Proton transfer is one of the fastest, ubiquitous reactions in biological systems. The fluorescence of the green fluorescent protein (GFP) chromophore, for example, is the result of ultrafast excited state proton transfer (ESPT) from a photoexcited protonated A* state to a deprotonated I* state. Molecular vibrations of the GFP chromophore in the 1000�1800 cm-1 range have been used to study ESPT, but the lower-frequency collective vibrational motions that reflect overall structural change have not been extensively examined. Using femtosecond stimulated Raman spectroscopy, three modes below 800 cm-1 were characterized for a GFP-calmodulin complex in which ESPT is dependent on Ca2+ binding. In the Ca2+-free protein, ESPT occurs with ~30 ps time constant; while in the Ca2+-bound protein, ESPT is blocked. Double peaks at ~462 and 510 cm-1 blueshift ~5 cm-1 in the first 2 ps and show sub-ps initial decay in the Ca2+-free protein. This is mostly due to a change in resonance enhancement in the Franck-Condon region. The second decay time constant increases dramatically upon Ca2+ binding that is consistent with inhibition of ESPT. The excited state mode at 462 (472) cm-1 in the Ca2+-free (bound) protein is particularly sensitive to the chromophore environment and likely involves significant motion at the phenolic end of the chromophore prior to ESPT. A 720 cm-1 deformation mode of the conjugated ring system with a 1.3 ps first decay time is prominent in both chromophores but manifest different decay dynamics on the hundreds of ps timescale upon Ca2+ binding. To gain more structural dynamics insights during and after ESPT reaction, we plan to perform tunable Raman experiments to specifically enhance the deprotonated I* modes. Experiments involving the chromophore phenolic-ring modification with functional groups are also underway. We can then infer how characteristic atomic motions modulate the fluorescence properties of the protein complex.

Characterization of low-frequency vibrational motions in excited state proton transfer for a green fluorescent protein-calmodulin sensor

248th National Meeting (2014)

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