title: A Q63E Rhodobacter sphaeroides AppA BLUF domain mutant is locked in a pseudo-light-excited signaling state.
abstract: The AppA BLUF photoreceptor from Rhodobacter sphaeroides contains a conserved key residue, Gln63, that is thought to undergo a shift in hydrogen-bonding interactions when a bound flavin is light excited. In this study we have characterized two substitution mutants of Gln63 (Q63E, Q63L) in the context of two constructs of the BLUF domain that have differing lengths, AppA1-126 and AppA17-133. Q63L mutations in both constructs exhibit a blue-shifted flavin absorption spectrum as well as a loss of the photocycle. Altered fluorescence emission and fluorescence quenching of the Q63L mutant indicate significant perturbations of hydrogen bonding to the flavin and surrounding amino acids which is confirmed by (1)H-(15)N HSQC NMR spectroscopy. The Q63E substitution mutant is constitutively locked in a lit signaling state as evidenced by a permanent 3 nm red shift of the flavin absorption, quenching of flavin fluorescence emission, analysis of (1)H-(15)N HSQC spectra, and the inability of full-length AppA Q63E to bind to the PpsR repressor. The significance of these findings on the mechanism of light-induced output signaling is discussed.
authors.affiliation: Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405, United States.
authors.forename: Vladimira
authors.initials: V
authors.lastname: Dragnea
authors.forename: Alphonse I
authors.initials: AI
authors.lastname: Arunkumar
authors.forename: Chul Won
authors.initials: CW
authors.lastname: Lee
authors.forename: David P
authors.initials: DP
authors.lastname: Giedroc
authors.forename: Carl E
authors.initials: CE
authors.lastname: Bauer
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002850:Chromatography, Gel; D005420:Flavoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D012242:Rhodobacter sphaeroides / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: C116785:AppA protein, Rhodobacter sphaeroides; D001426:Bacterial Proteins; D005420:Flavoproteins