| Autor: |
Maksimov EG; Department of Biophysics, Faculty of Biology, M.V. Lomonosov Moscow State University, 119992, Moscow, Russia. emaksimoff@yandex.ru., Moldenhauer M; Institute of Chemistry PC 14, Technical University of Berlin, Straße des 17. Juni 135, 10623, Berlin, Germany., Shirshin EA; Department of Quantum Electronics, Faculty of Physics, M.V. Lomonosov Moscow State University, 119992, Moscow, Russia., Parshina EA; Department of Biophysics, Faculty of Biology, M.V. Lomonosov Moscow State University, 119992, Moscow, Russia., Sluchanko NN; A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, 119071, Moscow, Russia., Klementiev KE; Department of Biophysics, Faculty of Biology, M.V. Lomonosov Moscow State University, 119992, Moscow, Russia., Tsoraev GV; Department of Biophysics, Faculty of Biology, M.V. Lomonosov Moscow State University, 119992, Moscow, Russia., Tavraz NN; Institute of Chemistry PC 14, Technical University of Berlin, Straße des 17. Juni 135, 10623, Berlin, Germany., Willoweit M; Institute of Chemistry PC 14, Technical University of Berlin, Straße des 17. Juni 135, 10623, Berlin, Germany., Schmitt FJ; Institute of Chemistry PC 14, Technical University of Berlin, Straße des 17. Juni 135, 10623, Berlin, Germany., Breitenbach J; Institute for Molecular Biosciences, Johann Wolfgang Goethe University of Frankfurt, Max-von-Laue-Straße 9, 60438, Frankfurt, Germany., Sandmann G; Institute for Molecular Biosciences, Johann Wolfgang Goethe University of Frankfurt, Max-von-Laue-Straße 9, 60438, Frankfurt, Germany., Paschenko VZ; Department of Biophysics, Faculty of Biology, M.V. Lomonosov Moscow State University, 119992, Moscow, Russia., Friedrich T; Institute of Chemistry PC 14, Technical University of Berlin, Straße des 17. Juni 135, 10623, Berlin, Germany., Rubin AB; Department of Biophysics, Faculty of Biology, M.V. Lomonosov Moscow State University, 119992, Moscow, Russia. |
| Abstrakt: |
Orange carotenoid protein (OCP) is a water-soluble photoactive protein responsible for a photoprotective mechanism of nonphotochemical quenching in cyanobacteria. Under blue-green illumination, OCP converts from the stable orange into the signaling red quenching form; however, the latter form could also be obtained by chemical activation with high concentrations of sodium thiocyanate (NaSCN) or point mutations. In this work, we show that a single replacement of tryptophan-288, normally involved in protein-chromophore interactions, by alanine, results in formation of a new protein form, hereinafter referred to as purple carotenoid protein (PCP). Comparison of resonance Raman spectra of the native photoactivated red form, chemically activated OCP, and PCP reveals that carotenoid conformation is sensitive to the structure of the C-domain, implicating that the chromophore retains some interactions with this part of the protein in the active red form. Combination of differential scanning fluorimetry and picosecond time-resolved fluorescence anisotropy measurements allowed us to compare the stability of different OCP forms and to estimate relative differences in protein rotation rates. These results were corroborated by hydrodynamic analysis of proteins by dynamic light scattering and analytical size-exclusion chromatography, indicating that the light-induced conversion of the protein is accompanied by a significant increase in its size. On the whole, our data support the idea that the red form of OCP is a molten globule-like protein in which, however, interactions between the carotenoid and the C-terminal domain are preserved. |
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