title: Spectroscopic studies of the AppA BLUF domain from Rhodobacter sphaeroides: addressing movement of tryptophan 104 in the signaling state.
abstract: Previous crystallographic studies of the AppA BLUF domain indicated that Trp104 is capable of undertaking alternate conformations depending on the length of the BLUF domain. A BLUF domain containing a C-terminal deletion (AppA1-126) reveals that Trp104 is partially solvent exposed while a BLUF domain containing a slightly longer carboxyl terminal region (AppA17-133) shows that Trp104 is deeply buried. This observation has led to a model proposing that Trp104 moves from a deeply buried position in the dark state to a solvent-exposed position in the light excited state. In this study we investigated whether there is indeed movement of Trp104 upon light excitation using a combination of NMR and absorption spectroscopy, steady-state fluorescence, and acrylamide quenching of tryptophan fluorescence. Our results indicate that AppA17-133 and AppA1-126 contain Trp104 in distinct alternate conformations in solution and that light absorption by the flavin causes partial movement/uncovering of Trp104. However, we conclude that light exposure does not cause dramatic change of Trp104 from "Trp-in" to "Trp-out" conformations (or vice versa) upon light absorption. These results do not support a model of Trp104 movement as a key output signal.
authors.affiliation: Departments of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405, USA.
authors.forename: Vladimira
authors.initials: V
authors.lastname: Dragnea
authors.forename: Alphonse I
authors.initials: AI
authors.lastname: Arunkumar
authors.forename: Hua
authors.initials: H
authors.lastname: Yuan
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* / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009154:Mutation; D017434:Protein Structure, Tertiary; D012242:Rhodobacter sphaeroides / Q000378:metabolism*; D015398:Signal Transduction; D014364:Tryptophan / Q000737:chemistry*
chemical_list: C116785:AppA protein, Rhodobacter sphaeroides; D001426:Bacterial Proteins; D005420:Flavoproteins; D014364:Tryptophan