title: Ultrafast Structural Evolution and Chromophore Inhomogeneity inside a Green-Fluorescent-Protein-Based Ca(2+) Biosensor.
abstract: Understanding excited-state structural dynamics of fluorescent-protein-based biosensors for Ca(2+) imaging is crucial for developing new in vivo Ca(2+) indicators and advancing bioimaging. We implemented wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) with a 530 nm Raman pump to uncover the working mechanism of an intensiometric fluorescent-protein biosensor, G-GECO1.1, highlighting the deprotonation process of its embedded chromophore. Besides confirming the dynamic difference of excited-state proton transfer (ESPT) in the Ca(2+)-free/bound protein, we revealed a chromophore two-ring twisting process with time constants of 36/60 ps that competes with ESPT. In contrast with FSRS data collected using the 800 nm Raman pump, the bluer Raman pump enables us to access a subset of reactant population with partially deprotonated character that exhibits an additional ESPT component on the ∼5 ps time scale. These findings provide deep mechanistic insights into the inhomogeneity and subpopulation-specific conformational dynamics of biosensor chromophores, which will guide the rational design of improved biosensors for metal ion imaging.
authors.affiliation: Department of Chemistry, Oregon State University , Corvallis, Oregon 97331, United States.
authors.forename: Longteng
authors.initials: L
authors.lastname: Tang
authors.affiliation: Department of Chemistry, Oregon State University , Corvallis, Oregon 97331, United States.
authors.forename: Weimin
authors.initials: W
authors.lastname: Liu
authors.affiliation: Department of Chemistry, Oregon State University , Corvallis, Oregon 97331, United States.
authors.forename: Yanli
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Chemistry, Oregon State University , Corvallis, Oregon 97331, United States.
authors.forename: Liangdong
authors.initials: L
authors.lastname: Zhu
authors.affiliation: Department of Chemistry, Oregon State University , Corvallis, Oregon 97331, United States.
authors.forename: Fangyuan
authors.initials: F
authors.lastname: Han
authors.affiliation: Department of Chemistry, Oregon State University , Corvallis, Oregon 97331, United States.
authors.forename: Chong
authors.initials: C
authors.lastname: Fang
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D002118:Calcium / Q000032:analysis*; D002413:Cations, Divalent / Q000032:analysis; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D008958:Models, Molecular; D013059:Spectrum Analysis, Raman / Q000379:methods*
chemical_list: D002413:Cations, Divalent; D049452:Green Fluorescent Proteins; D002118:Calcium