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Excited State Structural Events of a Dual-Emission Fluorescent Protein Biosensor for Ca²⁺ Imaging Studied by Femtosecond Stimulated Raman Spectroscopy

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  • Fluorescent proteins (FPs) are luminescent biomolecules that emit characteristic hues upon irradiation. A group of calmodulin (CaM)-green FP (GFP) chimeras have been previously engineered to enable the optical detection of calcium ions (Ca²⁺). We investigate one of these genetically encoded Ca²⁺ biosensors for optical imaging (GECOs), GEM-GECO1, which fluoresces green without Ca²⁺ but blue with Ca²⁺, using femtosecond stimulated Raman spectroscopy (FSRS). The time-resolved FSRS data (<800 cm⁻¹) reveal that initial structural evolution following 400-nm photoexcitation involves small-scale coherent proton motions on both ends of the chromophore two-ring system with a <250 fs time constant. Upon Ca²⁺ binding, the chromophore adopts a more twisted conformation in the protein pocket with increased hydrophobicity, which inhibits excited-state proton transfer (ESPT) by effectively trapping the protonated chromophore in S₁. Both the chromophore photoacidity and local environment form the ultrafast structural dynamics basis for the dual-emission properties of GEM-GECO1. Its photochemical transformations along multidimensional reaction coordinates are evinced by distinct stages of FSRS spectral evolution, particularly related to the ~460 and 504 cm⁻¹ modes. The direct observation of lower frequency modes provides crucial information about the nuclear motions preceding ESPT, which enriches our understanding of photochemistry and enables the rational design of new biosensors.
  • This article is part of the Photoinduced Proton Transfer in Chemistry and Biology Symposium special issue. This is an author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by the American Chemical Society and can be found at: http://pubs.acs.org/journal/jpcbfk
  • Keywords: Femtosecond stimulated Raman spectroscopy, Excited state proton transfer, Ultrafast conformational dynamics, Calcium-sensing fluorescent proteins, Fluorescence modulation mechanism
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  • Wang, Y., Tang, L., Liu, W., Zhao, Y., Oscar, B. G., Campbell, R. E., & Fang, C. (2015). Excited state structural events of a dual-emission fluorescent protein biosensor for Ca²⁺ imaging studied by femtosecond stimulated Raman spectroscopy. The Journal of Physical Chemistry B, 119(6), 2204-2218. doi:10.1021/jp505698z
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  • 119
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  • 6
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  • This work was supported by the Oregon State University Faculty Research Startup Grant and General Research Fund Award (to C.F.), Natural Sciences and Engineering Research Council of Canada & Canadian Institutes of Health Research (toR.E.C.), and a University of Alberta fellowship & an Alberta Innovates scholarship (to Y.Z.).R.E.C. holds a Tier II Canada Research Chair.
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