excited State Proton Transfer in Aqueous Solution Revealed by Femtosecond Stimulated Raman Spectroscopy. J. Phys. Chem. B 2012, 116, 10535-10550.
(2) Zhu, L.; Liu, W.; Fang, C. A Versatile Femtosecond Stimulated Raman Spectroscopy Setup with Tunable Pulses in the Visible to Near Infrared. Appl. Phys. Lett. 2014, 105, 041106.
(3) Liu, W.; Wang, Y.; Tang, L.; Oscar, B. G.; Zhu, L.; Fang, C. Panoramic Portrait of Primary Molecular Events Preceding Excited State Proton Transfer in Water. Chem. Sci. 2016, 7, 5484-5494.
(4) Liu, W.; Tang, L.; Oscar, B. G.; Wang, Y.; Chen, C.; Fang, C. Tracking Ultrafast Vibrational Cooling During Excited State Proton Transfer Reaction with Anti-Stokes and Stokes Femtosecond Stimulated Raman Spectroscopy. J. Phys. Chem. Lett. 2017, 8, 997–1003.
(5) Fang, C.; Frontiera, R. R.; Tran, R.; Mathies, R. A. Mapping GFP Structure Evolution during Proton Transfer with Femtosecond Raman Spectroscopy. Nature 2009, 462, 200-204.
(6) Oscar, B. G.; Liu, W.; Zhao, Y.; Tang, L.; Wang, Y.; Campbell, R. E.; Fang, C. Excited-State Structural Dynamics of a Dual-Emission Calmodulin-Green Fluorescent Protein Sensor for Calcium Ion Imaging. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 10191-10196.