- 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.
- 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
|Funding Statement (additional comments about funding)
- 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 (to
R.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.