Curated Optogenetic Publication Database

Abstract: Light sensing by photoreceptors controls phototropism, chloroplast movement, stomatal opening, and leaf expansion in plants. Understanding the molecular mechanism by which these processes are regulated requires a quantitative description of photoreceptor dynamics. We focus on a light-driven signal transduction mechanism in the LOV2 domain (LOV, light, oxygen, voltage) of the blue light photoreceptor phototropin 1 from Avena sativa (oat). High-resolution crystal structures of the dark and light states of an oat LOV2 construct including residues Leu404 through Leu546 (LOV2 (404-546)) have been determined at 105 and 293 K. In all four structures, LOV2 (404-546) exhibits the typical Per-ARNT-Sim (PAS) fold, flanked by an additional conserved N-terminal turn-helix-turn motif and a C-terminal flanking region containing an amphipathic Jalpha helix. These regions dock on the LOV2 core domain and bury several hydrophobic residues of the central beta-sheet of the core domain that would otherwise be exposed to solvent. Light structures of LOV2 (404-546) reveal that formation of the covalent bond between Cys450 and the C4a atom of the flavin mononucleotide (FMN) results in local rearrangement of the hydrogen-bonding network in the FMN binding pocket. These rearrangements are associated with disruption of the Asn414-Asp515 hydrogen bond on the surface of the protein and displacement of the N- and C-terminal flanking regions of LOV2 (404-546), both of which constitute a structural signal.

Abstract: Phototropins (phot1 and phot2) are blue light receptor kinases that control a range of photoresponses that serve to optimize the photosynthetic efficiency of plants. Light sensing by the phototropins is mediated by a repeated motif at the N-terminal region of the protein known as the LOV domain. Bacterially expressed LOV domains bind flavin mononucleotide noncovalently and are photochemically active in solution. Irradiation of the LOV domain results in the formation of a flavin-cysteinyl adduct (LOV390) which thermally relaxes back to the ground state in the dark, effectively completing a photocycle that serves as a molecular switch to control receptor kinase activity. We have employed a random mutagenesis approach to identify further amino acid residues involved in LOV-domain photochemistry. Escherichia coli colonies expressing a mutagenized population of LOV2 derived from Avena sativa (oat) phot1 were screened for variants that showed altered photochemical reactivity in response to blue light excitation. One variant showed slower rates of LOV390 formation but exhibited adduct decay times 1 order of magnitude faster than wild type. A single Ile --> Val substitution was responsible for the effects observed, which removes a single methyl group found in van der Waals contact with the cysteine sulfur involved in adduct formation. A kinetic acceleration trend was observed for adduct decay by decreasing the size of the isoleucine side chain. Our findings therefore indicate that the steric nature of this amino acid side chain contributes to stabilization of the C-S cysteinyl adduct.

Abstract: Light plays a crucial role in activating phototropins, a class of plant photoreceptors that are sensitive to blue and UV-A wavelengths. Previous studies indicated that phototropin uses a bound flavin mononucleotide (FMN) within its light-oxygen-voltage (LOV) domain to generate a protein-flavin covalent bond under illumination. In the C-terminal LOV2 domain of Avena sativa phototropin 1, formation of this bond triggers a conformational change that results in unfolding of a helix external to this domain called Jalpha [Harper, S. M., et al. (2003) Science 301, 1541-1545]. Though the structural effects of illumination were characterized, it was unknown how these changes are coupled to kinase activation. To examine this, we made a series of point mutations along the Jalpha helix to disrupt its interaction with the LOV domain in a manner analogous to light activation. Using NMR spectroscopy and limited proteolysis, we demonstrate that several of these mutations displace the Jalpha helix from the LOV domain independently of illumination. When placed into the full-length phototropin protein, these point mutations display constitutive kinase activation, without illumination of the sample. These results indicate that unfolding of the Jalpha helix is the critical event in regulation of kinase signaling for the phototropin proteins.

Abstract: For single-cell and multicellular systems to survive, they must accurately sense and respond to their cellular and extracellular environment. Light is a nearly ubiquitous environmental factor, and many species have evolved the capability to respond to this extracellular stimulus. Numerous photoreceptors underlie the activation of light-sensitive signal transduction cascades controlling these responses. Here, we review the properties of the light, oxygen, or voltage (LOV) family of blue-light photoreceptor domains, a subset of the Per-ARNT-Sim (PAS) superfamily. These flavin-binding domains, first identified in the higher-plant phototropins, are now shown to be present in plants, fungi, and bacteria. Notably, LOV domains are coupled to a wide array of other domains, including kinases, phosphodiesterases, F-box domains, STAS domains, and zinc fingers, which suggests that the absorption of blue light by LOV domains regulates the activity of these structurally and functionally diverse domains. LOV domains contain a conserved molecular volume extending from the flavin cofactor, which is the locus for light-driven structural change, to the molecular surface. We discuss the role of this conserved volume of structure in LOV-regulated processes.