title: Ultrafast infrared spectroscopy of an isotope-labeled photoactivatable flavoprotein.
abstract: The blue light using flavin (BLUF) domain photosensors, such as the transcriptional antirepressor AppA, utilize a noncovalently bound flavin as the chromophore for photoreception. Since the isoalloxazine ring of the chromophore is unable to undergo large-scale structural change upon light absorption, there is intense interest in understanding how the BLUF protein matrix senses and responds to flavin photoexcitation. Light absorption is proposed to result in alterations in the hydrogen-bonding network that surrounds the flavin chromophore on an ultrafast time scale, and the structural changes caused by photoexcitation are being probed by vibrational spectroscopy. Here we report ultrafast time-resolved infrared spectra of the AppA BLUF domain (AppA(BLUF)) reconstituted with isotopically labeled riboflavin (Rf) and flavin adenine dinucleotide (FAD), which permit the first unambiguous assignment of ground and excited state modes arising directly from the flavin carbonyl groups. Studies of model compounds and DFT calculations of the ground state vibrational spectra reveal the sensitivity of these modes to their environment, indicating that they can be used as probes of structural dynamics.
authors.affiliation: Department of Chemistry, Stony Brook University, Stony Brook, NY 11794-3400, USA.
authors.forename: Allison
authors.initials: A
authors.lastname: Haigney
authors.forename: Andras
authors.initials: A
authors.lastname: Lukacs
authors.forename: Rui-Kun
authors.initials: RK
authors.lastname: Zhao
authors.forename: Allison L
authors.initials: AL
authors.lastname: Stelling
authors.forename: Richard
authors.initials: R
authors.lastname: Brust
authors.forename: Ryu-Ryun
authors.initials: RR
authors.lastname: Kim
authors.forename: Minako
authors.initials: M
authors.lastname: Kondo
authors.forename: Ian
authors.initials: I
authors.lastname: Clark
authors.forename: Michael
authors.initials: M
authors.lastname: Towrie
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Greetham
authors.forename: Boris
authors.initials: B
authors.lastname: Illarionov
authors.forename: Adelbert
authors.initials: A
authors.lastname: Bacher
authors.forename: Werner
authors.initials: W
authors.lastname: Römisch-Margl
authors.forename: Markus
authors.initials: M
authors.lastname: Fischer
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Meech
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Tonge
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry* / Q000378:metabolism*; D007553:Isotope Labeling; D007700:Kinetics; D008027:Light; D017434:Protein Structure, Tertiary; D012242:Rhodobacter sphaeroides; D013055:Spectrophotometry, Infrared / Q000379:methods*; D014732:Vibration
chemical_list: C116785:AppA protein, Rhodobacter sphaeroides; D001426:Bacterial Proteins; D005420:Flavoproteins; D005182:Flavin-Adenine Dinucleotide