Chromophore Exchange in the LOV2 Domain of the Plant Photoreceptor Phototropin1 from Oat

Abstract

Phototropins are a family of plant photoreceptors mediating blue light responses such as phototropism, leaf expansion, chloroplast relocation, and stomatal opening. Characteristic for phototropins are two LOV domains which, when expressed in heterologous systems, each carry a single flavin mononucleotide (FMN) chromophore. Here we describe removal of FMN from the LOV2 domain of Avena sativa using a hydrophobic matrix and successful incorporation of flavin adenine dinucleotide (FAD), riboflavin, and 5'-malonyl-riboflavin into the resulting apoprotein; 5-deaza-FMN was not incorporated under the applied conditions. The chromoproteins reconstituted with the various flavins showed absorption spectra and photocycle almost identical to those of the native LOV2 domain and that reconstituted with FMN except for the kinetics: LOV2-riboflavin and LOV2-5'-malonyl-riboflavin showed more rapid regeneration in the dark. LOV2-FAD can be hydrolyzed to LOV2-FMN with phosphodiesterase, indicating that the adenosine part extrudes from the protein. Together with the data from the X-ray structure (Crosson, S., and Moffat, K. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 2995-3000), the results allow us to decide which of the chromophore-protein interactions are essential for the reconstitution process.

Influence of a Joining Helix on the BLUF Domain of the YcgF Photoreceptor from Escherichia coli

“…The photochemical properties were also not significantly influenced by the chemical identity of the flavin chromophore as reported for the R. sphaeroides AppA BLUF, [9,11] E. gracilis PACa F2 [23] and the Avena sativa LOV2 domains. [54] Accordingly, the observed protein-to-chromophore ratios, the refolding yields or the red-shifts of light-adapted flavin chromophores were not found to depend on the chosen flavin species. Most of the associated structural changes of the YcgF BLUF domain hence take place at the flavin isoalloxazine ring, and neither the adenosine monophosphate moiety nor the phosphate group of FAD is required for formation of the signaling state.…”

“…In this case, where the Ja-helix is missing, the phosphate group might interact via salt bridges with the YcgF BLUF photoreceptor as described for AppA [13] and the LOV2 domain. [54,55] Quite contrary to this, in the presence of the Ja-helix, the lightadapted state of the BLUF domain is mostly stabilized if bound to FAD. This specific stabilization might arise either from the dimeric association of the BLUF domain, which is more meaningful for the situation in the intact YcgF photoreceptor than the tetra-/pentameric state of the stand-alone YcgF domain, or from an indirect interaction across the entire YcgF BLUF domain between the C-terminal Ja helix and the jutting AMP moiety.…”

Blue Light Induces Radical Formation and Autophosphorylation in the Light-sensitive Domain of Chlamydomonas Cryptochrome

“…It has been shown for the homologous DNA photolyase from E. coli that riboflavin and FMN fail to bind to the apoprotein (44). These findings are in contrast to other blue light receptor domains such as the light-, oxygen-, and voltagesensitive (LOV) domains of phototropin, where reconstitution with flavin analogs has been achieved (45), or the sensors of blue light using FAD (BLUF) domain, where expression conditions determine the chromophore composition (34).…”

The Switch that Does Not Flip: The Blue-Light Receptor YtvA from Bacillus subtilis Adopts an Elongated Dimer Conformation Independent of the Activation State as Revealed by a Combined AUC and SAXS Study

“…Reconstitution of apoprotein and chromophore exchange to achieve a sample with FMN as the sole chromophore was done with FMN (Sigma-Aldrich Chemie GmbH, Munich, Germany) according to a protocol for LOV2 from Avena sativa. 39 FMN was purified using ion-exchange chromatography prior to usage. Chromophore homogeneity was checked by HPLC.…”