Spectroscopic studies of the AppA BLUF domain from Rhodobacter sphaeroides: addressing movement of tryptophan 104 in the signaling state.
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.
Dragnea
Vladimira
V
Departments of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405, USA.
Arunkumar
Alphonse I
AI
Yuan
Hua
H
Giedroc
David P
DP
Bauer
Carl E
CE
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*
C116785:AppA protein, Rhodobacter sphaeroides; D001426:Bacterial Proteins; D005420:Flavoproteins; D014364:Tryptophan