The excited- and ground-state photodynamics of wild-type Green Fluorescent Protein (wtGFP) are contrast to the S65T/H148D and S65T/H148E mutants with femtosecond dispersed pump-dump-probe measurements. Discrete transient intermediates with specific spectral properties are identified to describe the evolution in the mutant variants with greater complexity than in wtGFP. For the first time, excited- and ground state structural evolution of the chromophore is observed as non-stationary dumping kinetics. The proton transfer kinetics observed in wtGFP is accelerated two orders of magnitude faster in S65T/H148D presumably due to a low-barrier hydrogen bond between the intrinsic chromophore and the introduced aspartate residue. Here we follow the same strategy to employ an extra carefully timed laser pulses to remove or transfer part of the excited state population to ground state, where protons at different residing sites may be distinguished by choosing distinct back proton transfer pathways. Dump induced signals resolve a bifurcation of the proton transfer kinetic resulting in multiple resting spots. The excited and ground-state photo induced dynamics can be simultaneously resolved by using broadband PDP transient spectroscopy, combined with relevant models that underlie connectivity scheme in system dependent dynamics. The PDP photodynamics of S65T/H148D and S65T/H128E mutants were contrasted to wtGFP. Here the same multipulse transient absorption measurement is employed to characterize ground-state proton transfer processes in above two GFP mutants, which have diverse ESPT behaviors to the wtGFP.

Proton shuttle kinetics in a GFP variant switched by pKs changes in a low barrier hydrogen bond

248th National Meeting (2014)

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