Light-oxygen-voltage Domain Research Articles

Deciphering the Allosteric Process of the Phaeodactylum tricornutum Aureochrome 1a LOV Domain.

The conformational-driven allosteric protein diatom Phaeodactylum tricornutum aureochrome 1a (PtAu1a) differs from other light-oxygen-voltage (LOV) proteins for its uncommon structural topology. The mechanism of signaling transduction in the PtAu1a LOV domain (AuLOV) including flanking helices remains unclear because of this dissimilarity, which hinders the study of PtAu1a as an optogenetic tool. To clarify this mechanism, we employed a combination of tree-based machine learning models, Markov state models, machine-learning-based community analysis, and transition path theory to quantitatively analyze the allosteric process. Our results are in good agreement with the reported experimental findings and reveal a previously overlooked Cα helix and protein linkers as important in promoting the protein conformational changes. This integrated approach can be considered as a general workflow and applied on other allosteric proteins to provide detailed information about their allosteric mechanisms.

New Light on the Mechanism of Phototransduction in Phototropin.

Phototropins are photoreceptor proteins that regulate blue light-dependent biological processes for efficient photosynthesis in plants and algae. The proteins consist of a photosensory domain that responds to the ambient light and an output module that triggers cellular responses. The photosensory domain of phototropin from Chlamydomonas reinhardtii contains two conserved LOV (light-oxygen-voltage) domains with flavin chromophores. Blue light triggers the formation of a covalent cysteine-flavin adduct and upregulates the phototropin kinase activity. Little is known about the structural mechanism that leads to kinase activation and how the two LOV domains contribute to this. Here, we investigate the role of the LOV1 domain from C.reinhardtii phototropin by characterizing the structural changes occurring after blue light illumination with nano- to millisecond time-resolved X-ray solution scattering. By structurally fitting the data with atomic models generated by molecular dynamics simulations, we find that adduct formation induces a rearrangement of the hydrogen bond network from the buried chromophore to the protein surface. In particular, the change in conformation and the associated hydrogen bonding of the conserved glutamine 120 induce a global movement of the β-sheet, ultimately driving a change in the electrostatic potential on the protein surface. On the basis of the change in the electrostatics, we propose a structural model of how LOV1 and LOV2 domains interact and regulate the full-length phototropin from C. reinhardtii. This provides a rationale for how LOV photosensor proteins function and contributes to the optimal design of optogenetic tools based on LOV domains.

Effects of Proline Substitutions on the Thermostable LOV Domain from Chloroflexus aggregans

Light-oxygen-voltage (LOV) domains are ubiquitous photosensory modules found in proteins from bacteria, archaea and eukaryotes. Engineered versions of LOV domains have found widespread use in fluorescence microscopy and optogenetics, with improved versions being continuously developed. Many of the engineering efforts focused on the thermal stabilization of LOV domains. Recently, we described a naturally thermostable LOV domain from Chloroflexus aggregans. Here we show that the discovered protein can be further stabilized using proline substitution. We tested the effects of three mutations, and found that the melting temperature of the A95P mutant is raised by approximately 2 °C, whereas mutations A56P and A58P are neutral. To further evaluate the effects of mutations, we crystallized the variants A56P and A95P, while the variant A58P did not crystallize. The obtained crystal structures do not reveal any alterations in the proteins other than the introduced mutations. Molecular dynamics simulations showed that mutation A58P alters the structure of the respective loop (Aβ-Bβ), but does not change the general structure of the protein. We conclude that proline substitution is a viable strategy for the stabilization of the Chloroflexus aggregans LOV domain. Since the sequences and structures of the LOV domains are overall well-conserved, the effects of the reported mutations may be transferable to other proteins belonging to this family.

Dimerization of LOV domains of Rhodobacter sphaeroides (RsLOV) studied with FRET and stopped-flow experiments.

The bacterium Rhodobacter sphaeroides has a short LOV (light-oxygen-voltage) domain, which is not connected to an effector domain but has an α-helix extension at the N-terminus as well as a helix-turn-helix (HTH) motiv at the C-terminus. These extensions offer possibilities for interactions with effector enzymes or DNA. Whereas many LOV domains show a tendency to form dimers in the light state, RsLOV is unique in that it is a dimer in the dark state but dissociates into monomers after blue-light excitation. We studied the kinetics of this dimerization process by a combination of FRET spectroscopy and stopped-flow experiments with a time resolution of ≈10 ms. Although excitation of the flavin chromophore in dye-labeled LOV domains leads to considerable FRET from flavin to the dye, the typical adduct formation between flavin and a nearby cysteine still occurs with considerable yield. We obtain a rate constant for LOV-LOV dimerization in the range (0.8-1.8) × 105 M-1 s-1, and an equilibrium constant of the dark-state dimer in the range (3.0-7.0) × 10-6 M. Dissociation of the dimers in the light state and reforming of dimers after return to the dark state was monitored using an anti-FRET effect caused by excitonic interaction between dye labels on different monomers. Reforming of the dark state dimers is slower than recovery of the flavin-cysteinyl adduct, indicating that light-induced conformational changes in the LOV domain persist for much longer time than the adduct lifetime.

Single mutation in a novel bacterial LOV protein yields a singlet oxygen generator.

Mr4511 from Methylobacterium radiotolerans is a 164 amino acid protein built of a flavin mononucleotide (FMN) binding, blue-light responsive LOV (Light, Oxygen, Voltage) core domain plus flanking regions. In contrast to the majority of LOV domains, Mr4511 lacks a tryptophan residue that was previously identified as a major quencher for the FMN triplet state in photosensitizers for singlet oxygen (SO) engineered from these photoreceptors. Here we show that for Mr4511 it is sufficient to only mutate the reactive cysteine responsible for the photocycle (Cys71) in the native protein to generate an efficient SO photosensitizer: both C71S and C71G variants exhibit SO quantum yields of formation, ΦΔ, around 0.2 in air-saturated solutions. Under oxygen saturated conditions, ΦΔ reaches ∼0.5 in deuterated buffer. The introduction of Trp112 in the canonical position for LOV domains dramatically lowers ΦΔ to values comparable to miniSOG, one of the early FMN binding proteins touted as a SO sensitizer. Besides its SO properties, Mr4511 is also exceedingly robust against denaturation with urea and is more photostable than free FMN.

Optical Control of Transcription: Genetically Encoded Photoswitchable Variants of T7 RNA Polymerase.

Light-sensing protein domains that link an exogenous light signal to the activity of an enzyme have attracted much attention for the engineering of new regulatory mechanisms into proteins and for studying the dynamic behavior of intracellular reactions and reaction cascades. Light-oxygen-voltage (LOV) photoreceptors are blue-light-sensing modules that have been intensely characterized for this purpose and linked to several proteins of interest. For the successful application of these tools, it is crucial to identify appropriate fusion strategies for combining sensor and enzyme domains that sustain activity and light-induced responsivity. Terminal fusion of LOV domains is the natural strategy; however, this is not transferrable to T7 RNA polymerase because both of its termini are involved in catalysis. It is shown herein that it is possible to covalently insert LOV domains into the polymerase protein, while preserving its activity and generating new light-responsive allosteric coupling.

A thermostable flavin-based fluorescent protein from Chloroflexus aggregans: a framework for ultra-high resolution structural studies.

Light-Oxygen-Voltage (LOV) domains are conserved parts of photoreceptors in plants, bacteria and fungi that bind flavins as chromophores and detect blue light. In the past, LOV domain variants have been developed as fluorescent reporter proteins (called flavin-based fluorescent proteins; FbFPs), which due to their ability to fluoresce under anaerobic conditions, fast folding kinetics and a small size of ∼12-16 kDa are a promising reporter system for quantitative real-time analysis of biological processes. Here, we present a small thermostable flavin-based fluorescent protein CagFbFP derived from a soluble LOV domain-containing histidine kinase from the thermophilic bacterium Chloroflexus aggregans. CagFbFP is composed of 107 amino acids with a molecular weight of 11.6 kDa and consists only of the conserved LOV core domain. The protein is thermostable with a melting point of about 68 °C. It crystallizes easily and its crystals diffract to 1.07 Å. Both the crystal structure and small angle scattering data show that the protein is a dimer. Unexpectedly, glutamine 148, which in LOV photoreceptor proteins is the key residue responsible for signal transduction, occupies two conformations. Molecular dynamics simulations show that the two conformations interconvert rapidly. The crystal structure of the wild-type Chloroflexus aggregans LOV domain determined at 1.22 Å resolution confirmed the presence of two alternative conformations of the glutamine 148 side chain. Overall, this protein, due to its stability and ease of crystallization, appears to be a promising model for ultra-high resolution structural studies of LOV domains and for application as a fluorescent reporter.

A Noncanonical Chromophore Reveals Structural Rearrangements of the Light-Oxygen-Voltage Domain upon Photoactivation

Light-oxygen-voltage (LOV) domains are increasingly used to engineer photoresponsive biological systems. While the photochemical cycle is well documented, the allosteric mechanism by which formation of a cysteinyl-flavin adduct leads to activation is unclear. Via replacement of flavin mononucleotide (FMN) with 5-deazaflavin mononucleotide (5dFMN) in the Aureochrome1a (Au1a) transcription factor from Ochromonas danica, a thermally stable cysteinyl-5dFMN adduct was generated. High-resolution crystal structures (<2 Å) under different illumination conditions with either FMN or 5dFMN chromophores reveal three conformations of the highly conserved glutamine 293. An allosteric hydrogen bond network linking the chromophore via Gln293 to the auxiliary A′α helix is observed. With FMN, a “flip” of the Gln293 side chain occurs between dark and lit states. 5dFMN cannot hydrogen bond through the C5 position and proved to be unable to support Au1a domain dimerization. Under blue light, the Gln293 side chain instead “swings” away in a conformation distal to the chromophore and not previously observed in existing LOV domain structures. Together, the multiple side chain conformations of Gln293 and functional analysis of 5dFMN provide new insight into the structural requirements for LOV domain activation.

Helical Contributions Mediate Light-Activated Conformational Change in the LOV2 Domain of Avena sativa Phototropin 1.

Algae, plants, bacteria, and fungi contain flavin-binding light-oxygen-voltage (LOV) domains that function as blue light sensors to control cellular responses to light. In the second LOV domain of phototropins, called LOV2 domains, blue light illumination leads to covalent bond formation between protein and flavin that induces the dissociation and unfolding of a C-terminally attached α helix (Jα) and the N-terminal helix (A′α). To date, the majority of studies on these domains have focused on versions that contain truncations in the termini, which creates difficulties when extrapolating to the much larger proteins that contain these domains. Here, we study the influence of deletions and extensions of the A′α helix of the LOV2 domain of Avena sativa phototropin 1 (AsLOV2) on the light-triggered structural response of the protein by Fourier-transform infrared difference spectroscopy. Deletion of the A′α helix abolishes the light-induced unfolding of Jα, whereas extensions of the A′α helix lead to an attenuated structural change of Jα. These results are different from shorter constructs, indicating that the conformational changes in full-length phototropin LOV domains might not be as large as previously assumed, and that the well-characterized full unfolding of the Jα helix in AsLOV2 with short A′α helices may be considered a truncation artifact. It also suggests that the N- and C-terminal helices of phot-LOV2 domains are necessary for allosteric regulation of the phototropin kinase domain and may provide a basis for signal integration of LOV1 and LOV2 domains in phototropins.

Progress in the application of LOV2 domain in protein interaction

Protein-protein interactions (PPIs) and protein complexes formed by interactions are the main accomplishers of various functions of cells. As an important component of biological networks, protein interaction plays an important role in determining life processes such as signal transduction in cells. Although the experimental methods for studying protein interactions are endless, there are still many shortcomings and gaps in the research on the interaction of precisely regulated protein molecules. LOV (Light Oxygen Voltage) is a light-sensitive protein domain originally discovered in the photo of plants for receiving blue light stimulation, transmitting signals, and causing a series of reactions in plants. The LOV domain belongs to a member of the PAS (Per-Arnt-Sim) superfamily and contains two subunits, LOV1 and LOV2. LOV2 is thought to cause a conformational change after receiving light stimulation, primarily responsible for activating the kinase domain. It plays an important role in intracellular signal transduction. In view of the light control and sensitivity of LOV2, which can be used as a research tool in protein interaction processes, the mechanism of action and structural advantages of signal transduction in LOV2 domain in protein interactions are reviewed.

An Open-Source Plate Reader.

Microplate readers are foundational instruments in experimental biology and bioengineering that enable multiplexed spectrophotometric measurements. To enhance their accessibility, we here report the design, construction, validation, and benchmarking of an open-source microplate reader. The system features full-spectrum absorbance and fluorescence emission detection, in situ optogenetic stimulation, and stand-alone touch screen programming of automated assay protocols. The total system costs less than $3500, a fraction of the cost of commercial plate readers, and can detect the fluorescence of common dyes at concentrations as low as ∼10 nM. Functional capabilities were demonstrated in the context of synthetic biology, optogenetics, and photosensory biology: by steady-state measurements of ligand-induced reporter gene expression in a model of bacterial quorum sensing and by flavin photocycling kinetic measurements of a LOV (light-oxygen-voltage) domain photoreceptor used for optogenetic transcriptional activation. Fully detailed guides for assembling the device and automating it using the custom Python-based API (Application Program Interface) are provided. This work contributes a key technology to the growing community-wide infrastructure of open-source biology-focused hardware, whose creation is facilitated by rapid prototyping capabilities and low-cost electronics, optoelectronics, and microcomputers.

The contribution of the White Collar complex to Cryptococcus neoformans virulence is independent of its light-sensing capabilities

The White Collar complex is responsible for sensing light and transmitting that signal in many fungal species. In Cryptococcus neoformans and C. deneoformans the complex is involved in protection against damage from ultraviolet (UV) light, repression of mating in response to light, and is also required for virulence. The mechanism by which the Bwc1 photoreceptor contributes to virulence is unknown. In this study, a bwc1 deletion mutant of C. neoformans was transformed with three versions of the BWC1 gene, the wild type, BWC1C605A or BWC1C605S, in which the latter two have the conserved cysteine residue replaced with either alanine or serine within the light-oxygen-voltage (LOV) domain that interacts with the flavin chromophore. The bwc1+ BWC1 strain complemented the UV sensitivity and the repression of mating in the light. The bwc1+ BWC1C605A and bwc1+ BWC1C605S strains were not fully complemented for either of the phenotypes, indicating that these BWC1 alleles impair the light responses for strains with them. Transcript analysis showed that neither of the mutated strains (bwc1+ BWC1C605A and bwc1+ BWC1C605S) showed the light-inducible expression pattern of the HEM15 and UVE1 genes as occurs in the wild type strain. These results indicate that the conserved flavin-binding site in the LOV domain of Bwc1 is required for sensing and responding to light in C. neoformans. In contrast to defects in light responses, the wild type, bwc1+ BWC1, bwc1+ BWC1C605A and bwc1+ BWC1C605S strains were equally virulent, whereas the bwc1 knock out mutant was less virulent. Furthermore, pre-exposure of the strains to light prior to inoculation had no influence on the outcome of infection. These findings define a division in function of the White Collar complex in fungi, in that in C. neoformans the role of Bwc1 in virulence is independent of light sensing.

Mechanistic Basis of the Fast Dark Recovery of the Short LOV Protein DsLOV from Dinoroseobacter shibae.

Light, oxygen, voltage (LOV) proteins, a ubiquitously distributed class of photoreceptors, regulate a wide variety of light-dependent physiological responses. Because of their modular architecture, LOV domains, i.e., the sensory domains of LOV photoreceptors, have been widely used for the construction of optogenetic tools. We recently described the structure and function of a short LOV protein (DsLOV) from the marine phototropic bacterium Dinoroseobacter shibae, for which, in contrast to other LOV photoreceptors, the dark state represents the physiologically relevant signaling state. Among bacterial LOV photoreceptors, DsLOV possesses an exceptionally fast dark recovery, corroborating its function as a "dark" sensor. To address the mechanistic basis of this unusual characteristic, we performed a comprehensive mutational, kinetic, thermodynamic, and structural characterization of DsLOV. The mechanistic basis of the fast dark recovery of the protein was revealed by mutation of the previously noted uncommon residue substitution at position 49 found in DsLOV. The substitution of M49 with different residues that are naturally conserved in LOV domains tuned the dark-recovery time of DsLOV over 3 orders of magnitude, without grossly affecting its overall structure or the light-dependent structural change observed for the wild-type protein. Our study thus provides a striking example of how nature can achieve LOV photocycle tuning by subtle structural alterations in the LOV domain active site, highlighting the easy evolutionary adaptability of the light sensory function. At the same time, our data provide guidance for the mutational photocycle tuning of LOV domains, with relevance for the growing field of optogenetics.

LOV Domains in the Design of Photoresponsive Enzymes.

In nature, a multitude of mechanisms have emerged for regulating biological processes and, specifically, protein activity. Light as a natural regulatory element is of outstanding interest for studying and modulating protein activity because it can be precisely applied with regard to a site of action, instant of time, or intensity. Naturally occurring photoresponsive proteins, predominantly those containing a light-oxygen-voltage (LOV) domain, have been characterized structurally and mechanistically and also conjugated to various proteins of interest. Immediate advantages of these new photoresponsive proteins such as genetic encoding, no requirement of chemical modification, and reversibility are paid for by difficulties in predicting the envisaged activity or type and site of domain fusion. In this article, we summarize recent advances and give a survey on currently available design concepts for engineering photoswitchable proteins.

Understanding photoreception in fungi and its role in fungal development with focus on phytopathogenic fungi

Like other environmental factors, light can influence various developmental processes in fungal species. Most of the fungi can respond to different wavelengths of light such as blue (475 nm), near UV (300–380 nm) and red light (650 nm). Blue light has been found to be the most associated light quality with fungal development. The most commonly investigated photoregulatory receptors in fungi are the white collar complex proteins (WC-1, WC-2) in the light oxygen voltage domain which sense and regulate the effects of blue light on fungal development and reproduction. The sequencing of several fungal genomes has led to the identification of other photoreceptor genes and their products, such as phytochromes (for red light), cryptochromes (for blue light) and blue and green light absorbing rhodopsins in some ascomycetes and basidiomycetes. Although similar types of regulatory mechanisms appear to be involved in signalling light responses in fungal species, nevertheless, there may be other hitherto undiscovered mechanisms responsible for regulating light perception. It warrants further investigations on photoreception in more fungal species including plant pathogenic fungi to better understand the underlying mechanisms controlling light perception at the metabolic level. Certain circadian rhythms have been found to play crucial role in fungal perception and adaptation to light. The information has led to the use of certain photoselective materials for control of some plant diseases.

Seeing the Light: The Roles of Red- and Blue-Light Sensing in Plant Microbes.

Plants collect, concentrate, and conduct light throughout their tissues, thus enhancing light availability to their resident microbes. This review explores the role of photosensing in the biology of plant-associated bacteria and fungi, including the molecular mechanisms of red-light sensing by phytochromes and blue-light sensing by LOV (light-oxygen-voltage) domain proteins in these microbes. Bacteriophytochromes function as major drivers of the bacterial transcriptome and mediate light-regulated suppression of virulence, motility, and conjugation in some phytopathogens and light-regulated induction of the photosynthetic apparatus in a stem-nodulating symbiont. Bacterial LOV proteins also influence light-mediated changes in both symbiotic and pathogenic phenotypes. Although red-light sensing by fungal phytopathogens is poorly understood, fungal LOV proteins contribute to blue-light regulation of traits, including asexual development and virulence. Collectively, these studies highlight that plant microbes have evolved to exploit light cues and that light sensing is often coupled with sensing other environmental signals.

Design, characterization and in vivo functioning of a light-dependent histidine protein kinase in the yeast Saccharomyces cerevisiae

Helical alignment of the α-helical linker of the LOV (light-oxygen-voltage) domain of YtvA from Bacillus subtilis with the α-helical linker of the histidine-protein kinase domain of the Sln1 kinase of the phospho-relay system for osmoregulation of Saccharomyces cerevisiae has been used to construct a light-modulatable histidine protein kinase. In vitro, illumination with blue light inhibits both the ATP-dependent phosphorylation of this hybrid kinase, as well as the phosphoryl transfer to Ypd1, the phosphoryl transfer domain of the Sln1 system. The helical alignment was carried out with conservation of the complete Jα helix of YtvA, as well as of the phosphorylatable histidine residue of the Sln1 kinase, with conservation of the hepta-helical motive of coiled-coil structures, recognizable in the helices of the two separate, constituent, proteins. Introduction of the gene encoding this hybrid histidine protein kinase into cells of S. cerevisiae in which the endogenous Sln1 kinase had been deleted, allowed us to modulate gene expression in the yeast cells with (blue) light. This was first demonstrated via the light-induced alteration of the expression level of the mannosyl-transferase OCH1, via a translational-fusion approach. As expected, illumination decreased the expression level of OCH1; the steady state decrease in saturating levels of blue light was about 40%. To visualize the in vivo functionality of this light-dependent regulation system, we fused the green fluorescent protein (GFP) to another regulatory protein, HOG1, which is also responsive to the Sln1 kinase. HOG1 is phosphorylated by the MAP-kinase-kinase Pbs2, which in turn is under control of the Sln1 kinase, via the phosphoryl transfer domain Ypd1. Fluorescence microscopy was used to show that illumination of cells that contained the combination of the hybrid kinase and the HOG1::GFP fusion protein, led to a persistent increase in the level of nuclear accumulation of HOG1, in contrast to salt stress, which—as expected—showed the well-characterized transient response. The system described in this study will be valuable in future studies on the role of cytoplasmic diffusion in signal transduction in eukaryotic cells.

Quantitative analyses of the equilibria among DNA complexes of a blue-light-regulated bZIP module, Photozipper.

Aureochrome1 is a blue-light-receptor protein identified in a stramenopile alga, Vaucheria frigida. Photozipper (PZ) is an N-terminally truncated, monomeric, V. frigida aureochrome1 fragment containing a basic leucine zipper (bZIP) domain and a light–oxygen–voltage (LOV)-sensing domain. PZ dimerizes upon photoexcitation and consequently increases its affinity for the target sequence. In the present study, to understand the equilibria among DNA complexes of PZ, DNA binding by PZ and mutational variants was quantitatively investigated by electrophoretic-mobility-shift assay and fluorescence-correlation spectroscopy in the dark and light states. DNA binding by PZ was sequence-specific and light-dependent. The half-maximal effective concentration of PZ for binding to the target DNA sequence was ~40 nM in the light, which was >10-fold less than the value in the dark. By contrast, the dimeric PZ-S2C variant (with intermolecular disulfide bonds) had higher affinity for the target sequence, with dissociation constants of ~4 nM, irrespective of the light conditions. Substitutions of Glu159 and Lys164 in the leucine zipper region decreased the affinity of PZ for the target sequence, especially in the light, suggesting that these residues form inter-helical salt bridges between leucine zipper regions, stabilizing the dimer–DNA complex. Our quantitative analyses of the equilibria in PZ–DNA-complex formation suggest that the blue-light-induced dimerization of LOV domains and coiled-coil formation by leucine zipper regions are the primary determinants of the affinity of PZ for the target sequence.

Electron transfer pathways in a light, oxygen, voltage (LOV) protein devoid of the photoactive cysteine

Blue-light absorption by the flavin chromophore in light, oxygen, voltage (LOV) photoreceptors triggers photochemical reactions that lead to the formation of a flavin-cysteine adduct. While it has long been assumed that adduct formation is essential for signaling, it was recently shown that LOV photoreceptor variants devoid of the photoactive cysteine can elicit a functional response and that flavin photoreduction to the neutral semiquinone radical is sufficient for signal transduction. Currently, the mechanistic basis of the underlying electron- (eT) and proton-transfer (pT) reactions is not well understood. We here reengineered pT into the naturally not photoreducible iLOV protein, a fluorescent reporter protein derived from the Arabidopsis thaliana phototropin-2 LOV2 domain. A single amino-acid substitution (Q489D) enabled efficient photoreduction, suggesting that an eT pathway is naturally present in the protein. By using a combination of site-directed mutagenesis, steady-state UV/Vis, transient absorption and electron paramagnetic resonance spectroscopy, we investigate the underlying eT and pT reactions. Our study provides strong evidence that several Tyr and Trp residues, highly conserved in all LOV proteins, constitute the eT pathway for flavin photoreduction, suggesting that the propensity for photoreduction is evolutionary imprinted in all LOV domains, while efficient pT is needed to stabilize the neutral semiquinone radical.

Hydrogen Bonding Environment of the N3-H Group of Flavin Mononucleotide in the Light Oxygen Voltage Domains of Phototropins.

The light oxygen voltage (LOV) domain is a flavin-binding blue-light receptor domain, originally found in a plant photoreceptor phototropin (phot). Recently, LOV domains have been used in optogenetics as the photosensory domain of fusion proteins. Therefore, it is important to understand how LOV domains exhibit light-induced structural changes for the kinase domain regulation, which enables the design of LOV-containing optogenetics tools with higher photoactivation efficiency. In this study, the hydrogen bonding environment of the N3-H group of flavin mononucleotide (FMN) of the LOV2 domain from Adiantum neochrome (neo) 1 was investigated by low-temperature Fourier transform infrared spectroscopy. Using specifically 15N-labeled FMN, [1,3-15N2]FMN, the N3-H stretch was identified at 2831 cm-1 for the unphotolyzed state at 150 K, indicating that the N3-H group forms a fairly strong hydrogen bond. The N3-H stretch showed temperature dependence, with a shift to lower frequencies at ≤200 K and to higher frequencies at ≥250 K from the unphotolyzed to the intermediate states. Similar trends were observed in the LOV2 domains from Arabidopsis phot1 and phot2. By contrast, the N3-H stretch of the Q1029L mutant of neo1-LOV2 and neo1-LOV1 was not temperature dependent in the intermediate state. These results seemed correlated with our previous finding that the LOV2 domains show the structural changes in the β-sheet region and/or the adjacent Jα helix of LOV2 domain, but that such structural changes do not take place in the Q1029L mutant or neo1-LOV1 domain. The environment around the N3-H group was also investigated.