Dataset Information

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.

SUBMITTER: Dragnea V

PROVIDER: S-EPMC2774281 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Spectroscopic studies of the AppA BLUF domain from Rhodobacter sphaeroides: addressing movement of tryptophan 104 in the signaling state.

Dragnea Vladimira V Arunkumar Alphonse I AI Yuan Hua H Giedroc David P DP Bauer Carl E CE

Biochemistry 20091001 42

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 i ...[more]

PMID: 19746968

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Project description:BackgroundPhotosynthetic (PS) gene expression in Rhodobacter sphaeroides is regulated in response to changes in light and redox conditions mainly by PrrB/A, FnrL and AppA/PpsR systems. The PrrB/A and FnrL systems activate the expression of them under anaerobic conditions while the AppA/PpsR system represses them under aerobic conditions. Recently, two mathematical models have been developed for the AppA/PpsR system and demonstrated how the interaction between AppA and PpsR could lead to a phenotype in which PS genes are repressed under semi-aerobic conditions. These models have also predicted that the transition from aerobic to anaerobic growth mode could occur via a bistable regime. However, they lack experimentally quantifiable inputs and outputs. Here, we extend one of them to include such quantities and combine all relevant micro-array data publically available for a PS gene of this bacterium and use that to parameterise the model. In addition, we hypothesise that the AppA/PpsR system alone might account for the observed trend of PS gene expression under semi-aerobic conditions.ResultsOur extended model of the AppA/PpsR system includes the biological input of atmospheric oxygen concentration and an output of photosynthetic gene expression. Following our hypothesis that the AppA/PpsR system alone is sufficient to describe the overall trend of PS gene expression we parameterise the model and suggest that the rate of AppA reduction in vivo should be faster than its oxidation. Also, we show that despite both the reduced and oxidised forms of PpsR binding to the PS gene promoters in vitro, binding of the oxidised form as a repressor alone is sufficient to reproduce the observed PS gene expression pattern. Finally, the combination of model parameters which fit the biological data well are broadly consistent with those which were previously determined to be required for the system to show (i) the repression of PS genes under semi-aerobic conditions, and (ii) bistability.ConclusionWe found that despite at least three pathways being involved in the regulation of photosynthetic genes, the AppA/PpsR system alone is capable of accounting for the observed trends in photosynthetic gene expression seen at different oxygen levels.

Dataset Information

Spectroscopic studies of the AppA BLUF domain from Rhodobacter sphaeroides: addressing movement of tryptophan 104 in the signaling state.

Publications

Spectroscopic studies of the AppA BLUF domain from Rhodobacter sphaeroides: addressing movement of tryptophan 104 in the signaling state.

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