Vibrational spectroscopy of an algal Phot-LOV1 domain probes the molecular changes associated with blue-light reception

The LOV1 domain of the blue light Phot1-receptor (phototropin homolog) from Chlamydomonas reinhardtii has been studied by vibrational spectroscopy. The FMN modes of the dark state of LOV1 were identified by preresonance Raman spectroscopy and assigned to molecular vibrations. By comparing the blue-light-induced FTIR difference spectrum with the preresonance Raman spectrum, most of the differences are due to FMN modes. Thus, we exclude large backbone changes of the protein that might occur during the phototransformation of the dark state LOV1-447 into the putative signaling state LOV1-390. Still, the presence of smaller amide difference bands cannot be excluded but may be masked by overlapping FMN modes. The band at 2567 cm(-1) is assigned to the S-H stretching vibration of C57, the residue that forms the transient thio-adduct with the chromophore FMN. The occurrence of this band is evidence that C57 is protonated in the dark state of LOV1. This result challenges conclusions from the homologous LOV2 domain from oat that the thiolate of the corresponding cysteine is the reactive species.     

Irreversible photoreduction of flavin in a mutated Phot-LOV1 domain

Phot photoreceptors make up an important protein family regulating biological processes in response to blue light. They contain two light, oxygen, and voltage sensitive (LOV) domains and a serine/threonine kinase domain. Both LOV domains noncovalently bind a flavin mononucleotide (FMN). Upon absorption of blue light, the LOV domains undergo a photocycle, transiently forming a covalent adduct of a cysteine residue and the FMN (LOV-390). The mechanism of formation of this flavin-thiol adduct is still unclear. We studied a mutant of the LOV1 domain from the green alga Chlamydomonas reinhardtii with a methionine replacing the reactive cysteine 57 (C57M). As in the wild type, irradiation leads to formation of a photoadduct, which, however, is irreversibly converted into a red absorbing species, C57M-675. On the basis of spectroscopic results and the 2.1 A resolution crystal structure, this highly unusual FMN species was assigned to a neutral flavin radical covalently attached to the apoprotein at the N(5) position. In contrast to other flavoprotein neutral radicals, C57M-675 is stable even under aerobic or denaturing conditions. Pathways for the photoinduced formation of the adduct are discussed for the C57M mutant as well as the wild-type LOV1 domain.     

Crystal structures and molecular mechanism of a light-induced signaling switch: The Phot-LOV1 domain from Chlamydomonas reinhardtii

Phot proteins (phototropins and homologs) are blue-light photoreceptors that control mechanical processes like phototropism, chloroplast relocation, or guard-cell opening in plants. Phot receptors consist of two flavin mononucleotide (FMN)-binding light, oxygen, or voltage (LOV) domains and a C-terminal serine/threonine kinase domain. We determined crystal structures of the LOV1 domain of Phot1 from the green alga Chlamydomonas reinhardtii in the dark and illuminated state to 1.9 A and 2.8 A resolution, respectively. The structure resembles that of LOV2 from Adiantum (Crosson, S. and K. Moffat. 2001. PROC: Natl. Acad. Sci. USA. 98:2995-3000). In the resting dark state of LOV1, the reactive Cys-57 is present in two conformations. Blue-light absorption causes formation of a proposed active signaling state that is characterized by a covalent bond between the flavin C4a and the thiol of Cys-57. There are differences around the FMN chromophore but no large overall conformational changes. Quantum chemical calculations based on the crystal structures revealed the electronic distribution in the active site during the photocycle. The results suggest trajectories for electrons, protons, and the active site cysteine and offer an interpretation of the reaction mechanism.     

The phot LOV2 domain and its interaction with LOV1

Phot proteins are homologs of the blue-light receptor phototropin. We report a comparative study of the photocycles of the isolated, light-sensitive domains LOV1 and LOV2 from Chlamydomonas reinhardtii phot protein, as well as the construct LOV1/2 containing both domains. Transient absorption measurements revealed a short lifetime of the LOV2-wt triplet state (500 ns), but a long lifetime (287 micros) of the triplet in the mutant LOV2-C250S, in which the reactive cysteine is replaced by serine. For LOV1, in comparison, corresponding numbers of 800 ns and 4 micros for the two conformers in LOV1-wt, and 27 micros for LOV1-C57S have been reported. The triplet decay kinetics in the mixed domains LOV1/2-wt, LOV1/2-C57S, and LOV1/2-C250S can be analyzed as the superposition of the behavior of the corresponding single domains. The situation is different for the slow, thermal reaction of the photoadduct back to the dark form. Whereas the individual domains LOV1 and LOV2 show two decay components, the double domains LOV1/2-C57S and LOV1/2-C250S both show only a single component. The interaction of the two domains does therefore not manifest itself during the lifetime of the triplet states, but changes the decay behavior of the adduct states.     

Characterization of a flavin radical product in a C57M mutant of a LOV1 domain by electron paramagnetic resonance

In the flavin mononucleotide-binding LOV1 domain of the Phot1-receptor from Chlamydomonas reinhardtii the photoreactive cysteine C57 has been replaced by methionine. Photoexcitation of this C57M mutant yields a metastable photoproduct (C57M-415) that thermally decomposes into a stable paramagnetic species (C57M-675) with extremely red-shifted absorption in the visible range. In this contribution, we describe the characterization of this radical by multi-frequency electron paramagnetic resonance and electron-nuclear double resonance. The main features of the spectra identify the paramagnetic species as a flavin neutral radical. However, detailed analysis shows that the isoalloxazine moiety of the flavin is alkyl substituted at N(5), rather than protonated as is usually the case. The implication of these observations on the likely mechanism of photoproduct generation in wild-type LOV domains is discussed.     

Phot-LOV1: Photocycle of a Blue-Light Receptor Domain from the Green Alga Chlamydomonas reinhardtii

The “Phot” protein family comprises blue-light photoreceptors that consist of two flavin mononucleotide (FMN)-binding LOV (light, oxygen, and voltage) domains and a serine/threonine kinase domain. We have investigated the LOV1 domain of Phot1 from Chlamydomonas reinhardtii by time-resolved absorption spectroscopy. Photoexcitation of the dark form, LOV1-447, causes transient bleaching and formation of two spectrally similar red-shifted intermediates that are both assigned to triplet states of the FMN. The triplet states decay with time constants of 800 ns and 4 μs with an efficiency of >90% into a blue-shifted intermediate, LOV1-390, that is attributed to a thiol adduct of cysteine 57 to FMN C(4a). LOV1-390 reverts to the dark form in hundreds of seconds, the time constant being dependent on pH and salt concentration. In the mutant C57S, where the thiol adduct cannot be formed, the triplet state displays an oxygen-dependent decay directly to the dark form. We present here a spectroscopic characterization of an algal sensory photoreceptor in general and of a LOV1 domain photocycle in particular. The results are discussed with respect to the behavior of the homologous LOV2 domain from oat.     

The photochemistry of the light-, oxygen-, and voltage-sensitive domains in the algal blue light receptor phot

Phot proteins are blue light photoreceptors in plants and algae that mainly regulate photomovement responses. They contain two light-, oxygen-, and voltage-sensitive (LOV) domains and a serine/threonine kinase domain. Both LOV domains noncovalently bind a flavin mononucleotide (FMN) as chromophore. Upon blue light illumination, the LOV domains undergo a photocycle, transiently forming a covalent adduct of the FMN moiety with a nearby cysteine residue. The presence of two light-sensitive domains in the photoreceptor raises the question about the differences in properties and function between LOV1 and LOV2. As a model system, the photocycles of the LOV1 and LOV2 domains from phot of the green alga Chlamydomonas reinhardtii have been studied in detail, both separately and in a tandem construct. Here we give an overview about the results on the individual behavior of the domains and their interaction. Furthermore, the current status in the understanding of the role of LOV1 in phot in general is presented.     

Heterogeneous environment of the S-H group of Cys966 near the flavin chromophore in the LOV2 domain of Adiantum neochrome1

The primary photochemistry of the blue-light sensor protein, phototropin, is adduct formation between the C4a atom of the flavin mononucleotide (FMN) chromophore and a nearby, reactive cysteine (Cys966), following decay of the triplet excited state of FMN. The distance between the C4a position of FMN and the sulfur atom of Cys966 is 4.2 A in the LOV2 domain of Adiantum neochrome 1 (neo1-LOV2), a fusion protein of phototropin containing the phytochrome chromophoric domain. We previously reported the presence of an unreactive fraction in neo1-LOV2 at low temperatures, which presumably originated from the heterogeneous environment of Cys966 [Iwata, T., Nozaki, D., Tokutomi, S., Kagawa, T., Wada, M., and Kandori, H. (2003) Biochemistry 42, 8183-8191]. The present study showed that (i) 28% forms an adduct at 77 K (state I), (ii) 50% forms an adduct at 150 K but not at 77 K (state II), and (iii) 22% does not form an adduct at 150 K (state III). By Fourier transform infrared (FTIR) spectroscopy, we observed the S-H stretching frequencies at 2570 and 2562 cm-1 for state I and at 2563 cm-1 for state II, suggesting that the microenvironment of the S-H group of Cys966 determines the reactivity at low temperatures. Adduct formation is more efficient for state I than for states II and III. Molecular dynamics simulation strongly suggests that the observed multiple structures originate from the isomeric forms of Cys966. We thus concluded that there are multiple local structures of FMN and cysteine in neo1-LOV2, each of which is thermally converted by protein fluctuation at physiological temperatures.     

Conformational analysis of the blue-light sensing protein YtvA reveals a competitive interface for LOV-LOV dimerization and interdomain interactions.

The Bacillus subtilis protein YtvA is related to plant phototropins in that it senses UVA-blue-light by means of the flavin binding LOV domain, linked to a nucleotide-binding STAS domain. The structural basis for interdomain interactions and functional regulation are not known. Here we report the conformational analysis of three YtvA constructs, by means of size exclusion chromatography, circular dichroism (CD) and molecular docking simulations. The isolated YtvA-LOV domain (YLOV, aa 25-126) has a strong tendency to dimerize, prevented in full-length YtvA, but still observed in YLOV carrying the N-terminal extension (N-YLOV, aa 1-126). The analysis of CD data shows that both the N-terminal cap and the linker region (aa 127-147) between the LOV and the STAS domain are helical and that the central beta-scaffold is distorted in the LOV domains dimers. The involvement of the central beta-scaffold in dimerization is supported by docking simulation of the YLOV dimer and the importance of this region is highlighted by light-induced conformational changes, emerging from the CD data analysis. In YtvA, the beta-strand fraction is notably less distorted and distinct light-driven changes in the loops/turn fraction are detected. The data uncover a common surface for LOV-LOV and intraprotein interaction, involving the central beta-scaffold, and offer hints to investigate the molecular basis of light-activation and regulation in LOV proteins.     

Dynamic switching mechanisms in LOV1 and LOV2 domains of plant phototropins

LOV domains are the light-sensitive portion of plant phototropins. They absorb light through a flavin cofactor, photochemically form a covalent bond between the chromophore and a cysteine residue in the protein, and proceed to mediate activation of an attached kinase domain. Although the photoreaction itself is now well-characterized experimentally and computationally, it is still unclear how the formation of the adduct leads to kinase activation. We have performed molecular dynamics simulations on the LOV1 domain of Chlamydomonas reinhardtii and the LOV2 domain of Avena sativa, both before and after the photoreaction, to answer this question. The extensive simulations, over 240 ns in duration, reveal significant differences in how the LOV1 and LOV2 domains respond to photoactivation. The simulations indicate that LOV1 activation is likely caused by a change in hydrogen bonding between protein and ligand that destabilizes a highly conserved salt bridge, whereas LOV2 activation seems to result from a change in the flexibility of a set of protein loops. Results of electrostatics calculations, principal component analysis, sequence alignments, and root mean-square deviation analysis corroborate the above findings.     

Structural basis for light-dependent signaling in the dimeric LOV domain of the photosensor YtvA

The photosensor YtvA binds flavin mononucleotide and regulates the general stress reaction in Bacillus subtilis in response to blue light illumination. It belongs to the family of light-oxygen-voltage (LOV) proteins that were first described in plant phototropins and form a subgroup of the Per-Arnt-Sim (PAS) superfamily. Here, we report the three-dimensional structure of the LOV domain of YtvA in its dark and light states. The protein assumes the global fold common to all PAS domains and dimerizes via a hydrophobic interface. Directly C-terminal to the core of the LOV domain, an alpha-helix extends into the solvent. Light absorption causes formation of a covalent bond between a conserved cysteine residue and atom C(4a) of the FMN ring, which triggers rearrangements throughout the LOV domain. Concomitantly, in the dark and light structures, the two subunits of the dimeric protein rotate relative to each other by 5 degrees . This small quaternary structural change is presumably a component of the mechanism by which the activity of YtvA is regulated in response to light. In terms of both structure and signaling mechanism, YtvA differs from plant phototropins and more closely resembles prokaryotic heme-binding PAS domains.     

Recording of blue light-induced energy and volume changes within the wild-type and mutated phot-LOV1 domain from Chlamydomonas reinhardtii

The time-resolved thermodynamics of the flavin mononucleotide (FMN)-binding LOV1 domain of Chlamydomonas reinhardtii phot (phototropin homolog) was studied by means of laser-induced optoacoustic spectroscopy. In the wild-type protein the early red-shifted intermediate LOV(715) exhibits a small volume contraction, DeltaV(715) = -1.50 ml/mol, with respect to the parent state. LOV(715) decays within few micro s into the covalent FMN-Cys-57 adduct LOV(390), that shows a larger contraction, DeltaV(390) = -8.8 ml/mol, suggesting a loss of entropy and conformational flexibility. The high energy content of LOV(390), E(390) = 180 kJ/mol, ensures the driving force for the completion of the photocycle and points to a strained photoreceptor conformation. In the LOV-C57S mutated protein the photoadduct is not formed and DeltaV(390) is undetected. Large effects on the measured DeltaVs are observed in the photochemically competent R58K and R58K/D31Q mutated proteins, with DeltaV(390) = -2.0 and -1.9 ml/mol, respectively, and DeltaV(715) approximately 0. The D31Q and D31N substitutions exhibit smaller but well-detectable effects. These results show that the photo-induced volume changes involve the protein region comprising Arg-58, which tightly interacts with the FMN phosphate group.     

Photochemical properties of the flavin mononucleotide-binding domains of the phototropins from Arabidopsis,rice, and Chlamydomonas reinhardtii

Phototropins (phot1 and phot2, formerly designated nph1 and npl1) are blue-light receptors that mediate phototropism, blue light-induced chloroplast relocation, and blue light-induced stomatal opening in Arabidopsis. Phototropins contain two light, oxygen, or voltage (LOV) domains at their N termini (LOV1 and LOV2), each a binding site for the chromophore flavin mononucleotide (FMN). Their C termini contain a serine/threonine protein kinase domain. Here, we examine the kinetic properties of the LOV domains of Arabidopsis phot1 and phot2, rice (Oryza sativa) phot1 and phot2, and Chlamydomonas reinhardtii phot. When expressed in Escherichia coli, purified LOV domains from all phototropins examined bind FMN tightly and undergo a self-contained photocycle, characterized by fluorescence and absorption changes induced by blue light (T. Sakai, T. Kagawa, M. Kasahara, T.E. Swartz, J.M. Christie, W.R. Briggs, M. Wada, K. Okada [2001] Proc Natl Acad Sci USA 98: 6969-6974; M. Salomon, J.M. Christie, E. Knieb, U. Lempert, W.R. Briggs [2000] Biochemistry 39: 9401-9410). The photocycle involves the light-induced formation of a cysteinyl adduct to the C(4a) carbon of the FMN chromophore, which subsequently breaks down in darkness. In each case, the relative quantum efficiencies for the photoreaction and the rate constants for dark recovery of LOV1, LOV2, and peptides containing both LOV domains are presented. Moreover, the data obtained from full-length Arabidopsis phot1 and phot2 expressed in insect cells closely resemble those obtained for the tandem LOV-domain fusion proteins expressed in E. coli. For both Arabidopsis and rice phototropins, the LOV domains of phot1 differ from those of phot2 in their reaction kinetic properties and relative quantum efficiencies. Thus, in addition to differing in amino acid sequence, the phototropins can be distinguished on the basis of the photochemical cycles of their LOV domains. The LOV domains of C. reinhardtii phot also undergo light-activated spectral changes consistent with cysteinyl adduct formation. Thus, the phototropin family extends over a wide evolutionary range from unicellular algae to higher plants.     

Comparative investigation of the LOV1 and LOV2 domains in Adiantum phytochrome3

Phototropin (phot) is a blue-light photoreceptor for phototropic responses, relocation of chloroplasts, and stomata opening in plants. Phototropin has two chromophore-binding domains named LOV1 and LOV2 in its N-terminal half, each of which binds a flavin mononucleotide (FMN) noncovalently. The C-terminal half is a Ser/Thr kinase. A transgenic study of Arabidopsis suggested that only LOV2 domain is necessary for the kinase activity, whereas X-ray crystallographic structures of LOV1 and LOV2 domains are almost identical. These facts imply that the detailed structures and/or structural changes are different between LOV1 and LOV2 domains. In this study, we compared light-induced structural changes of the LOV1 and LOV2 domains of a phototropin, Adiantum phytochrome3 (phy3), by means of UV-visible and Fourier transform infrared (FTIR) spectroscopy. Photochemical properties of an adduct formation between FMN and a cysteine are essentially similar between phy3-LOV1 and phy3-LOV2. On the other hand, the S-H group of the reactive cysteine forms a hydrogen bond in phy3-LOV1, which is strengthened at low temperatures. This is possibly correlated with the fact that no adduct formation takes place for phy3-LOV1 at 77 K as revealed by the UV-visible absorption spectra. The most prominent difference was seen in the amide-I vibration that monitors the secondary structure of peptide backbone. Protein structural changes in phy3-LOV2 involve the regions of loops, alpha-helices, and beta-sheets, which differ significantly among various temperatures. Extended protein structural changes are probably correlated with the signal transduction activity of LOV2. In contrast, protein structural changes were very small in phy3-LOV1, and they were almost temperature independent. The photocycle of phy3-LOV1 takes 3.1 h, being more than 100 times longer than that of phy3-LOV2. These facts suggest that Adiantum phy3-LOV1 does not work for light sensing, being consistent with the previous transgenic study of Arabidopsis. It is likely that plants utilize a unique protein architecture (LOV domain) for different functions by regulating their protein structural changes.     

Exploring the multiscale signaling behavior of phototropin1 from Chlamydomonas reinhardtii using a full‐residue space kinetic Monte Carlo molecular dynamics technique

Devising analysis tools for elucidating the regulatory mechanism of complex enzymes has been a challenging task for many decades. It generally requires the determination of the structural‐dynamical information of protein solvent systems far from equilibrium over multiple length and time scales, which is still difficult both theoretically and experimentally. To cope with the problem, we introduce a full‐residue space multiscale simulation method based on a combination of the kinetic Monte Carlo and molecular dynamics techniques, in which the rates of the rate‐determining processes are evaluated from a biomolecular forcefield on the fly during the simulation run by taking into account the full space of residues. To demonstrate its reliability and efficiency, we explore the light‐induced functional behavior of the full‐length phototropin1 from Chlamydomonas reinhardtii (Cr‐phot1) and its various subdomains. Our results demonstrate that in the dark state the light oxygen voltage‐2‐Jα (LOV2‐Jα) photoswitch inhibits the enzymatic activity of the kinase, whereas the LOV1‐Jα photoswitch controls the dimerization with the LOV2 domain. This leads to the repulsion of the LOV1‐LOV2 linker out of the interface region between both LOV domains, which results in a positively charged surface suitable for cell–membrane interaction. By contrast, in the light state, we observe that the distance between both LOV domains is increased and the LOV1‐LOV2 linker forms a helix–turn–helix (HTH) motif, which enables gene control through nucleotide binding. Finally, we find that the kinase is activated through the disruption of the Jα‐helix from the LOV2 domain, which is followed by a stretching of the activation loop (A‐loop) and broadening of the catalytic cleft of the kinase. Proteins 2014; 82:2018–2040. © 2014 Wiley Periodicals, Inc.     

FMN Binding and Photochemical Properties of Plant Putative Photoreceptors Containing Two LOV Domains,LOV/LOV Proteins

LOV domains function as blue light-sensing modules in various photoreceptors in plants, fungi, algae, and bacteria. A LOV/LOV protein (LLP) has been found from Arabidopsis thaliana (AtLLP) as a two LOV domain-containing protein. However, its function remains unknown. We isolated cDNA clones coding for an LLP homolog from tomato (Solanum lycopersicum) and two homologs from the moss Physcomitrella patens. The tomato LLP (SlLLP) contains two LOV domains (LOV1 and LOV2 domains), as in AtLLP. Most of the amino acids required for association with chromophore are conserved in both LOV domains, except that the amino acid at the position equivalent to the cysteine essential for cysteinyl adduct formation is glycine in the LOV1 domain as in AtLLP. When expressed in Escherichia coli, SlLLP binds FMN and undergoes a self-contained photocycle upon irradiation of blue light. Analyses using mutant SlLLPs revealed that SlLLP binds FMN in both LOV domains, although the LOV1 domain does not show spectral changes on irradiation. However, when Gly⁶⁶ in the LOV1 domain, which is located at the position equivalent to the essential cysteine of LOV domains, is replaced by cysteine, the mutated LOV1 domain shows light-induced spectral changes. In addition, all four LOV domains of P. patens LLPs (PpLLP1 and PpLLP2) show the typical features of LOV domains, including the reactive cysteine in each. This study shows that plants have a new LOV domain-containing protein family with the typical biochemical and photochemical properties of other LOV domain-containing proteins such as the phototropins.     

Signals of LOV1: a computer simulation study on the wildtype LOV1-domain of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> and its mutants

Phototropins are photoreceptors regulating the blue-light response in plants and bacteria. They consist of two LOV (light oxygen voltage sensitive) domains each containing a non-covalently bound flavin-mononucleotide (FMN) chromophore, which are connected to a serine/threonine-kinase. Upon illumination, the LOV-domains undergo conformational changes, triggering a signal cascade in the organism through kinase activation. Here, we present results from molecular dynamics simulations in which we investigate the signal transduction pathway of the wildtype LOV1-domain of Chlamydomonas reinhardtii and a methyl-mercaptan (MM) adduct of its Cys57Gly-mutant at the molecular level. In particular, we analyzed the effect of covalent-bond formation between the reactive cysteine Cys57 and the FMN-reaction center, as well as the subsequent charge redistribution, on the spatio-dynamical behavior of the LOV1-domain. We compare the calculation results with experimental data and demonstrate that these adduct state characteristics have an important influence on the response of this photosensor. The light-induced changes implicate primarily an alteration of the surface charge distribution through rearrangement of the highly flexible Cα-, Dα- and Eα-helices including the Glu51-Lys91-salt bridge on the hydrophilic side of the protein domain and a β-sheet tightening process via coupling of the Aβ- and Bβ-strands. Our findings confirm the aptitude of the LOV1-domain to function as a dimerization partner, allowing the green alga to adapt its reproduction and growth speed to the environmental conditions.     

Light-induced structural changes in the LOV2 domain of Adiantum phytochrome3 studied by low-temperature FTIR and UV-visible spectroscopy

Phototropin (Phot) is a blue-light receptor in plants. The molecule has two FMN (flavin mononucleotide) binding domains named LOV (light-, oxygen-, and voltage-sensing), which is a subset of the PAS (Per-Arnt-Sim) superfamily. Illumination of the phot-LOV domains in the dark state (D447) produces a covalent C(4a) flavin-cysteinyl adduct (S390) via a triplet excited state (L660), which reverts to D447 in the dark. In this work, we studied the light-induced structural changes in the LOV2 domain of Adiantum phytochrome3 (phy3), which is a fusion protein of phot containing the phytochrome chromophoric domain, by low-temperature UV-visible and FTIR spectroscopy. UV-visible spectroscopy detected only one intermediate state, S390, in the temperature range from 77 to 295 K, indicating that the adduct is produced even at temperatures as low as 77 K, although a portion of D447 cannot be converted to S390 at low temperatures possibly because of motional freezing. In the whole temperature range, FTIR spectra in the S-H stretching frequency region showed that Cys966 of phy3-LOV2 is protonated in D447 and unprotonated on illumination, supporting adduct formation. The pK(a) of the S-H group in D447 is estimated to be >10. FTIR spectra also showed the light-induced appearance of a positive peak around 3621 cm(-1) in the whole temperature range, indicating that adduct formation accompanies rearrangement of a hydrogen bond of a water molecule(s), which can be either water25, water45, or both, near the chromophore. In contrast to the weak temperature dependence of the spectral changes in the UV-visible absorption and the FTIR of both S-H and O-H stretching bands, light-induced changes in the amide I vibration that probes protein backbone structure vary significantly with the increase in temperature. The spectral changes suggest that light excitation of FMN loosens the local structure around it, particularly in turns, in the early stages and that another change subsequently takes place to tighten it, mainly in beta-structure, but some occur in the alpha-helical structure of the protein moiety as well. Interestingly, these changes proceed without altering the shape of UV-visible spectra, suggesting the presence of multiple conformation states in S390.     

Mutations in N-terminal flanking region of blue light-sensing light-oxygen and voltage 2 (LOV2) domain disrupt its repressive activity on kinase domain in the Chlamydomonas phototropin

Phototropin is a light-regulated kinase that mediates a variety of photoresponses such as phototropism, chloroplast positioning, and stomata opening in plants to increase the photosynthetic efficiency. Blue light stimulus first induces local conformational changes in the chromophore-bearing light-oxygen and voltage 2 (LOV2) domain of phototropin, which in turn activates the serine/threonine (Ser/Thr) kinase domain in the C terminus. To examine the kinase activity of full-length phototropin conventionally, we employed the budding yeast Saccharomyces cerevisiae. In this organism, Ser/Thr kinases (Fpk1p and Fpk2p) that show high sequence similarity to the kinase domain of phototropins exist. First, we demonstrated that the phototropin from Chlamydomonas reinhardtii (CrPHOT) could complement loss of Fpk1p and Fpk2p to allow cell growth in yeast. Furthermore, this reaction was blue light-dependent, indicating that CrPHOT was indeed light-activated in yeast cells. We applied this system to a large scale screening for amino acid substitutions in CrPHOT that elevated the kinase activity in darkness. Consequently, we identified a cluster of mutations located in the N-terminal flanking region of LOV2 (R199C, L202L, D203N/G/V, L204P, T207I, and R210H). An in vitro phosphorylation assay confirmed that these mutations substantially reduced the repressive activity of LOV2 on the kinase domain in darkness. Furthermore, biochemical analyses of the representative T207I mutant demonstrated that the mutation affected neither spectral nor multimerization properties of CrPHOT. Hence, the N-terminal flanking region of LOV2, as is the case with the C-terminal flanking Jα region, appears to play a crucial role in the regulation of kinase activity in phototropin.