title: The role of local and remote amino acid substitutions for optimizing fluorescence in bacteriophytochromes: A case study on iRFP.
abstract: Bacteriophytochromes are promising tools for tissue microscopy and imaging due to their fluorescence in the near-infrared region. These applications require optimization of the originally low fluorescence quantum yields via genetic engineering. Factors that favour fluorescence over other non-radiative excited state decay channels are yet poorly understood. In this work we employed resonance Raman and fluorescence spectroscopy to analyse the consequences of multiple amino acid substitutions on fluorescence of the iRFP713 benchmark protein. Two groups of mutations distinguishing iRFP from its precursor, the PAS-GAF domain of the bacteriophytochrome P2 from Rhodopseudomonas palustris, have qualitatively different effects on the biliverdin cofactor, which exists in a fluorescent (state II) and a non-fluorescent conformer (state I). Substitution of three critical amino acids in the chromophore binding pocket increases the intrinsic fluorescence quantum yield of state II from 1.7 to 5.0% due to slight structural changes of the tetrapyrrole chromophore. Whereas these changes are accompanied by an enrichment of state II from ~40 to ~50%, a major shift to ~88% is achieved by remote amino acid substitutions. Additionally, an increase of the intrinsic fluorescence quantum yield of this conformer by ~34% is achieved. The present results have important implications for future design strategies of biofluorophores.
authors.affiliation: Technische Universität Berlin, Institut für Chemie, Sekr. PC14, Straβe des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Buhrke
authors.affiliation: Technische Universität Berlin, Institut für Chemie, Sekr. PC14, Straβe des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Francisco
authors.initials: F
authors.lastname: Velazquez Escobar
authors.affiliation: Technische Universität Berlin, Institut für Chemie, Sekr. PC14, Straβe des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Luisa
authors.initials: L
authors.lastname: Sauthof
authors.affiliation: Technische Universität Berlin, Institut für Chemie, Sekr. PC14, Straβe des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Svea
authors.initials: S
authors.lastname: Wilkening
authors.affiliation: Technische Universität Berlin, Institut für Chemie, Sekr. PC14, Straβe des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Nico
authors.initials: N
authors.lastname: Herder
authors.affiliation: Technische Universität Berlin, Institut für Chemie, Sekr. PC14, Straβe des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Neslihan N
authors.initials: NN
authors.lastname: Tavraz
authors.affiliation: Technische Universität Berlin, Institut für Chemie, Sekr. PC14, Straβe des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Mario
authors.initials: M
authors.lastname: Willoweit
authors.affiliation: Technische Universität Berlin, Institut für Chemie, Sekr. PC14, Straβe des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Anke
authors.initials: A
authors.lastname: Keidel
authors.affiliation: Technische Universität Berlin, Institut für Chemie, Sekr. PC14, Straβe des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Tillmann
authors.initials: T
authors.lastname: Utesch
authors.affiliation: Technische Universität Berlin, Institut für Chemie, Sekr. PC14, Straβe des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Maria-Andrea
authors.initials: MA
authors.lastname: Mroginski
authors.affiliation: Technische Universität Berlin, Institut für Chemie, Sekr. PC14, Straβe des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Franz-Josef
authors.initials: FJ
authors.lastname: Schmitt
authors.affiliation: Technische Universität Berlin, Institut für Chemie, Sekr. PC14, Straβe des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Hildebrandt
authors.affiliation: Technische Universität Berlin, Institut für Chemie, Sekr. PC14, Straβe des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Friedrich
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites; D008958:Models, Molecular; D010834:Phytochrome / Q000737:chemistry / Q000235:genetics*; D045663:Quantum Dots; D012241:Rhodopseudomonas / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D001426:Bacterial Proteins; D010834:Phytochrome