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

Downloadable Content

Download PDF

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:

This article is part of the Photoinduced Proton Transfer in Chemistry and Biology Symposium special issue.


Attribute NameValues
Alternative Title
  • 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.
Resource Type
Date Available
Date Issued
  • 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
Journal Title
Journal Volume
  • 119
Journal Issue/Number
  • 6
Rights Statement
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.
Peer Reviewed



This work has no parents.