Blue Light-Induced Conformational Changes in a Light-Regulated Transcription Factor, Aureochrome-1

Abstract

Aureochrome-1 (AUREO1) is a blue light (BL) receptor that mediates the branching response in the stramenopile alga, Vaucheria frigida. AUREO1 harbors a basic leucine zipper (bZIP) domain at the N-terminus and a light-oxygen-voltage-sensing (LOV) domain within the C-terminal region, and has been suggested to function as a light-regulated transcription factor. To understand the molecular mechanism of AUREO1, we have prepared three recombinant proteins: a full-length AUREO1 (FL), an N-terminal truncated construct containing bZIP and LOV (ZL) and a LOV-only (LOV) construct. The constructs showed the same absorption and fluorescent spectra in the dark state and underwent the characteristic cyclic reaction as previously observed in LOV domains upon BL excitation. FL and ZL bound to DNA in a sequence-specific manner. BL appeared to induce a shift of the α-helical structure of the LOV domain to a β-sheet structure, but did not alter the hydrodynamic radius (R(H)) of this domain. ZL formed a dimer possibly through disulfide linkages in the bZIP and the linker region between bZIP and LOV. BL induced an approximately 5% increase in the R(H) of ZL, although its secondary structure was unchanged. These results support a schema where BL-induced changes in the LOV domain may cause conformational changes in the bZIP and/or the linker of a dimeric ZL molecule. Since a 5% increase of the R(H) was also observed with the FL construct, BL may induce global conformational changes similar to those observed for ZL, and formation of the FL dimer may facilitate DNA binding.

Blue Light-induced Dimerization of Monomeric Aureochrome-1 Enhances Its Affinity for the Target Sequence

“…Preparation of AUREO1 Recombinant Proteins-Recombinant AUREO1 proteins were prepared as previously described (20,21). In brief, Escherichia coli cells expressing recombinant AUREO1 proteins were harvested by centrifugation and disrupted by sonication.…”

“…For DTT treatment, the recombinant AUREO1 proteins were incubated in loading buffer containing 50 mM DTT at 25°C for 16 -32 h in the dark. The purity of AUREO1 recombinant proteins were confirmed by SDS-PAGE, and the concentrations were determined from the absorbance at 447 nm using an extinction coefficient of 13,000 M Ϫ1 cm Ϫ1 (21). All experimental manipulations were performed under a yellow fluorescent lamp FLR40SY-IC/M (Mitsubishi Co., Ltd) or a yellow LED lamp with maximum wavelength ( max ) at 590 nm.…”

“…Spectroscopic Measurements-The AUREO1 recombinant proteins were diluted to 5 M (3 M for DTT-FL) in loading buffer containing 1 or 50 mM DTT, and ultraviolet visible (UVvisible) absorption spectra were measured using a V550 spectrophotometer (JASCO) (21). Spectral changes accompanying the regeneration of recombinant AUREO1 proteins were monitored after termination of BL illumination ( max at 470 nm for 1 min) at 25°C in the dark.…”

“…The transient grating method showed BL-induced S390 formation of AUREO1-ZL and -LOV with a time constant of 2.8 s and subsequent changes in the diffusion constant with time constants of 140 -160 ms (20). BL appeared to induce a shift of the ␣-helical structure of the LOV domain to a ␤-sheet structure, without affecting the hydrodynamic radius (R H ) of this domain, and a ϳ5% increase in the R H of ZL, without changing the contents of its secondary structure (21). Moreover, from the analyses of site-directed mutants, ZL formed a dimer through disulfide linkages of two Cys residues, Cys 162 and Cys 182 , in the bZIP and linker region between bZIP and LOV.…”

“…To elucidate the molecular mechanism of AUREO1, we have previously reported the preparation of six recombinant AUREO1 proteins with a histidine tag at the N or C terminus, full-length AUREO1 (AUREO1-FL), and N terminally truncated AUREO1 (AUREO1-ZL), consisting of the bZIP and LOV domains, and AUREO1-LOV, consisting of the LOV domain, and have investigated global conformational changes in these recombinant proteins using the transient grating technique, size exclusion chromatography (SEC), circular dichroism (CD) spectropolarimetry, and dynamic light scattering (DLS) (20,21). The transient grating method showed BL-induced S390 formation of AUREO1-ZL and -LOV with a time constant of 2.8 s and subsequent changes in the diffusion constant with time constants of 140 -160 ms (20).…”