Blue light-induced dimerization of monomeric AUREO1

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

Osamu Hisatomi1, Yoichi Nakatani1, Ken Takeuchi1, Fumio Takahashi2,3, and Hironao Kataoka4

1Department of Earth and Space Science, Graduate School of Science, Osaka University, Toyonaka,

Osaka 560-0043, Japan 2Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga

525-8577, Japan 3PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan

4Botanical Gardens, Tohoku University, 12-2 Kawauchi, Aoba-ku, Sendai 980-0862, Japan

*Running title: Blue light-induced dimerization of monomeric AUREO1 To whom correspondence should be addressed: Osamu Hisatomi, Department of Earth and Space Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan, Tel.: +81-6-6850-5500; Fax: +81-6-6850-5500; E-mail: hisatomi@ess.sci.osaka-u.ac.jp Key words: aureochrome; bZIP; LOV; dynamic light scattering; size exclusion column chromatography Background: Aureochromes in stramenopiles are thought to function as light-regulated transcription factors, though the molecular mechanism is unknown. Results: Monomeric AUREO1 is present in reduced conditions and undergoes dimerization upon illumination. Conclusion: Blue light-induced dimerization enhances the affinity for the target sequence. Significance: AUREO1 is useful for understanding the blue light responses of stramenopiles, and for optogenetics and biophysical analyses. ABSTRACT:

Aureochrome-1 (AUREO1) is a blue light (BL) receptor that mediates the branching response in stramenopile alga, Vaucheria frigida. AUREO1 contains a basic leucine zipper (bZIP) domain in the central region and a light-oxygen-voltage-sensing (LOV) domain at the C-terminus, and has been suggested to function as a light-regulated transcription factor. We have previously reported that preparations of recombinant AUREO1 contained the complete coding sequence (FL) and N-terminal truncated protein (ZL) containing bZIP and LOV domains, and suggested that wild-type ZL (ZLwt2) was in a dimer form with intermolecular

disulfide-linkages at Cys162 and Cys182 [Hisatomi et al., (2013) Plant Cell Physiol., 54, 93-106]. In the present study, we report the photoreactions, oligomeric structures, and DNA binding of monomeric cysteine-to-serine mutated ZL (ZLC2S), DTT-treated ZL (DTT-ZL), and FL (DTT-FL). Recombinant AUREO1 showed similar spectral properties and dark regeneration kinetics to those of dimeric ZLwt2. Dynamic light scattering and size exclusion chromatography revealed that ZLC2S and DTT-ZL were monomeric in the dark state. Dissociation of intermolecular disulfide bonds of ZLwt2 was in equilibrium with a midpoint oxidation-redox potential of approximately −245 ± 15 mV. BL induced the dimerization of monomeric ZL, which subsequently increased its affinity for the target sequence. Also, DTT-FL was monomeric in the dark state and underwent BL-induced dimerization, which led to formation of the FL2/DNA complex. Taken together, our results suggest that monomeric AUREO1 is present in vivo, with dimerization playing a key role in its role as a BL-regulated transcription factor. Light plays an important role in triggering various physiological processes for almost all organisms. Many organisms possess various photoreceptors that bind different chromophores that respond to different wavelengths of light (1).

http://www.jbc.org/cgi/doi/10.1074/jbc.M114.554618The latest version is at JBC Papers in Press. Published on May 1, 2014 as Manuscript M114.554618

Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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Phototropin is one of the most well-studied blue light (BL) receptor proteins in plants, and has been reported to mediate phototropism, chloroplast movement, stomatal opening, and leaf photomorphogenesis, to maximize the efficiency of photosynthesis (2-6). Phototropin has two light-oxygen-voltage-sensing (LOV) domains (7), and is a subset of the Per-ARNT-Sim superfamily. LOV acts as a light sensory domain located in the N-terminal region, and regulates the activity of a Ser/Thr protein kinase domain located at the C-terminus. LOV domains possess a non-covalently attached flavin adenine mononucleotide (FMN) as a chromophore. Upon BL excitation, FMN in the dark state (D450) forms a cysteinyl adduct with a highly conserved cysteine residue (adduct state, S390) (8-10). S390 reverts to D450 with characteristic time constants, and the types of LOV domains reflect their functions (11,12). The photochemical reactions induce conformational changes in the protein moiety that are thought to regulate the activities of various signal output domains. Besides the phototropin-type receptors with two LOV domains, there are many proteins consisting of one LOV domain and a variety of signal output modules (13). Some of them have been reported to possess a DNA-binding motif (14-16), and have been thought to regulate expression of genes in a BL-dependent manner. Aureochrome (AUREO) was first discovered in stramenopile alga, Vaucheria frigida, and has two homologs named aureochrome-1 (AUREO1) and aureochrome-2 (AUREO2) (14). RNA interference studies suggested that AUREO1 and AUREO2 play roles during blue light-induced branching and during development of a sex organ, respectively (14). Both AUREO1 and AUREO2 have a LOV domain located in the C-terminal region and a basic leucine zipper (bZIP) domain on the N-terminal side of the LOV domain. The bZIP domain is an α-helical DNA-binding motif found among eukaryotic transcription factors (17). Recombinant AUREO1 showed an absorption around 450 nm and was reported to bind the target sequence TGACGT in a light-dependent manner. It was therefore suggested that the AUREO proteins function as BL-regulated transcription factors (14). AUREO orthologs have been found only in photosynthetic stramenopiles such as brown algae, some diatoms, Ochromonas,

Chattonella, and Aureococcus (18). Interestingly, the AUREO proteins lack the effector domains at their C-terminus and the Glu-Lys salt bridges that are conserved in most other LOV domains. Crystal structures of aureochrome-1 LOV have been reported, and light-induced conformational changes have been reported to occur in the quaternary structure of the LOV dimer and in Phe298 of the Hβ strand in the core (19). However, the molecular mechanism of AUREOs during regulation of gene expression remains unclear. 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 (TG) technique, size exclusion chromatography (SEC), circular dichroism (CD) spectropolarimetry, and dynamic light scattering (DLS) (20,21). The TG method showed BL-induced S390 formation of AUREO1-ZL and -LOV with a time constant of 2.8 microseconds and subsequent changes in the diffusion constant with time constants of 140–160 milliseconds (20). BL appeared to induce a shift of the α-helical structure of the LOV domain to a β-sheet structure, without affecting the hydrodynamic radius (RH) of this domain, and a ~5% increase in the RH 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, Cys162 and Cys182, in the bZIP and the linker region between bZIP and LOV. In the present study, we investigated the spectral changes, oligomeric structures, and DNA binding of FL and ZL and its cysteine-to-serine mutant, ZL-C162S/C182S, using spectroscopic, DLS, and SEC analyses. Our results suggested that AUREO1 exists as a monomer in reducing conditions and BL induces dimerization of monomeric AUREO1, which subsequently increases affinity for the target DNA sequence. Consistent with these results, we also propose a model describing the molecular mechanism of the BL-regulated transcription factor, AUREO1.

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EXPERIMENTAL PROCEDURES

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. After cell debris were removed by centrifugation, recombinant proteins were purified twice with a Ni-NTA column (Ni SepharoseTM6 Fast Flow, GE Healthcare) and with a HiTrap Heparin HP column (GE Healthcare), according to the manufacturer’s instructions. After exchanging the solvents to the loading buffer (400 mM NaCl, 20 mM Tris-HCl, pH 7.0) containing 2 mM DTT using a gel filtration column (PD-10, GE Healthcare), the recombinant proteins were stored at 4 °C until use. 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−1cm−1 (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 (λmax) at 590 nm. Spectroscopic measurements − The AUREO1 recombinant proteins were diluted to 5 µM (3 µM for DTT-FL) in the loading buffer containing 1 mM or 50 mM DTT, and ultraviolet-visible (UV-vis) 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 (λmax at 470 nm for 1 min) at 25 °C in the dark. The regeneration curves of D450 were obtained from the absorbance at 447 nm. Each absorbance difference curve was fit by a single exponential using the formula, ∆Amax(1-e-kt), where ∆Amax was the maximum absorption difference and k was the rate constant of the reaction. Fluorescence emission spectra were measured at 25 °C with an excitation at 450 nm using an F-7000 fluorescence spectrophotometer (Hitach HiTech). DLS measurements − DLS of the AUREO1

recombinant protein solutions were measured with a Zetasizer-µV system (Malvern Instruments) in the automatic mode at 25 °C, and the z-average molecular sizes expressed as RH-app in solution were determined using Zetasizer Software (Version 6.20) as previously described (21). Briefly, the stock protein solutions were diluted to 40−100 µM with loading buffer containing 1 or 50 mM DTT. After removing the aggregates by centrifugation, light scattering was detected using the refractive index of the solution, 1.334, and the viscosity of the loading buffer, 0.9166 × 10-3 Pa·s. DLS of the recombinant proteins was measured several times in the dark (dark state), and immediately after the termination of BL illumination for 1 min (light state). For measuring DLS in the light-dark state, illuminated samples were incubated for 1 h in the dark after illumination. RH-app of ZLs was plotted versus the concentration, and RH was obtained from the extrapolated value at 0 µM protein concentration (21). For detection of the RH-app change during dark regeneration, ZLC2S or hZLC2S in the loading buffer containing 1 mM DTT was illuminated with BL for 1 min, and DLS measurements were performed every 2.7 min for 40 min. The RH-app change was fitted by a single exponential using the formula, RH-app = RH-app(t=∞) + (RH-app(t=0) − RH-app(t=∞))e-kt, where RH-app(t=0) and RH-app(t=∞) are the RH-app values at t=0 and t=∞, respectively, and k is the rate constant of the reaction. To investigate the effect of DTT, the ZLwt2 samples in the loading buffer, containing 50 mM DTT, were incubated in the sample holder, and light scattering was monitored for 20 h at 25 °C. In these experiments, DLS data resulting in a polydispersity index larger than 0.2 were omitted. Standard deviations of all the RH-app values obtained from the multiple measurements were less than 0.05 nm. SDS-PAGE in non-reducing conditions − Solutions containing approximately 50 pmol of recombinant ZLwt2 were added to 4 volumes of acetone and incubated at −20 ºC for more than 2 h. DTT-treated ZLwt2 was precipitated by centrifugation at 13,000×g for 5 min, and dissolved in DTT-free sample buffer (21). SDS-PAGE was run in a cold chamber maintained at 4 °C, and the gels were stained with Quick CBB plus (Wako Co. Ltd.). SEC measurements − SEC was performed

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using an AKTA purifier column chromatography system (GE Healthcare) with a Superdex 75 30/10 column (GE Healthcare), using an SEC buffer containing 300 mM or 250 mM NaCl and 20 mM Tris-HCl (pH 7.0) with or without 5 mM DTT at 25 ± 1 °C, with a flow rate of 0.5 mL/min. Three nanomoles of recombinant AUREO1 proteins were incubated in 600 µL of the SEC buffer for 1 h at 25 °C in the dark, and one third of the mixture was applied to the column (dark state). The remaining 400-µL sample solutions were illuminated for 2 min with BL, and 200 µL of the solutions were applied to the column continuously illuminated with a LED light (FJ1460; Fuji Corp.) to obtain elution profiles in the light state. The residual illuminated mixtures were then kept in the dark for 1 h at 25 °C, and applied to the column (light-dark state). The state of the samples was checked by measuring the absorption spectra of the eluted samples or by monitoring the absorbance at 390 nm and 450 nm. The concentrations of recombinant AUREO1 proteins in the eluted fractions were approximately 1 µM. To determine the effect of DTT and oxidation-redox potential (ORP) on disulfide bonds, 1 nmol of ZLwt2 was incubated in the ORP buffer containing 300 mM NaCl, 3–10 mM DTT, and 100 mM Tris-HCl (pH 7.0) at 25 ± 1 ºC in the dark. BL illuminations (λmax at 470 nm) were performed at 0.8 W/m2 (3 µmol photons m−2 s−1 at the sample position) or 38 W/m2 (150 µmol photons m−2 s−1), and illuminated samples were kept in the dark for 1 h at 25 ºC to regenerate the D450 state before analysis. Sample mixtures were eluted with SEC buffer with or without 5 mM DTT, and monitored at 250 nm and 280 nm. The monomer vs. dimer ratios were calculated from the absorbance at 280 nm, and oxidized DTT (DTTox) fractions were corrected and quantified using a molar extinction coefficient at 283 nm of 273 M-1cm-1 (22,23). The ORP of the mixtures was calculated using the Nernst equation, E = E0 + (RT/nF) ln[(DTTox)/(DTTred)2], where E0 = -332 mV (the standard potential at pH 7.0), R = 8.314 J mol−1 K−1, T was the absolute temperature, F = 95485 JV-1 mol-1, n = 2, and (DTTred) and (DTTox) were molar concentrations of reduced and oxidized DTT, respectively (24). To investigate the binding of AUREO1 to the target sequence, Cy3-labeled control Cp-oligo (Cpo) containing no target sequence and Cy3-labeled Ap-oligo (Apo)

containing the target sequence TGACGT were prepared (21), and each oligonucleotide was annealed to form the double-stranded Cpo and Apo. Then, 0.3 nmol of double-stranded Cpo or Apo was mixed with 3 nmol of recombinant AUREO1 in SEC buffer, incubated for 1 h at 25 °C, and one-third of each mixture was subjected to analysis in each state. For MW calibration of the recombinant proteins, spherical proteins in the gel filtration calibration kits for LMW (GE Healthcare), which included ribonuclease A (13.7 kDa), carbonic anhydrase (29.0 kDa), ovalbumin, (43.0 kDa), and conalbumin (75.0 kDa), were used as size markers, using a column volume of 24 mL and a void volume of 7.65 mL. RESULTS Recombinant AUREO1 proteins − Figure 1 shows a schematic drawing of recombinant AUREO1 protein, AUREO1-FLh (FL), containing the full coding sequence lacking Asn2 (N2), and N-terminally truncated AUREO1-ZL (21). The dimeric wild-type ZLh and the site-directed mutant, ZLh-C162S/C182S, in which Cys162 and Cys182 were substituted with serine, were designated as ZLwt2 and ZLC2S, respectively. The ZLC2S protein harboring a histidine tag at the N-terminus (hZLC2S) was used to investigate the effect of the tag on oligomeric structure, and on DNA binding. Previous studies showed that the histidine tag had negligible effects on the spectroscopic properties and photoreaction of recombinant AUREO1 proteins (21). FL and ZLwt2 treated with 50 mM DTT were denoted as DTT-FL and DTT-ZL, respectively, to discriminate them from untreated proteins.

Absorption and fluorescent emission spectra − To elucidate the effect of the cysteine-to-serine mutation and DTT treatment on the spectroscopic properties, we measured UV-vis absorption spectra of recombinant AUREO1 in the presence of 1 mM or 50 mM DTT. The absorption spectra of ZLwt2, ZLC2S, and hZLC2S at 1 mM DTT, and DTT-ZL and -FL at 50 mM DTT are superimposed in Fig. 2A. The absorption spectra in the visible region were almost identical to that of ZLwt2, with λmax at 447 ± 1 nm (Fig. 2A inset) (21). Figure 2B shows fluorescence spectra measured with an excitation at 450 nm, showing an emission maximum at 495 ± 1 nm with a

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shoulder at approximately 535 nm. The absorption spectral changes of ZLC2S (Fig. 2C), hZLC2S (Fig. 2D), DTT-ZL (Fig. 2E), and DTT-FL (Fig. 2F), before (thick solid line), just after BL illumination (dashed line), during dark regeneration (thin line), and at 48 min after regeneration (dotted line) were measured at 25 °C. The time course of the regeneration reaction of these recombinant AUREO1s monitored at 447 nm were superimposed in Fig. 2G. The half-life time (τ1/2 ) of each reaction was calculated as 6.8–7.8 min, which was comparable to that of ZLwt2 (τ1/2 = 7.2 ± 0.4 min) (21). From the Arrhenius plot of the regeneration reaction of ZLC2S, the activation energy was calculated as 110 ± 2 kJ/mol, which is within the error range of ZLwt2 (109 ± 2 kJ/mol). Taken together, because these recombinant AUREO1s showed nearly identical spectra and photoreaction kinetics, the cysteine-to-serine substitution and DTT treatment had negligible effects on the π-electron state and photoreaction of the LOV domain in the recombinant proteins. Oligomeric structures of ZLs in the dark state − DLS of the ZL solutions were measured to obtain their hydrodynamic radii, RH, in the dark state (Fig. 3A). Similar to RH-app of ZLwt2 (gray line), the RH-app of ZLC2S (red bars and line) and hZLC2S (blue bars and line) depended quasi-linearly on the concentration (40–100 µM), as expected from the relationship considering interparticle and hydrodynamic interactions RH-app ~ RH (1 + θC), where C is the concentration and θ is a constant (21). The RH of ZLC2S in the dark state was determined as 3.1 nm by extrapolation of the RH-app to a protein concentration of zero, which was approximately 86% (corresponding to 59% in volume) to that of ZLwt2 (3.6 nm) in the dark state. This indicated that ZLC2S was monomeric in the dark state, as expected from the results of our SDS-PAGE analyses under non-reducing conditions (21).

The RH-app of DTT-ZL at 40 or 50 µM protein concentrations (green circles) were plotted with those of ZLC2S (red triangles) and hZLC2S (blue triangles) in the presence of 50 mM DTT. RH-app appeared smaller than ZLC2S with 1 mM DTT, probably because of the high concentration of DTT, which affected the diffusion of proteins in solution. However, the RH-app of DTT-ZL was almost identical to that of ZLC2S at each

concentration, suggesting that DTT-ZL, as well as ZLC2S, were monomeric. Figure 3B shows the change in RH-app of ZLwt2 in the presence of 50 mM DTT at 40 and 50 µM protein concentrations. RH-app gradually decreased and reached a plateau at 10 h in the dark, suggesting that the dissociation of intermolecular disulfide bonds was slow under these conditions. The oligomeric structures of the ZLs were confirmed by SEC measurements (Fig. 3C). Elution profiles of ZLC2S (upper), hZLC2S (middle), and DTT-ZL (lower) were superimposed with those of ZLwt2 (gray lines). The elution profile of ZLwt2 shows a single species with an elution peak at 10.0 mL (open inverted triangle), corresponding to a MW of 67 kDa (Fig. 3D). This value is consistent with our previous finding of 65 kDa at 175 and 250 µM (20), and is similar to the dimer size calculated from the amino acid sequence (56 kDa). However, the elution peak of ZLC2S was 11.1 mL (filled inverted triangle), corresponding to a MW of 40 kDa (Fig. 3D). In addition, a single peak at 11.1 mL was detected for DTT-ZL eluted with SEC buffer containing 5 mM DTT. Together, the SEC results are consistent with DLS results, indicating that ZLC2S and DTT-ZL were monomeric in the dark state. Intermolecular disulfide bonds of ZLwt2 − To characterize intermolecular disulfide bonds, ZLwt2 was incubated for 16–32 h at 25 °C in ORP buffer containing 5 mM DTT (5 mM DTT-treated ZLwt2), and subjected to SEC analysis (Fig. 4A). Two peaks corresponding to the dimeric and monomeric species were detected in the elution profiles (upper). Similar profiles were observed when analyses were performed with the SEC elution buffer with (black lines) or without (gray lines) 5 mM DTT. The initial rate of DTTox formation in this condition was estimated less than 0.05 mM/h by using the molar extinction coefficient of 273 M-1cm-1 (data not shown). This data suggested that DTT in the SEC buffer had only a minor effect on disulfide bonds during the SEC analyses, which was consistent with the DLS data showing that the RH-app decrease of ZLwt2 was slow, even in the presence of 50 mM DTT. Whether these molecular species were dimers or monomers, was determined by SDS-PAGE under non-reducing conditions (Fig. 4B). Doublet bands appeared in the dimeric species caused by the difference of the intermolecular disulfide bonds,

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Cys162-Cys162 or Cys182-Cys182. The amount of monomeric species increased by adding 5 mM DTT to get a final concentration of 10 mM DTT and incubated for 24 h in the dark (Fig. 4A, middle), and decreased when the mixture was incubated with 10 mM H2O2 for 24 h (lower), indicating that dissociation and formation of disulfide bonds was likely during equilibrium. Interestingly, the dimeric species in the dark-regenerated samples increased, depending on the intensity and duration of illumination (Fig. 4C and 4D). Prolonged illumination with strong light as well as treatment with 10 mM H2O2 caused aggregation of the protein, (Fig. 4D, lane 5). To examine whether dimerization was directly induced by BL or indirectly affected through oxidation of the solution, the ORP of the mixtures was calculated from the amount of DTTox fractionated in the SEC analyses (Fig. 5A and 5B) (22-24). Figure 5C shows the relationship between the ORP and the monomeric ratio of 3–10 mM DTT-treated ZLwt2. Illuminated samples (open circles) have higher ORP values in comparison with non-illuminated samples (filled circles). Similar trends between ORP and monomeric ratios both in illuminated and in non-illuminated samples suggested that the shift of equilibrium was primarily through the oxidation of the solution. Because the DTT oxidation is accelerated upon illumination of 5 mM FMN in the ORP buffer (Fig. 5A, middle), the change in ORP was probably because of the photocatalytic activity of FMN (25,26). When excluding the data of the samples containing aggregated protein with H2O2 (Fig. 5C, open triangles), the midpoint ORP for dissociation of disulfide bonds in ZLwt2 was estimated to be approximately −245 ± 15 mV. DLS studies of illuminated ZLs − Next, we investigated the RH-app of monomeric ZLs in light (dashed lines) and light-dark (dotted lines) states (Fig. 6A). Surprisingly, the RH-app of ZLC2S (red lines and bars) and hZLC2S (blue lines and bars) in the light state were almost identical to those of ZLwt2 in the light state (gray dashed lines). The estimated RH of ZLC2S (3.8 nm) corresponded to 123% of the RH in the dark state (184% in volume). When illuminated ZLC2S solutions were kept in the dark for 1 h (the light-dark state), the RH-app returned to 3.1 nm as it did in the dark state. In addition, the RH-app of DTT-ZL (green circles) in the light state increased in a similar manner to

those of ZLC2S in the presence of 50 mM DTT (open symbols), and this RH-app change was reversible (closed symbols). Figure 6B shows the time course of the change in RH-app for ZLC2S (red bars and line) and hZLC2S (blue bars and line) at 40 µM after removing the BL illumination. The decrease in RH-app from 3.9 to 3.3 nm can be fitted by a single exponential with τ1/2 = 7.0–8.0 min. This time constant is in agreement with the recovery of absorbance at 447 nm during dark regeneration. We have previously reported that ZLwt2 is dimeric, irrespective of the light conditions and even at high concentrations (250 µM) (20), and that BL induced approximately a 5% change in RH because of the structural change of ZLwt2 (21). The BL-induced RH changes of monomeric ZLs were much larger than that of ZLwt2. The DLS data therefore suggested that monomeric ZLs formed dimers in the light state and reversibly dissociated into monomeric forms along with dark regeneration of D450 from S390. SEC studies of illuminated ZLs − The elution profiles of monomeric ZLs in the light (dashed line) and light-dark (dotted line) states were compared with those in the dark state (solid line). The elution peak of ZLC2S in the light state was detected at almost the same position as that of ZLwt2 (Fig. 7A). When ZLC2S was incubated for 1 h in the dark, the elution peak returned to 11.1 mL, as it did in the dark state. The estimated MW of ZLC2S in the light state (68 kDa) was comparable to that of ZLwt2, and 1.7 times larger than that in the dark state (Fig. 7B). Similar changes in elution profiles are observed for hZLC2S in the dark, light, and light-dark states (Fig. 7C), suggesting that the histidine tag does not affect the monomer-dimer transition of ZLC2S. DTT-ZL was then subjected to SEC analyses with SEC buffer containing 5 mM DTT (Fig. 7D). The elution peak showed a shift in the light state, and returned to the initial position in the light-dark state. Together, SEC results are consistent with DLS results, clearly indicating that monomeric ZLs associated into dimers in the light state, and that this monomer-dimer transition was reversible. DNA binding of ZLwt2 − To evaluate the DNA binding of AUREO1, we used two Cy3-labeled oligonucleotides, Cy3-labeled control Cp-oligo (Cpo) without the target sequence and Cy3-labeled Ap-oligo (Apo) containing the target sequence TGACGT of AUREO1 (14,21). In the

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absence of protein, both Cpo and Apo were detected as major double-stranded species with an elution peak at 11.7 mL, and a minor single-stranded species (less than 10 % of the total amount of each oligonucleotide) with an elution peak at 13.3 mL (data not shown). Figure 8A shows the elution profiles of ZLwt2 incubated with Cpo, monitored at 260 nm for detection of protein and DNA (gray solid line), 390 nm for detection of protein harboring FMN (black solid line), and 550 nm for detection of Cy3-labeled oligonucleotides (dotted line). In the dark state, the elution pattern shows two species with elution peaks at 9.9 mL and 11.9 mL (upper). The retention volumes of these peaks are within the error ranges of ZLwt2 and double-stranded Cpo (black asterisk). Similar profiles were observed in the light (middle) and light-dark (lower) states, suggesting that the ZLwt2/Cpo complex was not formed, irrespective of the light conditions. Figure 8B shows the elution profiles of ZLwt2 incubated with Apo. ZLwt2, and Apo co-eluted with a peak at 9.6 mL. The MW estimated from the elution peak (77 kDa) was consistent with that of the ZLwt2/Apo complex (Fig. 8C). These results suggested that ZLwt2 binds to Apo in a sequence-specific manner to form a stable ZLwt2/Apo complex, irrespective of the light conditions. To reduce electrostatic interactions between ZLwt2 and DNA, we analyzed the ZLwt2 and Apo mixtures in the presence of 400 mM NaCl (Fig. 8D). Two main peaks assigned as ZLwt2 and Apo were separately observed, irrespective of the light conditions. At this ionic strength, the ZLwt2/Apo complex is likely unstable. However, the presence of broadened Apo peaks suggested some interaction between ZLwt2 and Apo, even in the presence of 400 mM NaCl. DNA binding of monomeric ZLC2S − We next investigated the binding of monomeric ZLC2S with Apo (Fig. 9A). In the dark state, two major peaks were detected with a minor third peak of single-stranded Apo (gray asterisk) that did not contribute to the formation of the complex (upper). The first peak at 11.1 mL and the 2nd peak at 12.0 mL were assigned as ZLC2S and Apo, respectively. In the light state (middle), the first peak shifted to the left, and the absorbance at 550 nm detected within the first peak indicated the formation of the ZLC2S2/Apo complex (estimated MW of 72 kDa).

The first peak returned to its initial size in the dark state, when the mixture of ZLC2S and Apo was kept in the dark for 1 h (lower). When Cpo was used instead of Apo, the ZLC2S2/Cpo complex was not detected, even in the light state (data not shown). These results suggested that BL-induced dimerization of monomeric ZLC2S leads to the formation of the ZLC2S2/Apo complex. To evaluate the effect of the histidine tag and DTT treatment on DNA binding, hZLC2S and DTT-ZL were subjected to SEC analysis. Figure 9B shows the elution profiles of hZLC2S incubated with Apo in SEC buffer containing 250 mM NaCl. The hZLC2S2/Apo complex was found in the light state (middle), and not detected in the dark (upper) and light-dark (lower) states. Furthermore, the elution profiles showed that hZLC2S does not form a complex with Cpo, irrespective of the light conditions (data not shown). When SEC buffer containing 300 mM NaCl was used, hZLC2S2/Apo complex formation was not observed, even in the light state (Fig. 9C). The electrostatic interaction of the histidine hexamer adjacent to the basic region probably interrupts its binding to DNA in the presence of 300 mM NaCl. Figure 9D shows the elution profile of DTT-ZL incubated with Apo in SEC buffer containing 300 mM NaCl. The DTT-ZL2/Apo complex was formed in the light state, but not in the dark or light-dark states. Similar to ZLC2S, DTT-ZL did not bind to Cpo, irrespective of light conditions (data not shown). Together, these results suggested that monomeric ZLs selectively bound to the target sequence in a light-dependent manner, although the amino acid sequence of the N-domain could modulate the affinity. SEC analyses of DTT-FL − SEC was used to determine whether the BL-induced monomer-dimer transition was observed for DTT-FL containing the full coding sequence. The elution profile of DTT-FL showed a peak at 10.2 mL (filled diamond) in the dark state (solid line) (Fig. 10A), which shifted to 9.5 mL (open diamond) in the light state (dashed line), and returned to the initial volume when illuminated DTT-FL was kept in the dark for 1 h (dotted line). The MW estimated from the elution peak in the dark and light-dark states was 59 kDa, which was larger than that of monomeric FL calculated from the amino acid sequence (40 kDa) (Fig. 10B).

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However, the estimated MW in the light state (80 kDa) was 136% of that in the dark state, and was comparable to 79 kDa of dimeric FL (FL2). Similar differences were also found for ZLs, whose monomeric and dimeric forms had apparent MWs of 41 kDa and 67 kDa, respectively. The large MW deduced from the elution peak of DTT-FL in the dark state was probably because of the non-spherical shape of monomeric DTT-FL. The 36% increase was too large to be explained only by a structural change. We therefore concluded that DTT-FL, as well as monomeric ZL, undergo a reversible dimerization upon BL illumination. To characterize DNA binding of DTT-FL, SEC buffer containing 250 mM NaCl was used, because only a small fraction of the FL2/Apo complex was detected with SEC buffer containing 300 mM NaCl (data not shown). Figure 10C shows the elution profile of DTT-FL incubated with Cpo in SEC buffer containing 250 mM NaCl. In the dark state, two major peaks were detected, in a similar manner to ZLC2S (upper). The first and second peaks could be attributed to monomeric DTT-FL and double-stranded Cpo, respectively. In the light state, the first peak showed a shift to 9.5 mL because of the formation of FL2, but absorbance at 550 nm was not detected with this peak (middle). The elution pattern resembled that in the dark state when the mixture was kept in the dark for 1 h (lower). These results suggested that the FL2/Cpo complex was not formed, irrespective of the light conditions. When DTT-FL was incubated with Apo (Fig. 10D), two major peaks assigned as monomeric DTT-FL and double-stranded Apo were detected in the dark (upper) and light-dark (lower) states, respectively, whereas in the light state, absorbance at 550 nm of the first peak suggested that BL-induced dimerization of DTT-FL induced the formation of the FL2/Apo complex. Based upon our results, we propose a mode of action of AUREO1 as shown in Fig. 11. In reduced conditions, AUREO1 exists as a monomer, and BL induces the dimerization of monomeric AUREO1, which subsequently enhances its affinity for the target DNA sequence. In oxidizing conditions, intermolecular disulfide bond formation between the monomers increases the affinity of AUREO1 for the target sequence. AUREO1 may therefore aggregate when the redox

state strongly deviates from the normal cellular state. DISCUSSION Consistent with our previous study, the present results indicate that ZLwt2 is dimeric at a low DTT concentration. The midpoint ORP of the redox-regulated AP2/DREB-type transcription factor Rap2.4a has been reported to be -269 mV (27), and those of glutathione and thioredoxin are −240 mV and -270 mV, respectively (28,29). The midpoint ORP for the formation of the disulfide bonds in the aureochrome-1 dimer (−245 ± 15 mV) are quite close to the redox conditions in the cell. The status of the disulfide bonds would depend on the redox conditions in the cell. It has been suggested that moderate oxidation of the glutathione pool activates Rap2.4a-dependent gene expression, whereas wider deviations from the normal cellular redox state inactivates Rap2.4a by aggregate formation (27). In response to oxidative stimulus, homo- and heterodimerization of the bHLH transcription factor, Twist1, have also been reported (30). Shaikhali et al. (2012) have suggested that environmentally induced changes in the redox state regulate DNA binding activity of the G-group of bZIP transcription factors in Arabidopsis (31). Moreover, 13 among 20 AUREO sequences found in the database possessed cysteine residues in the bZIP domain and linker region between the bZIP and LOV domains. AUREO1 could be involved in both light- and redox-sensing, as reported in the blue light receptor (AppA) of an anoxygenic phototrophic bacterium, Rhodobacter sphaeroides (32). BL-induced dimerization has been reported for a LOV-HTH transcription factor of a marine bacterium EL222 and a circadian clock regulator of Neurospora VVD. The light-driven structural change leads to dimerization of EL222 on DNA, but the relatively short lifetime of the EL222 photoexcited state complicates its characterization (15,33). The N-terminally truncated VVD forms a dimer with the Kd value of 2-10 µM in the light state (34). Considering the concentrations of recombinant AUREO1s in the eluted fractions of our SEC experiments were estimated as approximately 1 µM, the upper limit on the Kd of the monomer-dimer equilibria in the light state are

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placed at 1 µM. Also, our DLS data suggest that the Kd of the equilibria in the dark state are greater than 100 µM. Although the concentration of aureochrome-1 in the cell has not been estimated so far, BL-induced dimerization may contribute rapid adaptation to light condition. Recent progress has been made in understanding the physiological function of aureochrome 1a in the diatom, Pheaodactylum tricornutum. Costa et al. (2013) suggested that aureochrome 1a is involved in photoacclimination (35). Furthermore, Huysman et al. (2013) reported that it mediates induction of diatom-specific cyclin dsCYC2 and controls the onset of cell division, and suggested that it functions synergistically with the bZIP transcription factor bZIP10 to induce dsCYC2 transcription (36). In other organisms, it has been postulated that overexpression of NgAUREO1, the gene coding for aureochrome 1 from Nannochloropsis gaditana, in Saccharomyces, leads to an increase of lipid accumulation in yeast cells (37). Monomeric AUREO1 may form heterodimers with other bZIP transcription factors and contribute to the regulation of various genes in a BL-dependent manner. Regarding the LOV domain, Herman et al. (2013) recently reported that BL-induced unfolding of the Jα helix facilitated dimerization of aureochrome-LOV of the diatom, P. tricornutum (38). BL-induced dimerization of the LOV domain can possibly drive the dimerization of monomeric AUREO1 from V. frigida. Using sedimentation velocity, Mitra et al. (2012) reported that aureochrome-LOV was dimeric in solution at concentrations of 18.6 µM and 7.15 µM, and suggested that the upper limit of the dimer-monomer dissociation equilibrium constant

was approximately 4 µM (19). In previous SEC studies, we have suggested that N-terminally truncated AUREO1-LOV had an apparent MW of 25 kDa (calculated MW of 18 kDa), and existed as a monomer at 175 µM, and partly as a dimer at 350 µM, in the dark (20,21). Although the number of amino acids and the conditions of these experiments were different, this inconsistency should be addressed in future studies. In contrast to the bZIP and LOV domains, amino acid sequences of the N-domains are more dissimilar among reported AUREOs. Recently, Deng et al. have reported that the aureochrome gene from Saccharina japonica lacked the N-domain (39). The N-domains may have significantly diverse functions but have weak functional constraints, as in tuning affinity, for the target sequence, as shown by the relatively low affinity of hZLC2S and DTT-FL for the target sequence. Paradoxically, the common functions among all AUREO proteins conserved during the evolution of stramenopiles should be located in the C-terminal side, containing bZIP and LOV domains. Our present study suggests that the bZIP-LOV domain functions as a BL-induced dimerizing module, or “photodimerizer.” In addition to the monomeric D450 form, our N-terminally truncated ZLC2S was also stable in the dimeric S390 form, probably because of synergistic interactions between the LOV and bZIP domains that tend to form a dimer for DNA binding. Based upon these results, we therefore suggest that our ZL proteins could be useful molecular tools for BL-regulated “bio-switches,” as well as for model molecules to characterize the function and assembly of various biological macromolecules.

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30. Danciu, T. E. and Whitman, M. (2010) Oxidative stress drives disulfide bond formation between basic helix-loop-helix transcription factors. J. Cell Biochem. 109, 417-424

31. Shaikhali, J., Norén, L., de Dios Barajas-López, J., Srivastava, V., König, J., Sauer, U. H., Wingsle, G., Dietz, K. J., and Strand, Å. (2012) Redox-mediated mechanisms regulate DNA binding activity of the G-group of basic region leucine zipper (bZIP) transcription factors in Arabidopsis. J. Biol. Chem. 287, 27510-27525

32. Masuda, S., Bauer, C. E. (2002) AppA is a blue light photoreceptor that antirepresses photosynthesis gene expression in Rhodobacter sphaeroides. Cell 110, 613-623

33. Zoltowski, B. D., Motta-Mena, L. B., and Gardner, K. H. (2013) Blue light-induced dimerization of a bacterial LOV-HTH DNA-binding protein. Biochemistry 52, 6653-6661

34. Zoltowski, B. D. and Crane, B. R. (2008) Light activation of the LOV protein vivid generates a rapidly exchanging dimer. Biochemistry 47, 7012-7019

35. Costa, B. S., Sachse, M., Jungandreas, A., Bartulos, C. R., Gruber, A., Jakob, T., Kroth, P. G., and Wilhelm, C. (2013) Aureochrome 1a is involved in the photoacclimation of the diatom Phaeodactylum tricornutum. PLoS One. 8: e74451. doi: 10.1371/journal.pone.0074451.

36. Huysman, M. J., Fortunato, A. E., Matthijs, M., Costa, B. S., Vanderhaeghen, R., van den Daele, H., Sachse, M., Inzé, D., Bowler, C., Kroth, P. G., Wilhelm, C., Falciatore, A., Vyverman, W., and De Veylder, L. (2013) AUREOCHROME1a-mediated induction of the diatom-specific cyclin dsCYC2 controls the onset of cell division in diatoms (Phaeodactylum tricornutum). Plant Cell 25, 215-228

37. Huang, Y. J., Wang, L., Zheng, M. G., Zheng, L., Tong, Y. L., and Li, Y. Overexpression of NgAUREO1, the gene coding for aurechrome 1 from Nannochloropsis gaditana, into Saccharomyces cerevisiae leads to a 1.6-fold increase in lipid accumulation. Biotechnol. Lett. in press.

38. Herman, E., Sachse, M., Kroth, P. G., and Kottke, T. (2013) Blue-light-induced unfolding of the Jα helix allows for the dimerization of aureochrome-LOV from the diatom Phaeodactylum tricornutum.

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Biochemistry 52, 3094-3101 39. Deng, Y., Yao, J., Fu, G., Guo, H., and Duan, D. (2014) Isolation, Expression, and Characterization

of Blue Light Receptor AUREOCHROME Gene From Saccharina japonica (Laminariales, Phaeophyceae). Mar. Biotechnol. (NY) 16, 135-143

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Acknowledgments−The authors thank Dr. Yoshinori Shichida (Kyoto University), Dr. Mamiko Ozaki and Dr. Fumio Hayashi (Kobe University), and Dr. Norio Hamada and Dr. Ryosuke Nakamura (Osaka University), for helpful discussions. FOOTNOTES *This research was partly supported by a Grant-in-Aid for Scientific Research on Innovative Areas (No. 23120517 to O.H.) from The Ministry of Education, Culture, Sports, Science and Technology, and by a Grant-in-Aid for Scientific Research (C) (No. 22570162 to O.H.) from the Japan Society for the Promotion of Science. 1To whom correspondence should be addressed: Osamu Hisatomi, Department of Earth and Space Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan, Tel.: +81-6-6850-5500; Fax: +81-6-6850-5500; E-mail: hisatomi@ess.sci.osaka-u.ac.jp 2The abbreviations used are: BL, blue light; FMN, flavin adenine mononucleotide; LOV, light-oxygen-voltage-sensing; ARNT, aryl hydrocarbon receptor nuclear translocator; AUREO, aureochrome; bZIP, basic region/leucine zipper; TG, transient-grating; DLS, dynamic light scattering; RH, hydrodynamic radius (radii); RH-app, apparent hydrodynamic radius (radii); λmax, maximum wavelength; MW, molecular weight; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; DTT, dithiothreitol; SEC, size exclusion chromatography; ORP, oxidation-redox potential; DTTred, reduced form of DTT; DTTox, oxidized form of DTT

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FIGURE LEGENDS FIGURE 1. Schematic drawing of AUREO1 recombinant proteins. FL, ZLwt2, ZLC2S, and hZLC2S are shown with the location of cysteine residues. The numbering of each amino acid is based on full-length AUREO1. FIGURE 2. Spectroscopic measurements of recombinant AUREO1s. Ultraviolet-visible absorption (A) and fluorescence emission (B) spectra of the recombinant AUREO1s at 5 µM (for ZLC2S, hZLC2S, and DTT-ZL) or 3 µM (for DTT-FL) in loading buffer containing 1 mM (for ZLC2S and hZLC2S) or 50 mM (for DTT-ZL and DTT-FL) DTT at 25 °C. Spectra of ZLC2S (black solid lines), hZLC2S (black dotted lines), DTT-ZL (black dashed lines), and DTT-FL (gray dashed lines) are superimposed with that of ZLwt2 (gray solid lines). The absorption and the fluorescent emission spectra are shown by normalizing both the maximal absorbance at 447 nm and the maximal emission at 495 nm as 1.0. Inset designates the enlarged absorption spectra from 350 nm to 550 nm. Spectral changes during dark regeneration of D450 from S390 for ZLC2S (C), hZLC2S (D), DTT-ZL (E) and DTT-FL (F) at 25 °C. The spectra were measured at the initial dark state (solid line), immediately (dashed line), 2, 4, 8, 16, 32 (thin solid lines), and 48 min (thick dotted lines) after the termination of BL illumination. (G) Normalized fitted curves of the dark regeneration for each AUREO1 using the formula, ∆Amax(1-e-kt), where ∆Amax is a maximum absorption difference, and k is the rate constant of the reaction. The line types are the same as in panel A. FIGURE 3. DLS and SEC measurements of ZLs in the dark state. (A) Dependence of RH-app on the concentrations of ZLC2S (red bar and lines) and hZLC2S (blue bar and lines) superimposed with the regression line of dimeric ZLwt2 (gray lines) in the dark state. Red triangle, blue triangle, and green circle indicate the RH-app of ZLC2S, hZLC2S, and DTT-ZL, respectively, at 40 or 50 µM in the presence of 50 mM DTT. (B) The time-course of RH-app change for ZLwt2 at 40 µM (black circle) or 50 µM (gray circle) protein concentrations in loading buffer containing 50 mM DTT, in the dark. (C) Elution profiles of ZLC2S (upper), hZLC2S (middle), and DTT-ZL (lower) superimposed with those of ZLwt2 (gray lines) in the dark state, monitored by absorption at 280 nm. Open and closed inverted triangles indicate the elution peak volumes of ZLwt2 and ZLC2S, respectively. (D) The MW of dimeric ZLwt2 (open inverted triangle) and monomeric ZLs (closed inverted triangle), estimated from SEC data. Circles indicate the MW of proteins used for column calibration. FIGURE 4. Monomer-dimer equilibrium of 5 mM DTT-treated ZLwt2 in the D450 state. (A) SEC profiles of 5 mM DTT-treated ZLwt2, eluted with SEC buffer containing 5 mM DTT (upper), added 5 mM DTT to get a final concentration of 10 mM DTT (middle), or added 10 mM H2O2 (lower) and incubated for 4 h (dotted lines) and 18 h (solid lines) at 25 °C. Each profile is superimposed with that of 5 mM DTT-treated ZLwt2 eluted with DTT-free SEC buffer (gray line). (B) SDS-PAGE in non-reducing conditions: ZLwt2 (lane 1); ZLC2S (lane 2); DTT-ZL (lane 3); 5 mM DTT-treated ZLwt2 (lane 4); 5 mM DTT-treated ZLwt2 added 5 mM DTT to get a final concentration of 10 mM DTT (lane 5) or incubated added 10 mM H2O2 (lane 6) and incubated for 18 h. Open and filled arrowheads indicate dimeric and monomeric species of ZL, respectively. (C) SEC profiles of 5 mM DTT-treated ZLwt2 in the regenerated D450 state, after BL illumination at 0.8 W/m2 (upper) or 38 W/m2 (middle) for 4 h (dotted line) and 18 h (solid line). Each profile is superimposed with that of 5 mM DTT-treated ZLwt2, in the dark state. SEC profiles of 5 mM DTT-treated ZLwt2 incubated for 1 h (dotted line) and 14 h (solid line), in the dark after BL illumination at 38 W/m2 for 4 h (lower). (D) Non-reducing SDS-PAGE of 5 mM DTT-treated ZLwt2, in the dark state (lane 1), and in the regenerated D450 state after BL illumination at 0.8 W/m2 for 4 h (lane 2) and 18 h (lane 3), or at 38 W/m2 for 4 h (lane 4) and for 18 h (lane 5). Arrowheads as in panel B. FIGURE 5. DTT fractions in elution profiles and the midpoint ORP for dissociation of disulfide bonds. (A) SEC profiles of ORP mixtures, non-containing (upper) or containing (middle) 5 mM FMN after BL-illumination at 38 W/m2 for 18 h, and containing 5 mM DTT-treated ZLwt2 (lower) in the dark state,

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monitored at 250 (gray lines), 280 (black solid lines) and 450 (black dotted line) nm. Inverted triangles indicate the peak elution volumes of dimeric (open) and monomeric (closed) forms of ZL. Open arrow at 26 ml and closed arrow at 32 ml indicate the DTT fractions. (B) Absorption spectra of the DTT fractions eluted at 26 ml and 32 ml coincide with those of, respectively, DTTred (gray line) and DTTox (black line) reported previously (22). (C) The monomeric ratio of 3–10 mM DTT-treated ZLwt2 plotted against calculated ORP. Closed and open circles indicate non-illuminated and illuminated samples, respectively, and closed and open triangles indicate the samples added 5 mM DTT (to get a final concentration of 10 mM DTT) and 10 mM H2O2, respectively, and incubated for 18 h. FIGURE 6. DLS measurements of ZLs in the light and light-dark states. (A) Dependence of RH-app on the concentrations of ZLC2S (red bars and lines) and hZLC2S (blue bars and lines) superimposed with the regression lines of dimeric ZLwt2 (gray line). The regression lines for the light (dashed line) and dark-light (dotted line) states are shown with those in the dark state (thin solid line). The RH-app of ZLC2S (red triangles), hZLC2S (blue triangles), and DTT-ZL (green circles) at 40 µM or 50 µM just after BL illumination (open symbols) are shown with those in the light-dark state (filled symbols). (B) The time-course of RH-app changes for ZLC2S (red bars and line) and hZLC2S (blue bars and line) with standard deviations after BL illumination, and single exponential fitted curves (see Experimental Procedures). FIGURE 7. Elution profiles of ZLs. (A) Elution profiles of ZLC2S (black lines) superimposed with those of ZLwt2 (gray lines) in SEC buffer, monitored by absorbance at 280 nm, in the dark state (upper), the light state (middle), and the light-dark state (lower). Inverted triangles indicate the peak elution volumes of dimeric ZLwt2 (open) and monomeric ZLC2S (closed). (B) The MW estimated from of the elution peaks in the dark (closed inverted triangle) and the light (open inverted triangle) states. Circles indicate the MW of the proteins used for column calibration. (C) Elution profiles of hZLC2S with SEC buffer in the dark (solid lines), the light (dashed lines), and the light-dark (dotted lines) states. (D) Elution profiles of DTT-ZL with SEC buffer containing 5 mM DTT. Line types and symbols are the same as in panels A and B. FIGURE 8. Elution profiles of ZLwt2 with Cy3-labeled oligonucleotides. Elution profiles of ZLwt2 incubated with Cpo (A) and Apo (B), monitored at 260 nm (gray solid lines), 390 nm (black solid lines), and 550 nm (black dotted lines) in the dark (upper), in the light (middle), and in the light-dark (lower) states. Open inverted triangle and open rectangle indicate the elution volumes of ZLwt2 and the ZLwt2/Apo complex, respectively, and asterisks designate the peak volumes of double-stranded (black) and single-stranded (gray) Cy3-labeled oligonucleotides. (C) The MW was estimated from the peak volumes of ZLwt2 (open inverted triangle) and ZLwt2/Apo complex (open rectangle). Circles indicate the MW of the proteins used for column calibration. (D) Elution profiles of ZLwt2 incubated with Apo in the presence of 400 mM NaCl in the dark (upper), in the light (middle), and in the light-dark (lower) states. Monitoring wavelengths, line types and symbols are the same as in panel A. FIGURE 9. Elution profiles of monomeric ZLs with Apo. Elution profiles of ZLC2S (A), hZLC2S (B, C), and DTT-ZL (D) incubated with Apo in the presence of 300 mM (A, C, D) or 250 mM (B) NaCl, in the dark (upper), in the light (middle), and in the light-dark (lower) states. Monitoring wavelengths are 260 nm (gray solid lines), 390 nm (black solid lines), and 550 nm (black dotted lines). Inverted triangles indicate the peak elution volumes of dimeric ZLwt2 (open) and monomeric ZLC2S (closed), and asterisks designate the peak volumes of double-stranded (black) and single-stranded (gray) Apo. FIGURE 10. Elution profiles of DTT-FL. (A) Elution profiles of DTT-FL in SEC buffer containing 250 mM NaCl and 5 mM DTT, in the dark (solid lines), the light (dashed lines), and the light-dark (dotted lines) states. Diamonds indicate elution volumes of the peaks in the dark (closed) and the light (open) states. (B) The MW estimated from the elution peaks in the dark (closed diamond) and the light (open diamond) states. Circles indicate the MW of the proteins used for column calibration. Elution profiles of

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Blue light-induced dimerization of monomeric AUREO1

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DTT-FL incubated with Cpo (C) and Apo (D) in the dark (upper), the light (middle), and the light-dark (lower) states. Diamonds are the same as in panel (A), and the monitoring wavelengths, asterisks, and type of the lines are the same as in Fig. 9. FIGURE 11. A schematic model of AUREO1 in the oxidizing and reduced conditions. In the oxidizing condition (upper), AUREO1 forms a dimer with intermolecular disulfide linkages at Cys162 and Cys182, and binds to the target DNA sequence with a high affinity. BL induces a global conformational change that increases its RH by 5% (21). AUREO1 may aggregate when the redox state strongly deviates from the normal cellular state. In the reduced condition (lower), AUREO1 is monomeric in the dark state. BL induces the dimerization of monomeric AUREO1, which enhances affinity for the target sequence. LOV, LOV domain; bZIP, bZIP domain; N, N-domain; S, photoadduct; C, cysteine residues forming (in red circles) and not forming (gray circles) intermolecular disulfide bonds.

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Fig. 1

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Osamu Hisatomi, Yoichi Nakatani, Ken Takeuchi, Fumio Takahashi and Hironao Kataokafor the Target Sequence

Blue Light-induced Dimerization of Monomeric Aureochrome-1 Enhances Its Affinity

published online May 1, 2014J. Biol. Chem. 

  10.1074/jbc.M114.554618Access the most updated version of this article at doi:

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