LOV Domain Research Articles

Unusual photodynamic characteristics of the light-oxygen-voltage domain of phototropin linked to terrestrialadaptation of Klebsormidium nitens.

Phototropin (Phot), a blue light-sensing LOV domain protein, mediates blue light responses and is evolutionarily conserved across the green lineage. Klebsormidium nitens, a green terrestrial alga, presents a valuable opportunity to study adaptive responses from aquatic to land habitat transitions. We determined the crystal structure of Klebsormidium nitens Phot LOV1 domain (KnLOV1) in the dark and engineered different mutations (R60K, Q122N, and D33N) to modulate the lifetime of the photorecovery cycle. We observed unusual, slow recovery kinetics in the wild-type KnLOV1 domain (τ = 41 ± 3 min) compared to different mutants (R60K: τ = 2.0 ± 0.1 min, Q122N: τ = 1.7 ± 0.1 min, D33N: τ = 9.6 ± 0.1 min). Crystal structures of wild-type KnLOV1 and mutants revealed subtle but critical changes near the protein chromophore that is responsible for modulating protein dark recovery time. Our findings shed light on the unique structural and biochemical characteristics of the newly studied KnLOV1 and its evolutionary importance for phototropin-mediated physiology.

The structure of Chlamydomonas LOV1 as revealed by time-resolved serial synchrotron crystallography.

The photo-reaction of the LOV1 domain of the Chlamydomonas reinhardtii phototropin is investigated by room-temperature time-resolved serial crystallography. A covalent adduct forms between the C4a atom of the central flavin-mononucleotide chromophore and a protein cysteine. The structure of the adduct is very similar to that of LOV2 determined 23 years ago from the maidenhair fern Phy3.

LOV1 protein of Pseudomonas cichorii JBC1 modulates its virulence and lifestyles in response to blue light

Bacteria perceive light signals via photoreceptors and modulate many physiological and genetic processes. The impacts played by light, oxygen, or voltage (LOV) and blue light (BL) photosensory proteins on the virulence-related traits of plant bacterial pathogens are diverse and complex. In this study, we identified LOV protein (Pc-LOV1) from Pseudomonas cichorii JBC1 (PcJBC1) and characterized its function using LOV1-deficient mutant (JBC1Δlov1). In the dark state, the recombinant Pc-LOV1 protein showed an absorption band in UV-A region with a double peak at 340 nm and 365 nm, and within the blue-region, it exhibited a main absorption at 448 nm along with two shoulder peaks at 425 nm and 475 nm, which is a typical feature of oxidized flavin within LOV domain. The adduct-state lifetime (τrec) of Pc-LOV1 was 67.03 ± 4.34 min at 25 °C. BL negatively influenced the virulence of PcJBC1 and the virulence of JBC1Δlov1 increased irrespective of BL, indicating that Pc-LOV1 negatively regulates PcJBC1 virulence. Pc-LOV1 and BL positively regulated traits relevant to colonization on plant surface, such as adhesion to the plant tissue and biofilm formation. In contrast, swarming motility, exopolysaccharide production, and siderophore synthesis were negatively controlled. Gene expression supported the modulation of bacterial features by Pc-LOV1. Overall, our results suggest that the LOV photosensory system plays crucial roles in the adaptive responses and virulence of the bacterial pathogen PcJBC1. The roles of other photoreceptors, sensing of other wavelengths, and signal networking require further investigation.

Design of AsLOV2 domain as a carrier of light-induced dissociable FMN photosensitizer.

Flavin mononucleotide (FMN) is a highly efficient photosensitizer (PS) yielding singlet oxygen (1 O2 ). However, its 1 O2 production efficiency significantly decreases upon isoalloxazine ring encapsulation into the protein matrix in genetically encoded photosensitizers (GEPS). Reducing isoalloxazine ring interactions with surrounding amino acids by protein engineering may increase 1 O2 production efficiency GEPS, but at the same time weakened native FMN-protein interactions may cause undesirable FMN dissociation. Here, in contrast, we intentionally induce the FMN release by light-triggered sulfur oxidation of strategically placed cysteines (oxidation-prone amino acids) in the isoalloxazine-binding site due to significantly increased volume of the cysteinyl side residue(s). As a proof of concept, in three variants of the LOV2 domain of Avena sativa (AsLOV2), namely V416C, T418C, and V416C/T418C, the effective 1 O2 production strongly correlated with the efficiency of irradiation-induced FMN dissociation (wild type (WT) < V416C < T418C < V416C/T418C). This alternative approach enables us: (i) to overcome the low 1 O2 production efficiency of flavin-based GEPSs without affecting native isoalloxazine ring-protein interactions and (ii) to utilize AsLOV2, due to its inherent binding propensity to FMN, as a PS vehicle, which is released at a target by light irradiation.

Photoactivation of LOV domains with chemiluminescence.

Optogenetics has opened new possibilities in the remote control of diverse cellular functions with high spatiotemporal precision using light. However, delivering light to optically non-transparent systems remains a challenge. Here, we describe the photoactivation of light-oxygen-voltage-sensing domains (LOV domains) with in situ generated light from a chemiluminescence reaction between luminol and H2O2. This activation is possible due to the spectral overlap between the blue chemiluminescence emission and the absorption bands of the flavin chromophore in LOV domains. All four LOV domain proteins with diverse backgrounds and structures (iLID, BcLOV4, nMagHigh/pMagHigh, and VVDHigh) were photoactivated by chemiluminescence as demonstrated using a bead aggregation assay. The photoactivation with chemiluminescence required a critical light-output below which the LOV domains reversed back to their dark state with protein characteristic kinetics. Furthermore, spatially confined chemiluminescence produced inside giant unilamellar vesicles (GUVs) was able to photoactivate proteins both on the membrane and in solution, leading to the recruitment of the corresponding proteins to the GUV membrane. Finally, we showed that reactive oxygen species produced by neutrophil like cells can be converted into sufficient chemiluminescence to recruit the photoswitchable protein BcLOV4-mCherry from solution to the cell membrane. The findings highlight the utility of chemiluminescence as an endogenous light source for optogenetic applications, offering new possibilities for studying cellular processes in optically non-transparent systems.

Reduction midpoint potential of a paradigm light–oxygen–voltage receptor and its modulation by methionine residues

The introduction of methionine residues into the Avena sativa phototropin 1 LOV2 domain raises the reduction midpoint of its flavin-nucleotide chromophore.

Dimerization of iLID optogenetic proteins observed using 3D single-molecule tracking in live E. coli

3D single-molecule tracking microscopy has enabled measurements of protein diffusion in living cells, offering information about protein dynamics and cellular environments. For example, different diffusive states can be resolved and assigned to protein complexes of different size and composition. However, substantial statistical power and biological validation, often through genetic deletion of binding partners, are required to support diffusive state assignments. When investigating cellular processes, real-time perturbations to protein spatial distributions is preferable to permanent genetic deletion of an essential protein. For example, optogenetic dimerization systems can be used to manipulate protein spatial distributions that could offer a means to deplete specific diffusive states observed in single-molecule tracking experiments. Here, we evaluate the performance of the iLID optogenetic system in living E. coli cells using diffraction-limited microscopy and 3D single-molecule tracking. We observed a robust optogenetic response in protein spatial distributions after 488 nm laser activation. Surprisingly, 3D single-molecule tracking results indicate activation of the optogenetic response when illuminating with high-intensity light with wavelengths at which there is minimal photon absorbance by the LOV2 domain. The preactivation can be minimized through the use of iLID system mutants, and titration of protein expression levels.

A noncommutative combinatorial protein logic circuit controls cell orientation in nanoenvironments.

Single-protein-based devices that integrate signal sensing with logical operations to generate functional outputs offer exceptional promise for monitoring and modulating biological systems. Engineering such intelligent nanoscale computing agents is challenging, as it requires the integration of sensor domains into a functional protein via intricate allosteric networks. We incorporate a rapamycin-sensitive sensor (uniRapR) and a blue light-responsive LOV2 domain into human Src kinase, creating a protein device that functions as a noncommutative combinatorial logic circuit. In our design, rapamycin activates Src kinase, causing protein localization to focal adhesions, whereas blue light exerts the reverse effect that inactivates Src translocation. Focal adhesion maturation induced by Src activation reduces cell migration dynamics and shifts cell orientation to align along collagen nanolane fibers. Using this protein device, we reversibly control cell orientation by applying the appropriate input signals, a framework that may be useful in tissue engineering and regenerative medicine.

Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells.

The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions and function with light. We integrated optogenetic control into proximity labeling, a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the proximity labeling enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. 'LOV-Turbo' works in multiple contexts and dramatically reduces background in biotin-rich environments such as neurons. We used LOV-Turbo for pulse-chase labeling to discover proteins that traffic between endoplasmic reticulum, nuclear and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by bioluminescence resonance energy transfer from luciferase, enabling interaction-dependent proximity labeling. Overall, LOV-Turbo increases the spatial and temporal precision of proximity labeling, expanding the scope of experimental questions that can be addressed with proximity labeling.

Allosteric inactivation of an engineered optogenetic GTPase

Optogenetics is a technique for establishing direct spatiotemporal control over molecular function within living cells using light. Light application induces conformational changes within targeted proteins that produce changes in function. One of the applications of optogenetic tools is an allosteric control of proteins via light-sensing domain (LOV2), which allows direct and robust control of protein function. Computational studies supported by cellular imaging demonstrated that application of light allosterically inhibited signaling proteins Vav2, ITSN, and Rac1, but the structural and dynamic basis of such control has yet to be elucidated by experiment. Here, using NMR spectroscopy, we discover principles of action of allosteric control of cell division control protein 42 (CDC42), a small GTPase involved in cell signaling. Both LOV2 and Cdc42 employ flexibility in their function to switch between "dark"/"lit" or active/inactive states, respectively. By conjoining Cdc42 and phototropin1 LOV2 domains into the bi-switchable fusion Cdc42Lov, application of light-or alternatively, mutation in LOV2 to mimic light absorption-allosterically inhibits Cdc42 downstream signaling. The flow and patterning of allosteric transduction in this flexible system are well suited to observation by NMR. Close monitoring of the structural and dynamic properties of dark versus "lit" states of Cdc42Lov revealed lit-induced allosteric perturbations that extend to Cdc42's downstream effector binding site. Chemical shift perturbations for lit mimic, I539E, have distinct regions of sensitivity, and both the domains are coupled together, leading to bidirectional interdomain signaling. Insights gained from this optoallosteric design will increase our ability to control response sensitivity in future designs.

Fine spectral tuning of a flavin-binding fluorescent protein for multicolor imaging

Flavin-binding fluorescent proteins are promising genetically encoded tags for microscopy. However, spectral properties of their chromophores (riboflavin, flavin mononucleotide, and flavin adenine dinucleotide) are notoriously similar even between different protein families, which limits applications of flavoproteins in multicolor imaging. Here, we present a palette of 22 finely tuned fluorescent tags based on the thermostable LOV domain from Chloroflexus aggregans. We performed site saturation mutagenesis of three amino acid positions in the flavin-binding pocket, including the photoactive cysteine, to obtain variants with fluorescence emission maxima uniformly covering the wavelength range from 486 to 512nm. We demonstrate three-color imaging based on spectral separation and two-color fluorescence lifetime imaging of bacteria, as well as two-color imaging of mammalian cells (HEK293T), using the proteins from the palette. These results highlight the possibility of fine spectral tuning of flavoproteins and pave the way for further applications of flavin-binding fluorescent proteins in fluorescence microscopy.

Molecular characterization of phototropin signalosome regulating photophysiological processes in green algae.

Phototropin is a blue light-sensing photoreceptor, comprises two LOV domains that non-covalently bind FMN as chromophore and a serine-threonine kinase domain at its C-terminus. Phototropin is well established in higher plants and aquatic algae. However, the photoreceptor biology of the evolutionary important alga has not yet been established. We characterized the phototropin LOV domain from evolutionary important algae and compared the changes with the aquatic green algae, Chlamydomonas reinhardtii phototropin LOV domain. The intraflagellar transport (IFT) machinery responsible for bidirectional movement of proteins inwithin cilia or flagella is involved in trafficking of Chlamydomonas phototropin (CrPhot). Through immunoblotting and immunolocalization experiments with a variety of IFT mutants, we investigated the molecular details of phototropin trafficking. The CrPhot knock-out strains showed reduced photomotility, decrease level of other predicted interacting proteins like ChRs and, 14-3-3 as compared to that of the wild type strain. Various approaches like protein profiling, expression profile (qRT-PCR), and proteomics were applied to delineate its role in light-mediated physiology in C. reinhardtii. We studied the photochemistry, oligomerization, and photodynamic behaviour of LOV1 domain of a novel terrestrial algal phototropin using UV-visible, fluorescence, CD, atomic force microscopy, and time-resolved fluorescence spectroscopy techniques. Our findings highlight the unique characteristics of LOV domain in terms of very long dark recovery time. We identified the key residues responsible for this recovery kinetics in novel LOV1 protein. Different variants of LOV1 protein were produced and photochemistry was compared with the respect to the wild type protein. We proposed optogenetic potential of the engineered LOV domain in modulation of diverse cellular signalling pathways such as ciliogenesis, cell cycle, and apoptosis.

Determining the mechanisms of the allosteric architecture of DHFR.

Our ability to rationally optimize allosteric regulation is limited by incomplete knowledge of the mechanisms by which mutations tune allostery. To examine this, we investigated the effects of allosteric residues in a synthetic allosteric switch in which Dihydrofolate reductase (DHFR) is regulated by a blue-light sensitive LOV2 domain. In prior work using saturation mutagenesis, we showed that less than 5% of mutations had a statistically significant influence on allostery. While allostery disrupting mutations were located near the LOV2 insertion site, we found that allostery enhancing mutations were widely dispersed and enriched on the protein surface. However, these experiments did not reveal the mechanism by which the allosteric signal is propagated or how those mutations tune it. To understand these effects, we are characterizing the light-dependent changes in conformational dynamics for the chimeric enzyme by solution nuclear magnetic resonance spectroscopy and analysis of correlations between residue pairs in long timescale molecular dynamics. We have also measured the entropic and enthalpic contribution of select mutations towards the allosteric effect through kinetics assays. From these data, we aim to understand the mechanisms by which the global map of allosteric contributions inside the enzyme operates and provide insight into how allostery can be evolutionarily optimized.

Towards next-generation optogenetic actuators of voltage-gated Ca2+ channels.

CaV1 channels (CaV1.2/1.3) are fundamentally involved in the normal function and pathophysiology of the heart and the brain. These channels convey Ca2+ influx that sculpts action potentials, triggers muscle contraction, and mediates excitation-transcription coupling. To orchestrate these diverse functions, CaV1 activity is fine-tuned by multiple regulatory processes, including Ca2+-dependent inactivation (CDI), which is mediated by the Ca2+-binding protein, calmodulin (CaM). Disruption of CaM regulation is linked to cardiac arrhythmias and neurodevelopmental disorders. However, the precise role of this modulation in various physiological settings is not fully understood. In part, this gap in understanding stems from a lack of approaches to dynamically alter CDI. Here, we engineer a novel optogenetic tool to acutely prevent CDI. To do so, we undertook a biomimetic approach by leveraging naturally occurring CaV1 regulatory mechanisms that modify CDI. Specifically, SH3 and cysteine-rich-domain (stac) protein was recently found to selectively prevent CaV1 CDI. Our previous work identified a short amino acid segment of stac called the U-domain that strongly inhibits CDI. Here, we attached the stac3 U-domain to a light-sensitive Lov2 domain to optically switch its accessibility for modulating CaV1 channels, yielding an actuator known as LovU-CaV1. Whole-cell electrophysiological recordings showed that co-expression of LovU-CaV1 resulted in low basal inhibition of CDI of CaV1.2. However, upon light activation of LovU-CaV1, we observed a rapid reduction in CDI that was then reversible. LovU-CaV1 provides a new avenue to optically manipulate endogenous Ca channels in both the heart and the brain. Future studies will employ this tool to dissect the importance of L-type channel inactivation in both cardiomyocytes and in neurons.

Light triggered functional dynamics of AsLOV2 by time-resolved EPR at high magnetic fields.

Proteins are molecular machines that power life. To understand their operation as machines it is necessary to investigate the evolution of their conformational changes in time, triggered by an external stimulus. We chose to focus on the photoswitchable protein AsLOV2. The current state of understanding is that the structures of the dark and the lit states exist but the non-equilibrium trajectories that connect the states are unknown. The main impact of this knowledge gap is felt in the optogenetics community where there is a significant demand for improved light-driven actuators. To guide knowledge-driven engineering of LOV proteins, measurements of time-resolved structural changes are needed. We present a unique method based on the remarkable EPR properties of Gd-sTPATCN used as a spin-label of AsLOV2. At 8.6 Tesla, the EPR spectrum is dominated by a single sharp line<5G, making it exquisitely sensitive to dipolar broadening by the magnetic field of nearby Gd(III) metal centers.Tracking the EPR lineshape as a function of time, we successfully detect that light activation causes an increase in distance between the Aα; and Jα; helices of AsLOV2 in the solution state. We also resolved the importance of Q513 residue in coupling light to mechanical motion in AsLOV2, which has been a topic of debate for a long time. We observed light-triggered slowed down mechanical motion between Aα; and Jα; helices even on the mutation of Q513 to non-polar alanine, signifying that other factors or residues also play a major role in the actuation of mechanical motion in AsLOV2.Next, we plan to film a “molecular movie” of AsLOV2 by measuring inter-spin inter-residue distances using rapid-scan transient cwEPR in the solution state. We acknowledge financial support from NSF MCB-2025860 and UCOP through MRI-19-601107.

Effect of tryptophan mutation on the structure of LOV1 domain of phototropin1 protein of Ostreococcus tauri: A combined molecular dynamics simulation and biophysical approach

BackgroundLight, oxygen and voltage (LOV) proteins detect blue light by formation of a covalent ‘photoadduct’ between the flavin chromophore and the neighboring conserved cysteine residue. LOV proteins devoid of this conserved photoactive cysteine are unable to form this ‘photoadduct’ upon light illumination, but they can still elicit functional response via the formation of neutral flavin radical. Recently, tryptophan residue has been shown to be the primary electron donors to the flavin excited state. MethodsPhotoactive cysteine (Cys42) and tryptophan (Trp68) residues in the LOV1 domain of phototropin1 of Ostreococcus tauri (OtLOV1) was mutated to alanine and threonine respectively. Effect of these mutations have been studied using molecular dynamics simulation and spectroscopic techniques. ResultsMolecular dynamics simulation indicated that W68T did not affect the structure of OtLOV1 protein, but C42A leads to some structural changes. An increase in the fluorescence lifetime and quantum yield values was observed for the Trp68 mutant. ConclusionsAn increase in the fluorescence lifetime and quantum yield of Trp68 mutant compared to the wild type protein suggests that Trp68 residue participates in quenching of the flavin excited state followed by photoexcitation. General significanceEnhanced photo-physical properties of Trp68 OtLOV1 mutant might enable its use for the optogenetic and microscopic applications.

Residue alterations within a conserved hydrophobic pocket influence light, oxygen, voltage photoreceptor dark recovery

Light, oxygen, voltage (LOV) photoreceptors are widely distributed throughout all kingdoms of life, and have in recent years, due to their modular nature, been broadly used as sensor domains for the construction of optogenetic tools. For understanding photoreceptor function as well as for optogenetic tool design and fine-tuning, a detailed knowledge of the photophysics, photochemistry, and structural changes underlying the LOV signaling paradigm is instrumental. Mutations that alter the lifetime of the photo-adduct signaling state represent a convenient handle to tune LOV sensor on/off kinetics and, thus, steady-state on/off equilibria of the photoreceptor (or optogenetic switch). Such mutations, however, should ideally only influence sensor kinetics, while being benign with regard to the nature of the structural changes that are induced by illumination, i.e., they should not result in a disruption of signal transduction. In the present study, we identify a conserved hydrophobic pocket for which mutations have a strong impact on the adduct-state lifetime across different LOV photoreceptor families. Using the slow cycling bacterial short LOV photoreceptor PpSB1-LOV, we show that the I48T mutation within this pocket, which accelerates adduct rupture, is otherwise structurally and mechanistically benign, i.e., light-induced structural changes, as probed by NMR spectroscopy and X-ray crystallography, are not altered in the variant. Additional mutations within the pocket of PpSB1-LOV and the introduction of homologous mutations in the LOV photoreceptor YtvA of Bacillus subtilis and the Avena sativa LOV2 domain result in similarly altered kinetics. Given the conserved nature of the corresponding structural region, the here identified mutations should find application in dark-recovery tuning of optogenetic tools and LOV photoreceptors, alike.Graphical abstract

Slow protein dynamics probed by time-resolved oscillation crystallography at room temperature.

The development of serial crystallography over the last decade at XFELs and synchrotrons has produced a renaissance in room-temperature macromolecular crystallography (RT-MX), and fostered many technical and methodological breakthroughs designed to study phenomena occurring in proteins on the picosecond-to-second timescale. However, there are components of protein dynamics that occur in much slower regimes, of which the study could readily benefit from state-of-the-art RT-MX. Here, the room-temperature structural study of the relaxation of a reaction intermediate at a synchrotron, exploiting a handful of single crystals, is described. The intermediate in question is formed in microseconds during the photoreaction of the LOV2 domain of phototropin 2 from Arabidopsis thaliana, which then decays in minutes. This work monitored its relaxation in the dark using a fast-readout EIGER X 4M detector to record several complete oscillation X-ray diffraction datasets, each of 1.2 s total exposure time, at different time points in the relaxation process. Coupled with in crystallo UV-Vis absorption spectroscopy, this RT-MX approach allowed the authors to follow the relaxation of the photoadduct, a thio-ether covalent bond between the chromophore and a cysteine residue. Unexpectedly, the return of the chromophore to its spectroscopic ground state is followed by medium-scale protein rearrangements that trigger a crystal phase transition and hinder the full recovery of the structural ground state of the protein. In addition to suggesting a hitherto unexpected role of a conserved tryptophan residue in the regulation of the photocycle of LOV2, this work provides a basis for performing routine time-resolved protein crystallography experiments at synchrotrons for phenomena occurring on the second-to-hour timescale.

Genome-Wide Identification and Analysis of the Aureochrome Gene Family in Saccharina japonica and a Comparative Analysis with Six Other Algae.

Aureochrome (AUREO) is a kind of blue light photoreceptor with both LOV and bZIP structural domains, identified only in Stramenopiles. It functions as a transcription factor that responds to blue light, playing diverse roles in the growth, development, and reproduction of Stramenopiles. Most of its functions are currently unknown, especially in the economically important alga S. japonica farmed on a large scale. This study provided a comprehensive analysis of the characteristics of AUREO gene families in seven algae, focusing on the AUREOs of S. japonica. AUREO genes were strictly identified from seven algal genomes. Then AUREO phylogenetic tree was constructed from 44 conserved AUREO genes collected. These AUREO genes were divided into five groups based on phylogenetic relationships. A total of 28 genes unnamed previously were named according to the phylogenetic tree. A large number of different cis-acting elements, especially bZIP transcription factors, were discovered upstream of AUREO genes in brown algae. Different intron/exon structural patterns were identified among all AUREOs. Transcriptomic data indicated that the expression of Sj AUREO varied significantly during the different development stages of S. japonica gametophytes. Periodic rhythms of light induction experiments indicate that Sj AUREO existed in a light-dependent circadian expression pattern, differing from other similar studies in the past. This may indicate that blue light affects gametophyte development through AUREO as a light signal receptor. This study systematically identified and analyzed the AUREO gene family in seven representative brown algae, which lay a good foundation for further study and understanding of AUERO functions in agal growth and development.

Dimerization processes for light-regulated transcription factor Photozipper visualized by high-speed atomic force microscopy

Dimerization is critical for transcription factors (TFs) to bind DNA and regulate a wide variety of cellular functions; however, the molecular mechanisms remain to be completely elucidated. Here, we used high-speed atomic force microscopy (HS-AFM) to observe the dimerization process for a photoresponsive TF Photozipper (PZ), which consists of light–oxygen–voltage-sensing (LOV) and basic-region-leucine-zipper (bZIP) domains. HS-AFM visualized not only the oligomeric states of PZ molecules forming monomers and dimers under controlled dark–light conditions but also the domain structures within each molecule. Successive AFM movies captured the dimerization process for an individual PZ molecule and the monomer–dimer reversible transition during dark–light cycling. Detailed AFM images of domain structures in PZ molecules demonstrated that the bZIP domain entangled under dark conditions was loosened owing to light illumination and fluctuated around the LOV domain. These observations revealed the role of the bZIP domain in the dimerization processes of a TF.