The photophysics and photochemistry of the Blue Fluorescent Protein mKalama1 was studied on the timescale ranging from subpicoseconds to seconds. This protein shows no excited-state proton transfer during its nanosecond excited-state singlet lifetime; however, its chromophore undergoes a deprotonation in the ground state after electronic relaxation. Such deprotonation causes distinct optical absorption changes in the broad UV-to-NIR spectral range (ca. 300 - 800 nm); the disappearance of the respective transient absorption signal has a complex nature and spans the whole microsecond-to-second time scale. The high-resolution crystal structure analysis of mKalama1 shows that the excited-state proton transfer pathways known in GFP are blocked in this protein and the conserved E222 residue serves as a primary ground-state proton acceptor. The mechanisms underlying the relaxation kinetics are discussed based on the electronic structure calculations of the proposed intermediates in the mKalama1 photocycle. The observed ground-state transient absorption features were assigned to the anionic, zwitterionic, and radical forms of the chromophore. The non-radiative branches in the excited-state decay include the 'hidden' ground-state intraprotein proton transfer coupled to the photoinduced chromophore isomerization and radical formation. The latter is induced by multiphoton ionization of the neutral chromophore coupled to the proton release through a well-defined water molecule chain. Our findings open a new perspective of the fluorescent protein dynamics as tracked by its optical transient absorption signature on the micro-to-millisecond time scale.

Hidden photoinduced proton transfer in the Blue Fluorescent Protein mKalama1: Theoretical insight

248th National Meeting (2014)

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