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 ß-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.

Identification of the C=O stretching vibrations of FMN and peptide backbone by 13C-labeling of the LOV2 domain of Adiantum phytochrome3.

Phototropin, a blue-light photoreceptor in plants, has two FMN-binding domains named LOV1 and LOV2. We previously observed temperature-dependent FTIR spectral changes in the C=O stretching region (amide-I vibrational region of the peptide backbone) for the LOV2 domain of Adiantum phytochrome3 (phy3-LOV2), suggesting progressive structural changes in the protein moiety (Iwata, T., Nozaki, D., Tokutomi, S., Kagawa, T., Wada, M., and Kandori, H. (2003) Biochemistry 42, 8183-8191). Because FMN also possesses two C=O groups, in this article, we aimed at assigning C=O stretching vibrations of the FMN and protein by using 13C-labeling. We assigned the C(4)=O and C(2)=O stretching vibrations of FMN by using [4,10a-13C2] and [2-13C] FMNs, respectively, whereas C=O stretching vibrations of amide-I were assigned by using 13C-labeling of protein. We found that both C(4)=O and C(2)=O stretching vibrations shift to higher frequencies upon the formation of S390 at 77-295 K, suggesting that the hydrogen bonds of the C=O groups are weakened by adduct formation. Adduct formation presumably relocates the FMN chromophore apart from its hydrogen-bonding donors. Temperature-dependent amide-I bands are unequivocally assigned by separating the chromophore bands. The hydrogen bond of the peptide backbone in the loop region is weakened upon S390 formation at low temperatures, while being strengthened at room temperature. The hydrogen bond of the peptide backbone in the alpha-helix is weakened regardless of temperature. On the other hand, structural perturbation of the beta-sheet is observed only at room temperature, where the hydrogen bond is strengthened. Light-signal transduction by phy3-LOV2 must be achieved by the progressive protein structural changes initiated by the adduct formation of the FMN.

Enhanced production of electron cyclotron resonance plasma by exciting selective microwave mode on a large-bore electron cyclotron resonance ion source with permanent magnet.

We are constructing a tandem type ECRIS. The first stage is large-bore with cylindrically comb-shaped magnet. We optimize the ion beam current and ion saturation current by a mobile plate tuner. They change by the position of the plate tuner for 2.45 GHz, 11-13 GHz, and multi-frequencies. The peak positions of them are close to the position where the microwave mode forms standing wave between the plate tuner and the extractor. The absorbed powers are estimated for each mode. We show a new guiding principle, which the number of efficient microwave mode should be selected to fit to that of multipole of the comb-shaped magnets. We obtained the excitation of the selective modes using new mobile plate tuner to enhance ECR efficiency.

Controlling precise magnetic field configuration around electron cyclotron resonance zone for enhancing plasma parameters and beam current.

Multi-charged ion source which has wide operating conditions is required in various application fields. We have constructed tandem type ECR ion source (ECRIS); one of the features of its main stage is an additional coil for controlling magnetic field distribution around the mirror bottom precisely. Here the effect of magnetic field variation caused by the additional coil is experimentally considered in terms of plasma parameters and beam current as the first investigation of the main stage plasma. Furthermore, behavior of magnetic lines of force flowing from the ECR zone is calculated, and is compared with measurement results aiming for better understanding of interrelationship between plasma production and ion beam generation on the ECRIS.

New tandem type ion source based on electron cyclotron resonance for universal source of synthesized ion beams.

A new tandem type source has been constructed on the basis of electron cyclotron resonance (ECR) plasma for producing synthesized ion beams. We investigate feasibility and hope to realize the device which has wide range operation window in a single device to produce many kinds of ion beams based on ECR ion source (ECRIS). It is considered that ECR plasmas are necessary to be available to individual operations with different plasma parameters. Both of analysis of ion beams and investigation of plasma parameters are conducted on produced plasmas. We describe construction of the new tandem type ion source based on ECRIS with wide operation window for aiming at producing synthesized ion beams as this new source can be a universal source.

Formation of multi-charged ion beams by focusing effect of mid-electrode on electron cyclotron resonance ion source.

We are constructing a tandem type electron cyclotron resonance ion source (ECRIS) and a beam line for extracting ion beams. The ion beam is extracted from the second stage by an accel-decel extraction system with a single-hole and the ion beam current on each electrode is measured. The total ion beam current is measured by a faraday cup downstream the extraction electrodes. We measure these currents as a function of the mid-electrode potential. We also change the gap length between electrodes and perform similar measurement. The behaviors of these currents obtained experimentally against the mid-electrode potential show qualitatively good agreement with a simple theoretical consideration including sheath potential effects. The effect of mid-electrode potential is very useful for decreasing the beam loss for enhancing ion beam current extracted from ECRIS.

Dependence of ion beam current on position of mobile plate tuner in multi-frequencies microwaves electron cyclotron resonance ion source.

We are constructing a tandem-type electron cyclotron resonance ion source (ECRIS). The first stage of this can supply 2.45 GHz and 11-13 GHz microwaves to plasma chamber individually and simultaneously. We optimize the beam current I(FC) by the mobile plate tuner. The I(FC) is affected by the position of the mobile plate tuner in the chamber as like a circular cavity resonator. We aim to clarify the relation between the I(FC) and the ion saturation current in the ECRIS against the position of the mobile plate tuner. We obtained the result that the variation of the plasma density contributes largely to the variation of the I(FC) when we change the position of the mobile plate tuner.

Electron cyclotron resonance plasma production by using pulse mode microwaves and dependences of ion beam current and plasma parameters on the pulse condition.

We measure the ion beam current and the plasma parameters by using the pulse mode microwave operation in the first stage of a tandem type ECRIS. The time averaged extracted ion beam current in the pulse mode operation is larger than that of the cw mode operation with the same averaged microwave power. The electron density n(e) in the pulse mode is higher and the electron temperature T(e) is lower than those of the cw mode operation. These plasma parameters are considered to cause in the increase of the ion beam current and are suitable to produce molecular or cluster ions.

Profiles of ion beams and plasma parameters on a multi-frequencies microwaves large bore electron cyclotron resonance ion source with permanent magnets.

In order to contribute to various applications of plasma and beams based on an electron cyclotron resonance, a new concept on magnetic field with all magnets on plasma production and confinement has been proposed with enhanced efficiency for broad and dense ion beam. The magnetic field configuration consists of a pair of comb-shaped magnet surrounding plasma chamber cylindrically. Resonance zones corresponding for 2.45 GHz and 11-13 GHz frequencies are positioned at spatially different positions. We launch simultaneously multiplex frequencies microwaves operated individually, try to control profiles of the plasma parameters and the extracted ion beams, and to measure them in detail.

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.

Role of Gln1029 in the photoactivation processes of the LOV2 domain in adiantum phytochrome3.

Phototropin (phot) is a blue-light receptor in plants. The molecule has two FMN (flavin mononucleotide)-binding domains named the LOV (light-oxygen-voltage) domain, that is a subset of a PAS (per-arnt-sim) superfamily. Illumination of phot-LOV domains produces a covalent C(4a) flavin-cysteinyl adduct, which is called the S390 intermediate state. According to the crystal structures of the LOV2 domain of Adiantum phytochrome3 (phy3), a fusion protein of phot containing the phytochrome chromophoric domain, in the unphotolyzed and S390 states, and the side chain of Gln1029 switches hydrogen bonds with the FMN chromophore. Gln1029 is the hydrogen-bonding donor of the C(4)=O group of FMN in the unphotolyzed state, whereas Gln1029 is the hydrogen-bonding acceptor of the N(5)-H group of FMN in S390. In this paper, we measured the light-induced structural changes in the Q1029L mutant protein of phy3-LOV2 by means of low-temperature FTIR spectroscopy, and the obtained spectra are compared with those of the wild type. Low-temperature UV-visible spectroscopy of Q1029L detected only one intermediate state, S390, at 77-295 K, as well as the wild type. The C(4)=O stretch of FMN at 1710 cm(-1) is shifted to 1723 cm(-1) in Q1029L, presumably because of the lack of hydrogen bonds between Gln1029 and FMN. Upon formation of S390, the C(4)=O group hydrogen bond is weakened in both wild type and Q1029L. These observations are fully consistent with the X-ray crystal structures of the unphotolyzed and S390 states. On the other hand, the C(4)=O stretch of FMN and amide-I vibrations are temperature-independent in Q1029L, in contrast to wild type, in which highly temperature-dependent FTIR spectra are detected. Amide-I vibrations of Q1029L at room temperature are similar to those of the wild type at 77-150 K but not at room temperature. These facts imply that the Q1029L mutant protein lacks progressive protein structural changes following flavin-cysteinyl adduct formation in the wild type, which eventually alter structures of beta sheet and alpha helix in the protein moiety. Hydrogen-bonding interaction of Gln1029 with the FMN chromophore likely plays an important role in the protein structural changes of phy3-LOV2.

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.