Magerl, Kathrin and Dick, Bernhard (2020) Dimerization of LOV domains of Rhodobacter sphaeroides (RsLOV) studied with FRET and stopped-flow experiments. PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES, 19 (2). pp. 159-170. ISSN 1474-905X, 1474-9092
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The bacterium Rhodobacter sphaeroides has a short LOV (light-oxygen-voltage) domain, which is not connected to an effector domain but has an a-helix extension at the N-terminus as well as a helix-turn-helix (HTH) motiv at the C-terminus. These extensions offer possibilities for interactions with effector enzymes or DNA. Whereas many LOV domains show a tendency to form dimers in the light state, RsLOV is unique in that it is a dimer in the dark state but dissociates into monomers after blue-light excitation. We studied the kinetics of this dimerization process by a combination of FRET spectroscopy and stopped-flow experiments with a time resolution of approximate to 10 ms. Although excitation of the flavin chromophore in dye-labeled LOV domains leads to considerable FRET from flavin to the dye, the typical adduct formation between flavin and a nearby cysteine still occurs with considerable yield. We obtain a rate constant for LOV-LOV dimerization in the range (0.8-1.8) x 10(5) M-1 s(-1), and an equilibrium constant of the dark-state dimer in the range (3.0-7.0) x 10(-6) M. Dissociation of the dimers in the light state and reforming of dimers after return to the dark state was monitored using an anti-FRET effect caused by excitonic interaction between dye labels on different monomers. Reforming of the dark state dimers is slower than recovery of the flavin-cysteinyl adduct, indicating that light-induced conformational changes in the LOV domain persist for much longer time than the adduct lifetime.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | BLUE-LIGHT; STRUCTURAL BASIS; DNA-BINDING; AUREOCHROME-LOV; PROTEIN; OXYGEN; PHOTORECEPTOR; PHOTOTROPIN; PHOTOCYCLE; REVEALS; |
| Subjects: | 500 Science > 540 Chemistry & allied sciences |
| Divisions: | Chemistry and Pharmacy > Institut für Physikalische und Theoretische Chemie Chemistry and Pharmacy > Institut für Physikalische und Theoretische Chemie > Chair of Chemistry III - Physical Chemistry (Molecular Spectroscopy and Photochemistry) Chemistry and Pharmacy > Institut für Physikalische und Theoretische Chemie > Chair of Chemistry III - Physical Chemistry (Molecular Spectroscopy and Photochemistry) > Prof. Dr. Bernhard Dick |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 31 Mar 2021 07:03 |
| Last Modified: | 31 Mar 2021 07:03 |
| URI: | https://pred.uni-regensburg.de/id/eprint/45190 |
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