Curated Optogenetic Publication Database

Abstract: Phytochromes are photoreceptor proteins in plants, fungi, and bacteria. They can adopt two photochromic states with differential biochemical responses. The structural changes transducing the signal from the chromophore to the biochemical output modules are poorly understood due to challenges in capturing structures of the dynamic, full-length protein. Here, we present cryoelectron microscopy (cryo-EM) structures of the phytochrome from Pseudomonas aeruginosa (PaBphP) in its resting (Pfr) and photoactivated (Pr) state. The kinase-active Pr state has an asymmetric, dimeric structure, whereas the kinase-inactive Pfr state opens up. This behavior is different from other known phytochromes and we explain it with the unusually short connection between the photosensory and output modules. Multiple sequence alignment of this region suggests evolutionary optimization for different modes of signal transduction in sensor proteins. The results establish a new mechanism for light-sensing by phytochrome histidine kinases and provide input for the design of optogenetic phytochrome variants.

Abstract: Bacterial phytochrome photoreceptors usually belong to two-component signaling systems which transmit environmental stimuli to a response regulator through a histidine kinase domain. Phytochromes switch between red light-absorbing and far-red light-absorbing states. Despite exhibiting extensive structural responses during this transition, the model bacteriophytochrome from Deinococcus radiodurans (DrBphP) lacks detectable kinase activity. Here, we resolve this long-standing conundrum by comparatively analyzing the interactions and output activities of DrBphP and a bacteriophytochrome from Agrobacterium fabrum (Agp1). Whereas Agp1 acts as a conventional histidine kinase, we identify DrBphP as a light-sensitive phosphatase. While Agp1 binds its cognate response regulator only transiently, DrBphP does so strongly, which is rationalized at the structural level. Our data pinpoint two key residues affecting the balance between kinase and phosphatase activities, which immediately bears on photoreception and two-component signaling. The opposing output activities in two highly similar bacteriophytochromes suggest the use of light-controllable histidine kinases and phosphatases for optogenetics.

Abstract: Phototropins are photoreceptor proteins, which regulate blue light dependent biological processes for efficient photosynthesis in plants and algae. The proteins consist of a photosensory domain that responds to the ambient light and an output module that triggers cellular responses. The photosensory domain of phototropin from Chlamydomonas reinhardtii contains two conserved LOV (Light-Oxygen-Voltage) domains with flavin chromophores. Blue light triggers the formation of a covalent cysteine-flavin adduct and upregulates the phototropin kinase activity. Little is known about the structural mechanism which leads to kinase activation and how the two LOV domains contribute to this. Here, we investigate the role of the LOV1 domain from Chlamydomonas reinhardtii phototropin by characterizing the structural changes occurring after blue light illumination with nano- millisecond time-resolved X-ray solution scattering. By structurally fitting the data with atomic models generated by molecular dynamics simulations, we find that the adduct formation induces a rearrangement of the hydrogen bond network from the buried chromophore to the protein surface. Particularly, the change in conformation and associated hydrogen bonding of the conserved glutamine 120 induce a global movement of the β-sheet, ultimately driving a change in electrostatic potential on the protein surface. Based on the change of electrostatics, we propose a structural model of how LOV1 and LOV2 domains interact and regulate the full-length phototropin from Chlamydomonas reinhardtii. This provides a rationale for how LOV photosensor proteins function and contributes to the optimal design of optogenetic tools based on LOV domains.

Abstract: Bacterial phytochrome photoreceptors usually belong to two-component signaling systems which transmit environmental stimuli to a response regulator through a histidine kinase domain. Phytochromes switch between red light-absorbing and far-red light-absorbing states. Despite exhibiting extensive structural responses during this transition, the model bacteriophytochrome from Deinococcus radiodurans (DrBphP) lacks detectable kinase activity. Here, we resolve this long-standing conundrum by comparatively analyzing the interactions and output activities of DrBphP and a bacteriophytochrome from Agrobacterium fabrum (AgP1). Whereas AgP1 acts as a conventional histidine kinase, we identify DrBphP as a light-sensitive phosphatase. While AgP1 binds its cognate response regulator only transiently, DrBphP does so strongly, which is rationalized at the structural level. Our data pinpoint two key residues affecting the balance between kinase and phosphatase activities, which immediately bears on photoreception and two-component signaling. The opposing output activities in two highly similar bacteriophytochromes inform the use of light-controllable histidine kinases and phosphatases for optogenetics.

Abstract: Light-oxygen-voltage (LOV) receptors are sensory proteins controlling a wide range of organismal adaptations in multiple kingdoms of life. Because of their modular nature, LOV domains are also attractive for use as optogenetic actuators. A flavin chromophore absorbs blue light, forms a bond with a proximal cysteine residue, and induces changes in the surroundings. There is a gap of knowledge on how this initial signal is relayed further through the sensor to the effector module. To characterize these conformational changes, we apply time-resolved X-ray scattering to the homodimeric LOV domain from Bacillus subtilis YtvA. We observe a global structural change in the LOV dimer synchronous with the formation of the chromophore photoproduct state. Using molecular modeling, this change is identified as splaying apart and relative rotation of the two monomers, which leads to an increased separation at the anchoring site of the effector modules.

Abstract: The phytochrome family of light-switchable proteins has long been studied by biochemical, spectroscopic and crystallographic means, while a direct probe for global conformational signal propagation has been lacking. Using solution X-ray scattering, we find that the photosensory cores of several bacterial phytochromes undergo similar large-scale structural changes upon red-light excitation. The data establish that phytochromes with ordinary and inverted photocycles share a structural signaling mechanism and that a particular conserved histidine, previously proposed to be involved in signal propagation, in fact tunes photoresponse.