LOV-based optogenetic devices: light-driven modules to impart photoregulated control of cellular signaling

Abstract

The Light-Oxygen-Voltage domain family of proteins is widespread in biology where they impart sensory responses to signal transduction domains. The small, light responsive LOV modules offer a novel platform for the construction of optogenetic tools. Currently, the design and implementation of these devices is partially hindered by a lack of understanding of how light drives allosteric changes in protein conformation to activate diverse signal transduction domains. Further, divergent photocycle properties amongst LOV family members complicate construction of highly sensitive devices with fast on/off kinetics. In the present review we discuss the history of LOV domain research with primary emphasis on tuning LOV domain chemistry and signal transduction to allow for improved optogenetic tools.

Keywords: LOV domain; optogenetics; photobiology; photosensors; protein engineering.

Figure 1

LOV chemistry and structure. (A) Typical photocycle spectra of LOV containing proteins. Dark state proteins (black) demonstrate spectra consistent with oxidized flavin. Light activation (red) bleaches the 450 nm absorbing bands leaving a single 390 nm peak indicative of a C4a adduct. (B) LOV photocycles are characterized by a ground state oxidize flavin that form a flavin-cysteine C4a adduct following blue-light treatment. Adduct formation proceeds through an excited singlet state (LOV^*) that rapidly forms a Triplet species (LOV^T). The triplet abstracts an electron from C38 generating a radical pair. Radical recombination forms the C4a adduct (LOV390). The adduct decays to the ground state by either thermal decay (k_T) or UV-scission. (C–F) Structures of LOV proteins involved in optogenetic tools, AsLOV2 (C), VVD (D), YtvA (E), and EL222 (F). The LOV core is depicted in gray, with associated N-terminal caps (green) and C-terminal caps (salmon).

Figure 2

Sites for rate altering variants. (A) Sequence alignment and universal numbering scheme for LOV proteins and optogenetic tools. The numbering scheme (in parentheses for AsLOV2) used in this review references K413 of AsLOV2 as residue 1 of the core LOV domain. All residues are then numbered in reference to the alignment provided, where residue inserts (E-F loop) or deletions (YtvA) are ignored in the universal numbering system. Residues that have been targeted for rate altering effects are depicted in blue. (B) Steric interactions (blue residues) select for alternative conformations of C38. Conf2 places the thiol directly above the C4a position, where it is poised for C4a adduct formation. I4 juts in between the two conformations placing its methyl group only 4.0 Å away from Cβ. Rotation between the two conformations would require movement of I4. (C) A network of H-bonds in the pyrimidine ring stabilize the C4a adduct through electron withdrawing effects. (D) Full active site containing residues attenuating Conf1/2 (blue), residues at the re-face (red) and H-bonding residues (gray). Three residues, M54, L82, and M84 attenuate adduct decay pathways through steric and electronic regulation of the flavin.

Figure 3

LOV optogenetic tools. Existing LOV-based tools exploit one of two possible mechanisms. (A) LOV and effector are attached through a helical linker to create an inhibitory surface that is released following photoexcitation. (B) An effector molecule is split into two inactive components. Light activation induces LOV-mediated dimer formation to activate the effector molecule.

Figure 4

LOV signaling mechanisms. (A) Dark (gray) and light (cyan) state structures of AsLOV2. Photoactivation leads to rotation of Q101 to alter H-bonding contacts to N2. N2 undergoes a light driven interchange involving contacts with D103 and Q101. The H-bond switch affects Ncap structure to disorder the Jα helix. (B) Dark (gray) and light (cyan) state structures of VVD. C4a adduct formation promotes rotation of Q101 to alter H-bonds to A2. Movement of the Ncap reorientates C1 to disrupt contacts with D-2, leading to rearrangement of the Ncap and dimer formation. (C) (YtvA) and (D) (EL222) mechanisms are less understood but likely involve Q101 and H-bonding contacts (black dotted line) to neighboring residues.