title: Excited-state structural dynamics of a dual-emission calmodulin-green fluorescent protein sensor for calcium ion imaging.
abstract: 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 biosensor, GEM-GECO1, for calcium ion (Ca(2+)) sensing. This study reveals that, in the absence of Ca(2+), 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 Ca(2+) 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 Ca(2+) in physiologically relevant environments.
authors.affiliation: Department of Chemistry, Oregon State University, Corvallis, OR 97331; and.
authors.forename: Breland G
authors.initials: BG
authors.lastname: Oscar
authors.affiliation: Department of Chemistry, Oregon State University, Corvallis, OR 97331; and.
authors.forename: Weimin
authors.initials: W
authors.lastname: Liu
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada.
authors.forename: Yongxin
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Department of Chemistry, Oregon State University, Corvallis, OR 97331; and.
authors.forename: Longteng
authors.initials: L
authors.lastname: Tang
authors.affiliation: Department of Chemistry, Oregon State University, Corvallis, OR 97331; and.
authors.forename: Yanli
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Campbell
authors.affiliation: Department of Chemistry, Oregon State University, Corvallis, OR 97331; and Chong.Fang@oregonstate.edu.
authors.forename: Chong
authors.initials: C
authors.lastname: Fang
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002118:Calcium / Q000032:analysis*; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics; D002413:Cations, Divalent / Q000032:analysis; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013059:Spectrum Analysis, Raman
chemical_list: D002147:Calmodulin; D002413:Cations, Divalent; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002118:Calcium
keywords: calcium-sensing fluorescent protein; femtosecond Raman spectroscopy; fluorescence modulation mechanism; molecular movie