title: Fluorescence from Multiple Chromophore Hydrogen-Bonding States in the Far-Red Protein TagRFP675.
abstract: Far-red fluorescent proteins are critical for in vivo imaging applications, but the relative importance of structure versus dynamics in generating large Stokes-shifted emission is unclear. The unusually red-shifted emission of TagRFP675, a derivative of mKate, has been attributed to the multiple hydrogen bonds with the chromophore N-acylimine carbonyl. We characterized TagRFP675 and point mutants designed to perturb these hydrogen bonds with spectrally resolved transient grating and time-resolved fluorescence (TRF) spectroscopies supported by molecular dynamics simulations. TRF results for TagRFP675 and the mKate/M41Q variant show picosecond time scale red-shifts followed by nanosecond time blue-shifts. Global analysis of the TRF spectra reveals spectrally distinct emitting states that do not interconvert during the S1 lifetime. These dynamics originate from photoexcitation of a mixed ground-state population of acylimine hydrogen bond conformers. Strategically tuning the chromophore environment in TagRFP675 might stabilize the most red-shifted conformation and result in a variant with a larger Stokes shift.
authors.affiliation: JILA, University of Colorado and NIST , Boulder, Colorado 80309, United States.|Department of Chemistry & Biochemistry, University of Colorado , Boulder, Colorado 80309, United States.
authors.forename: Patrick E
authors.initials: PE
authors.lastname: Konold
authors.affiliation: Department of Chemistry, Pohang University of Science and Technology (POSTECH) , Pohang, South Korea 790-784.
authors.forename: Eunjin
authors.initials: E
authors.lastname: Yoon
authors.affiliation: Department of Chemistry, Pohang University of Science and Technology (POSTECH) , Pohang, South Korea 790-784.
authors.forename: Junghwa
authors.initials: J
authors.lastname: Lee
authors.affiliation: JILA, University of Colorado and NIST , Boulder, Colorado 80309, United States.|Department of Chemistry & Biochemistry, University of Colorado , Boulder, Colorado 80309, United States.
authors.forename: Samantha L
authors.initials: SL
authors.lastname: Allen
authors.affiliation: Department of Physics, Florida International University , Miami, Florida 33199, United States.
authors.forename: Prem P
authors.initials: PP
authors.lastname: Chapagain
authors.affiliation: Department of Physics, Florida International University , Miami, Florida 33199, United States.
authors.forename: Bernard S
authors.initials: BS
authors.lastname: Gerstman
authors.affiliation: Department of Physics, Florida International University , Miami, Florida 33199, United States.|Department of Physics, Virginia Tech , Blacksburg, Virginia 24061, United States.
authors.forename: Chola K
authors.initials: CK
authors.lastname: Regmi
authors.affiliation: Massachusetts Institute of Technology Media Lab, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Kiryl D
authors.initials: KD
authors.lastname: Piatkevich
authors.affiliation: Department of Anatomy & Structural Biology, Albert Einstein College of Medicine , Bronx, New York 10461, United States.
authors.forename: Vladislav V
authors.initials: VV
authors.lastname: Verkhusha
authors.affiliation: Department of Chemistry, Pohang University of Science and Technology (POSTECH) , Pohang, South Korea 790-784.
authors.forename: Taiha
authors.initials: T
authors.lastname: Joo
authors.affiliation: JILA, University of Colorado and NIST , Boulder, Colorado 80309, United States.|Department of Chemistry & Biochemistry, University of Colorado , Boulder, Colorado 80309, United States.
authors.forename: Ralph
authors.initials: R
authors.lastname: Jimenez
mesh_terms: D005453:Fluorescence; D006860:Hydrogen Bonding; D008164:Luminescent Proteins / Q000737:chemistry*; D008968:Molecular Conformation; D056004:Molecular Dynamics Simulation; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein