My research focuses on the structure and function of AppA, the anti-repressor of photosynthetic gene transcription. AppA is a flavoprotein, able to sense the presence of light and oxygen in the bacterial cell (Rb. sphaeroides ), and regulate the expression of photosynthetic genes as a function of these two factors. The protein assumes different conformations in the dark , when it is able to associate with the repressor PpsR in the cell, and after exposure to strong white light when this complex dissociates. The conformational changes are fully reversible. We have crystallized the BLUF domain of AppA (in the “dark” state) and solved the structure at 2.3 Å resolution. AppA BLUF domain is a dimer in solution and also in the crystal. Flavin is anchored between two a – helices and shielded by 5 b - sheets from one side. Most of the conserved amino acids are located in the flavin-binding pocket or making the contact between two monomers. As the protein in the crystal is still photoactive, we are now working on the “light” structure, or signaling state, of this protein. Currently we are also trying to crystallize AppA full-length protein and the complex AppA-PpsR. For functional studies, we use time-resolved fluorescence and pump-probe absorption spectroscopy on AppAwt and several mutants to elucidate molecular mechanism of light excitation and regulatory properties of this protein. We have demonstrated that electron transfer from conserved Tyr21 to the N5 of flavin is the first step in the light excitation process.

Recent publications:

Kraft, B. J., Masuda, S., Kikuchi, J., Dragnea, V., Tollin, G., Zaleski, J. M., and Bauer, C. E. (2003) Spectroscopic and mutational analysis of the blue-light photoreceptor AppA: a novel photocycle involving flavin stacking with an aromatic amino acid, Biochemistry 42, 6726-34.

Anderson, S., Dragnea, V., Masuda, S., Ybe, J., Moffat, K., and Bauer, C. (2005) Structure of a novel photoreceptor, the BLUF domain of AppA from Rhodobacter sphaeroides, Biochemistry 44, 7998-8005.

Dragnea, V., Waegele, M., Balascuta, S., Bauer, C., and Dragnea, B. (2005) Time-resolved spectroscopic studies of the AppA blue-light receptor BLUF domain from Rhodobacter sphaeroides, Biochemistry 44, 15978-85.