title: Tuning calcium biosensors with a single-site mutation: structural dynamics insights from femtosecond Raman spectroscopy.
abstract: Fluorescent protein biosensors are popular reporters for biological processes and life sciences, but their fundamental working mechanisms remain unclear. To characterize the functional fluorescence events on their native timescales, we implemented wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) to shed light on a blue-green emission-ratiometric fluorescent protein based Ca2+biosensor with a single Pro377Arg mutation. The transient Raman modes of the embedded chromophore from ca. 1000-1650 cm-1exhibit characteristic intensity and frequency dynamics which infer the underlying atomic motions and photochemical reaction stages. Our experimental study reveals the hidden structural inhomogeneity of the protein local environment upon Ca2+binding with the mutated arginine residue trapping multiple chromophore subpopulations, which manifest distinct time constants of ∼16 and 90 ps for excited state proton transfer (ESPT) following 400 nm photoexcitation. The altered ESPT reaction pathways and emission properties of the Ca2+biosensor represent the foundational step of rationally designing advanced fluorescent protein biosensors to tune their functionalities by site-specifically altering the local environment (e.g., the active site) of the embedded chromophore.
authors.affiliation: Oregon State University, Department of Chemistry, 263 Linus Pauling Science Centre (lab), 153 Gilbert Hall (office), Corvallis, OR 97331, USA. Chong.Fang@oregonstate.edu.
authors.forename: Sean R
authors.initials: SR
authors.lastname: Tachibana
authors.affiliation: Oregon State University, Department of Chemistry, 263 Linus Pauling Science Centre (lab), 153 Gilbert Hall (office), Corvallis, OR 97331, USA. Chong.Fang@oregonstate.edu.
authors.forename: Longteng
authors.initials: L
authors.lastname: Tang
authors.affiliation: Oregon State University, Department of Chemistry, 263 Linus Pauling Science Centre (lab), 153 Gilbert Hall (office), Corvallis, OR 97331, USA. Chong.Fang@oregonstate.edu.
authors.forename: Yanli
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Oregon State University, Department of Chemistry, 263 Linus Pauling Science Centre (lab), 153 Gilbert Hall (office), Corvallis, OR 97331, USA. Chong.Fang@oregonstate.edu and Department of Physics, 301 Weniger Hall, Corvallis, OR 97331, USA.
authors.forename: Liangdong
authors.initials: L
authors.lastname: Zhu
authors.affiliation: Oregon State University, Department of Chemistry, 263 Linus Pauling Science Centre (lab), 153 Gilbert Hall (office), Corvallis, OR 97331, USA. Chong.Fang@oregonstate.edu.
authors.forename: Weimin
authors.initials: W
authors.lastname: Liu
authors.affiliation: Oregon State University, Department of Chemistry, 263 Linus Pauling Science Centre (lab), 153 Gilbert Hall (office), Corvallis, OR 97331, USA. Chong.Fang@oregonstate.edu and Department of Physics, 301 Weniger Hall, Corvallis, OR 97331, USA.
authors.forename: Chong
authors.identifier: http://orcid.org/0000-0002-8879-1825
authors.initials: C
authors.lastname: Fang
mesh_terms: D001665:Binding Sites; D015374:Biosensing Techniques; D002118:Calcium / Q000032:analysis*; D006860:Hydrogen Bonding; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D013050:Spectrometry, Fluorescence; D013059:Spectrum Analysis, Raman
chemical_list: D008164:Luminescent Proteins; D002118:Calcium