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Hidden photoinduced reactivity of the blue fluorescent protein mKalama1

DOI: 10.1039/C5CP00887E DOI Help
PMID: 25805012 PMID Help

Authors: Russell B. Vegh (Georgia Institute of Technology) , Dmitry A. Bloch (Georgia Institute of Technology) , Andreas S. Bommarius (Georgia Institute of Technology) , Michael Verkhovsky (Georgia Institute of Technology) , Sergei Pletnev (Georgia Institute of Technology) , Hideo Iwai (University of Helsinki; Georgia Institute of Technology) , Anastasia V. Bochenkova (Georgia Institute of Technology) , Kyril M. Solntsev (Georgia Institute of Technology)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Physical Chemistry Chemical Physics , VOL 17 (19) , PAGES 12472 - 12485

State: Published (Approved)
Published: May 2015

Abstract: Understanding the photoinduced dynamics of fluorescent proteins is essential for their applications in bioimaging. Despite numerous studies on the ultrafast dynamics, the delayed response of these proteins, which often results in population of kinetically trapped dark states of various origins, is largely unexplored. Here, by using transient absorption spectroscopy spanning the time scale from picoseconds to seconds, we reveal a hidden reactivity of the bright blue-light emitting protein mKalama1 previously thought to be inert. This protein shows no excited-state proton transfer during its nanosecond excited-state lifetime; however, its tyrosine-based chromophore undergoes deprotonation coupled to non-radiative 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 transient absorption signal has a complex nature and spans the whole microsecond-to-second time scale. The mechanisms underlying the relaxation kinetics are disclosed based on the X-ray structural analysis of mKalama1 and the high-level electronic structure calculations of proposed intermediates in the photocycle. We conclude that the non-radiative excited-state decay includes two major branches: internal conversion coupled to intraprotein proton transfer, where a conserved residue E222 serves as the proton acceptor; and ionization induced by two consecutive resonant absorption events, followed by deprotonation of the chromophore radical cation to bulk solvent through a novel watermediated proton-wire pathway. Our findings open up new perspectives on the dynamics of fluorescent proteins as tracked by its optical transient absorption in the time domain extending up to seconds.

Subject Areas: Biology and Bio-materials


Instruments: I24-Microfocus Macromolecular Crystallography