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Dimerization of LOV domains of Rhodobacter sphaeroides (RsLOV) studied with FRET and stopped-flow experiments

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Abstract

The bacterium Rhodobacter sphaeroides has a short LOV (light-oxygen-voltage) domain, which is not connected to an effector domain but has an α-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 ≈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) × 105 M−1 s−1, and an equilibrium constant of the dark-state dimer in the range (3.0–7.0) × 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.

Graphical abstract: Dimerization of LOV domains of Rhodobacter sphaeroides (RsLOV) studied with FRET and stopped-flow experiments

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Article information


Submitted
24 Oct 2019
Accepted
27 Dec 2019
First published
27 Dec 2019

Photochem. Photobiol. Sci., 2020, Advance Article
Article type
Paper

Dimerization of LOV domains of Rhodobacter sphaeroides (RsLOV) studied with FRET and stopped-flow experiments

K. Magerl and B. Dick, Photochem. Photobiol. Sci., 2020, Advance Article , DOI: 10.1039/C9PP00424F

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