Poster: Photomorphogenesis

Abs # 509: Proton Transfers and Protein Conformation changes in the Photocycle of the LOV2 Domain of Phototropin 1

Presenter:	Bogomolni, Roberto A, bogo@chemistry.ucsc.edu
Authors	Bogomolni, Roberto A (A)   Corchnoy, Stephanie B (A)   Swartz, Trevor E (B) (A)  Szundi, Istvan  (A)   Lewis, James W (A)   Briggs, Winslow R (B)
Affiliations:	(A): University of California, Santa Cruz (B): Carnegie Institution of Washington

The phototropins (Phot1 and Phot2) are membrane-associated flavoproteins that function as photoreceptors in plant phototropism, chloroplast movements, stomatal opening, and leaf expansion. Phot1 has two FMN-binding domains, LOV1 and LOV2, within its N-terminal region and a C-terminal serine-threonine protein kinase domain. Light stimulation of LOV2 produces a cysteinyl adduct at the flavin C(4a) position, which decays thermally back to the dark state with a half-time of about 50s. We measured the pH-dependence and solvent kinetic isotope effects of the photocycle. Adduct formation and decay showed minimal pH dependence between pH 3.7 and pH 9.5 indicating that the pK values of mechanistically relevant groups are outside this range. LOV2 showed a nearly five-fold slowing of adduct formation in D2O indicating that the rate-limiting step involves proton transfer(s). Light-induced changes in the far-UV circular dichroism (CD) spectrum of LOV2 revealed putative protein structural perturbations during the back reaction. The light-minus-dark CD difference spectrum resembles an inverted a-helix spectrum, suggesting that a-helicity is reversibly lost upon light-irradiation. Decay kinetics for CD spectral changes in the far-UV region occur at the same rate as those in the visible region indicating synchronous relaxation of protein and chromophore structures, suggesting that an intramolecular proton transfer is a common rate-limiting step for both processes .