Kazunori Zikihara

Quaternary structure of LOV‐domain containing polypeptide of Arabidopsis FKF1 protein

Flavin‐binding, Kelch repeat, F‐box (FKF1) protein is a photoreceptor to regulate flowering of Arabidopsis. The protein has a light, oxygen and voltage (LOV)‐sensing domain binding a flavin mononucleotide. The photo‐activation of the domain is an indispensable step to initiate the cellular signaling for flowering. In the present study, a LOV‐containing polypeptide of FKF1 was prepared by an overexpression system, and the quaternary structure of it was studied by size exclusion chromatography and small‐angle X‐ray scattering. The apparent molecular weight from chromatography suggested a globular trimeric or an anisotropic‐shaped dimeric association of the polypeptide in solution. The scattering experiment demonstrated a dimeric association of the polypeptides with an elongated molecular shape displaying the radius of gyration of 27 Å and the maximum dimension of 94 Å. The molecular shape simulated from scattering profiles suggests an antiparallel association of the LOV domains in the dimer. Though the absorption spectrum of blue‐light irradiated polypeptide was stable in the photoactivated state for a long period, the scattering profiles showed very small changes between the dark and light conditions. Based on the homologies in the amino‐acid sequences and the scattering profiles, these results are discussed in connection with the structures and function of LOV domains of phototropin.

Involvement of Electron Transfer in the Photoreaction of Zebrafish Cryptochrome‐DASH†

Photoreaction of a blue‐light photoreceptor Cryptochrome‐DASH (Cry‐DASH), a new member of the Cryptochrome family, from zebrafish was studied by UV–visible absorption spectroscopy in aqueous solutions at 293u2003K. Zebrafish Cry‐DASH binds two chromophores, a flavin adenine dinucleotide (FAD) and a N5,N10‐methenyl‐5,6,7,8‐tetrahydrofolate (MTHF) noncovalently. The bound FAD exists in the oxidized form (FADox) in the dark. Blue light converts FADox to the neutral radical form (FADH?). Formed FADH? is transformed to the fully reduced form FADH2 (or FADH−) by successive light irradiation, or reverts to FADox. FADH2 (or FADH−) reverts to FADH? or possibly to FADox directly. The effect of dithiothreitol suggests a possible electron transfer between FAD in zebrafish Cry‐DASH and reductants in the external medium. This is the first report on the photoreaction pathway and kinetics of a vertebrate Cry‐DASH family protein.

Stability of Dimer and Domain-Domain Interaction of Arabidopsis Phototropin 1 LOV2

Transient grating signals after photoexcitation of Arabidopsis phototropin 1 light-oxygen-voltage 2 (phot1LOV2) domain without the linker were found to be very sensitive to temperature. In particular, the diffusion signal drastically increased with rising temperature. The signal was consistently explained by the superposition of the photo-induced dissociation and association reactions. This observation indicated the presence of an equilibrium between the monomer and dimer forms of the phot1LOV2 domain in the dark. The equilibrium was confirmed by a gel chromatographic technique. The equilibrium constants at various temperatures were calculated from the fraction of the dimer, and the stabilization enthalpy and entropy were determined. Interestingly, the transient grating signal of phot1LOV2 with the linker (phot1LOV2-linker), which exists as the monomer form, was also temperature dependent; the diffusion signal intensity decreased with increasing temperature. Because the diffusion signal reflects a conformation change of the linker upon photoexcitation, this temperature dependence indicated that there were two forms of the phot1LOV2-linker. One form exhibited a conformational change upon photoexcitation whereas the other form showed no change. These two forms are not distinguishable spectroscopically. The fraction of these species depended on the temperature. Considering the monomer-dimer equilibrium of the phot1LOV2 domain, we suggest that the nonreactive form possesses the linker region that is dissociated from the LOV2 domain. Because the dissociation of the linker region from the LOV2 domain is a key step for the conformation change of the phot1LOV2-linker to induce biological activity, we proposed that the phototropins could have a role as a temperature sensor.

Three Putative Photosensory Light, Oxygen or Voltage (LOV) Domains with Distinct Biochemical Properties from the Filamentous Cyanobacterium Anabaena sp. PCC 7120

Abstract Light, oxygen or voltage (LOV) domains function as blue-light sensors in the phototropin family of photoreceptors found in plants, algae and bacteria. We detected putative LOV domains (Alr3170-LOV, All2875-LOV and Alr1229-LOV) in the genome of a filamentous cyanobacterium, Anabaena sp. PCC 7120. These cyanobacterial LOV domains are closely clustered with the known LOV domains. Alr3170-LOV and All2875-LOV carry the conserved cysteine residue unique to the photoactive LOV, whereas Alr1229-LOV does not. We expressed these three LOV domains in Escherichia coli and purified them. In fact, Alr3170-LOV and All2875-LOV that are conserved in Nostoc punctiforme, a related species, bound flavin mononucleotide and showed spectral changes unique to known LOV domains on illumination with blue light. Alr3170-LOV was completely photoreduced and dark reversion was slow, whereas All2875-LOV was slowly photoreduced and dark reversion was rapid. For comparison, AvAll2875-LOV in a closely related A. variabilis was also studied as a homolog of All2875-LOV. Finally, we observed that Alr1229-LOV that is not conserved in N. punctiforme showed no flavin binding.