Excited state structural dynamics of a dual-emission calmodulingreen fluorescent protein sensor for calcium ion imaging Public Deposited

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  • Fluorescent proteins (FPs) have played a pivotal role in bioimaging and advancing biomedicine. The versatile fluorescence from engineered, genetically encodable FP variants greatly enhances cellular imaging capabilities, which are dictated by excited state structural dynamics of the embedded chromophore inside the protein pocket. Visualization of the molecular choreography of the photoexcited chromophore requires a spectroscopic technique capable of resolving atomic motions on the intrinsic timescale of femtosecond to picosecond. We use femtosecond stimulated Raman spectroscopy to study the excited state conformational dynamics of a recently developed FP-calmodulin sensor, GEM-GECO1, for calcium ion (Ca²⁺) sensing. This study reveals that, in the absence of Ca²⁺, the dominant skeletal motion is a ~170 cm⁻¹ phenol-ring in-plane rocking that facilitates excited-state proton transfer (ESPT) with a time constant of ~30 ps (6 times slower than wild-type GFP) to reach the green fluorescent state. The functional relevance of the motion is corroborated by molecular dynamics simulations. Upon Ca²⁺ binding, this in-plane rocking motion diminishes and blue emission from a trapped photoexcited neutral chromophore dominates because ESPT is inhibited. Fluorescence properties of site-specific protein mutants lend further support to functional roles of key residues including proline 377 in modulating the H-bonding network and fluorescence outcome. These crucial structural dynamics insights will aid rational design in bioengineering to generate versatile, robust, and more sensitive optical sensors to detect Ca²⁺ at physiologically relevant environments.
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  • Oscar, B. G., Liu, W., Zhao, Y., Tang, L., Wang, Y., Campbell, R. E., & Fang, C. (2014). Excited-state structural dynamics of a dual-emission calmodulin-green fluorescent protein sensor for calcium ion imaging. Proceedings of the National Academy of Sciences, 111(28), 10191-10196. doi:10.1073/pnas.1403712111
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  • 111
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  • 28
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  • Financial support is provided by the Oregon State University Faculty Startup Research Grant (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.
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