Light signal transduction pathway from flavin chromophore to the J alpha helix of Arabidopsis phototropin1.

Department of Frontier Materials, Nagoya Institute of Technology, Nagoya, Japan.
Biophysical Journal (Impact Factor: 3.83). 04/2009; 96(7):2771-8. DOI: 10.1016/j.bpj.2008.12.3924
Source: PubMed

ABSTRACT In the plant blue-light sensor phototropin, illumination of the chromophoric LOV domains causes activation of the serine/threonine kinase domain. Flavin mononucleotide (FMN) is a chromophore molecule in the two LOV domains (LOV1 and LOV2), but only LOV2 is responsible for kinase activation. Previous studies reported an important role of an additional helix connected to the C-terminal of LOV2 (Jalpha helix) for the function of phototropin; however, it remains unclear how the Jalpha helix affects light-induced structural changes in LOV2. In this study we compared light-induced protein structural changes of the LOV2 domain of Arabidopsis phot1 in the absence (LOV2-core) and presence (LOV2-Jalpha) of the Jalpha helix by Fourier-transform infrared spectroscopy. Prominent peaks were observed only in the amide-I region (1650 (-)/1625 (+) cm(-1)) of LOV2-Jalpha at physiological temperatures (>/=260 K), corresponding to structural perturbation of the alpha-helix. The peaks were diminished by point mutation of functionally important amino acids such as Phe-556 between FMN and the beta-sheet, Gln-575 being hydrogen-bonded with FMN, and Ile-608 on the Jalpha helix. We thus conclude that a light signal is relayed from FMN through these amino acids and eventually changes the interaction between LOV2-core and the Jalpha helix in Arabidopsis phot1.

1 Bookmark
  • [Show abstract] [Hide abstract]
    ABSTRACT: The photochemical reaction of the LOV1 (light-oxygen-voltage 1) domain of phototropin 1 from Arabidopsis thaliana was investigated by the time-resolved transient grating method. As with other LOV domains, an absorption spectral change associated with an adduct formation between its chromophore (flavin mononucleotide) and a cysteine residue was observed with a time constant of 1.1 μs. After this reaction, a significant diffusion coefficient (D) change (D of the reactant = 8.2 × 10(-11) m(2) s(-1), and D of the photoproduct = 6.4 × 10(-11) m(2) s(-1)) was observed with a time constant of 14 ms at a protein concentration of 270 μM. From the D value of the ground state and the peak position in size exclusion chromatography, we have confirmed that the phot1LOV1 domain exists as a dimer in the dark. The D-value and the concentration dependence of the rate indicated that the phot1LOV1 domain associates to form a tetramer (dimerization of the dimer) upon photoexcitation. We also found that the chromophore is released from the binding pocket of the LOV domain when it absorbs two photons within a pulse duration, which occurs in addition to the normal photocycle reaction. On the basis of these results, we discuss the molecular mechanism of the light dependent role of the phot1LOV1 domain.
    Photochemical and Photobiological Sciences 06/2013; · 2.92 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The spatio-temporal control of gene expression is fundamental to elucidate cell proliferation and deregulation phenomena in living systems. Novel approaches based on light-sensitive multi-protein complexes have recently been devised, showing promising perspectives for the noninvasive and reversible modulation of the DNA-transcriptional activity in vivo. This has lately been demonstrated in a striking way through the generation of the artificial protein construct LOV-TAP, in which the light-oxygen-voltage-2-Jα photoswitch of phototropin1 from Avena Sativa (AsLOV2-Jα) has been ligated to the tryptophan-repressor protein (TrpR) from Escherichia coli. Although tremendous progress has been achieved on the generation of such protein constructs, a detailed understanding of their functioning as opto-genetical tools is still in its infancy. Here, we elucidate the early stages of the light-induced regulatory mechanism of LOV-TAP at the molecular level, using the noninvasive molecular dynamics simulation technique. More specifically, we find that Cys450-FMN-adduct formation in the AsLOV2-Jα-binding pocket after photoexcitation induces the cleavage of the peripheral Jα-helix from the LOV core, causing a change of its polarity and electrostatic attraction of the photoswitch onto the DNA surface. This goes along with the flexibilization through unfolding of a hairpin-like helix-loop-helix region inter-linking the AsLOV2-Jα- and TrpR-domains, ultimately enabling the condensation of LOV-TAP onto the DNA surface. By contrast, in the dark state the AsLOV2-Jα photoswitch remains inactive and exerts a repulsive electrostatic force on the DNA surface. This leads to a distortion of the hairpin region, which finally relieves its tension by causing the disruption of LOV-TAP from the DNA. Proteins 2012. © 2012 Wiley Periodicals, Inc.
    Proteins Structure Function and Bioinformatics 10/2012; · 3.34 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Devising analysis tools for elucidating the regulatory mechanism of complex enzymes is a challenging task since 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 sub-domains. 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;. © 2014 Wiley Periodicals, Inc.
    Proteins Structure Function and Bioinformatics 03/2014; · 3.34 Impact Factor

Full-text (2 Sources)

Available from
Jun 2, 2014