title: Vibrational assignment of the ultrafast infrared spectrum of the photoactivatable flavoprotein AppA.
abstract: The blue light using flavin (BLUF) domain proteins, such as the transcriptional antirepressor AppA, are a novel class of photosensors that bind flavin noncovalently in order to sense and respond to high-intensity blue (450 nm) light. Importantly, the noncovalently bound flavin chromophore is unable to undergo large-scale structural change upon light absorption, and thus there is significant 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 isotopes of FAD, specifically [U-(13)C(17)]-FAD, [xylene-(13)C(8)]-FAD, [U-(15)N(4)]-FAD, and [4-(18)O(1)]-FAD both in solution and bound to AppA(BLUF). This allows for unambiguous assignment of ground- and excited-state modes arising directly from the flavin. Studies of model compounds and DFT calculations of the ground-state vibrational spectra reveal the sensitivity of these modes to their environment, indicating they can be used as probes of structural dynamics.
authors.affiliation: Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, United States.
authors.forename: Allison
authors.initials: A
authors.lastname: Haigney
authors.forename: Andras
authors.initials: A
authors.lastname: Lukacs
authors.forename: Richard
authors.initials: R
authors.lastname: Brust
authors.forename: Rui-Kun
authors.initials: RK
authors.lastname: Zhao
authors.forename: Michael
authors.initials: M
authors.lastname: Towrie
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Greetham
authors.forename: Ian
authors.initials: I
authors.lastname: Clark
authors.forename: Boris
authors.initials: B
authors.lastname: Illarionov
authors.forename: Adelbert
authors.initials: A
authors.lastname: Bacher
authors.forename: Ryu-Ryun
authors.initials: RR
authors.lastname: Kim
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* / Q000235:genetics / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000378:metabolism; D013055:Spectrophotometry, Infrared; D014732:Vibration
chemical_list: C116785:AppA protein, Rhodobacter sphaeroides; D001426:Bacterial Proteins; D005420:Flavoproteins; D011994:Recombinant Proteins