Curated Optogenetic Publication Database

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.

Abstract: Near-infrared light is favourable for imaging in mammalian tissues due to low absorbance of hemoglobin, melanin, and water. Therefore, fluorescent proteins, biosensors and optogenetic constructs for optimal imaging, optical readout and light manipulation in mammals should have fluorescence and action spectra within the near-infrared window. Interestingly, natural Bacterial Phytochrome Photoreceptors (BphPs) utilize the low molecular weight biliverdin, found in most mammalian tissues, as a photoreactive chromophore. Due to their near-infrared absorbance BphPs are preferred templates for designing optical molecular tools for applications in mammals. Moreover, BphPs spectrally complement existing genetically-encoded probes. Several BphPs were already developed into the near-infrared fluorescent variants. Based on the analysis of the photochemistry and structure of BphPs we suggest a variety of possible BphP-based fluorescent proteins, biosensors, and optogenetic tools. Putative design strategies and experimental considerations for such probes are discussed.

Abstract: Phytochromes are red/far-red photoreceptors using cysteine-linked linear tetrapyrrole (bilin) chromophores to regulate biological responses to light. Light absorption triggers photoisomerization of the bilin between the 15Z and 15E photostates. The related cyanobacteriochromes (CBCRs) extend the photosensory range of the phytochrome superfamily to shorter wavelengths of visible light. Several subfamilies of CBCRs have been described. Representatives of one such subfamily, including AnPixJ and NpR6012g4, exhibit red/green photocycles in which the 15Z photostate is red-absorbing like that of phytochrome but the 15E photoproduct is instead green-absorbing. Using recombinant expression of individual CBCR domains in Escherichia coli, we fully survey the red/green subfamily from the cyanobacterium Nostoc punctiforme. In addition to 14 new photoswitching CBCRs, one apparently photochemically inactive protein exhibiting intense red fluorescence was observed. We describe a novel orange/green photocycle in one of these CBCRs, NpF2164g7. Dark reversion varied in this panel of CBCRs; some examples were stable as the 15E photoproduct for days, while others reverted to the 15Z dark state in minutes or even seconds. In the case of NpF2164g7, dark reversion was so rapid that reverse photoconversion of the green-absorbing photoproduct was not significant in restoring the dark state, resulting in a broadband response to light. Our results demonstrate that red/green CBCRs can thus act as sensors for the color or intensity of the ambient light environment.

Abstract: Phytochromes are photoreceptors in phototropic organisms that respond to light conditions by changing interactions between a response regulator and DNA. Bacterial phytochromes (BphPs) comprise an input photosensory core domain (PCD) and an output transducing domain (OTD). We report the structure of a BphP containing both PCD and the majority of its OTD, and demonstrate interaction with its cognate repressor. The OTD of RpBphP1, from Rhodopseudomonas palustris, is composed of a PAS/PAC domain and, to our knowledge, a hitherto unrecognized two-helix output sensor (HOS) domain. Unlike canonical BphPs, it does not transmit phosphorelay signals but forms a complex with the transcriptional repressor RpPpsR2 on photoconversion with far-red light. We show that HOS is essential for complex formation and that the anti-parallel dimer geometry is crucial in achieving HOS domain activation and protomer swapping under the control of light. These results provide insights into the steps taken by a two-component signaling system.

Abstract: Phytochromes are red/far-red photosensory proteins that regulate adaptive responses to light via photoswitching of cysteine-linked linear tetrapyrrole (bilin) chromophores. The related cyanobacteriochromes (CBCRs) extend the photosensory range of the phytochrome superfamily to shorter wavelengths of visible light. CBCRs and phytochromes share a conserved Cys residue required for bilin attachment. In one CBCR subfamily, often associated with a blue/green photocycle, a second Cys lies within a conserved Asp-Xaa-Cys-Phe (DXCF) motif and is essential for the blue/green photocycle. Such DXCF CBCRs use isomerization of the phycocyanobilin (PCB) chromophore into the related phycoviolobilin (PVB) to shorten the conjugated system for sensing green light. We here use recombinant expression of individual CBCR domains in Escherichia coli to survey the DXCF subfamily from the cyanobacterium Nostoc punctiforme. We describe ten new photoreceptors with well-resolved photocycles and three additional photoproteins with overlapping dark-adapted and photoproduct states. We show that the ability of this subfamily to form PVB or retain PCB provides a powerful mechanism for tuning the photoproduct absorbance, with blue-absorbing dark states leading to a broad range of photoproducts absorbing teal, green, yellow, or orange light. Moreover, we use a novel green/teal CBCR that lacks the blue-absorbing dark state to demonstrate that PVB formation requires the DXCF Cys residue. Our results demonstrate that this subfamily exhibits much more spectral diversity than had been previously appreciated.

Abstract: Cyanobacteriochromes are phytochrome homologues in cyanobacteria that act as sensory photoreceptors. We compare two cyanobacteriochromes, RGS (coded by slr1393) from Synechocystis sp. PCC 6803 and AphC (coded by all2699) from Nostoc sp. PCC 7120. Both contain three GAF (cGMP phosphodiesterase, adenylyl cyclase and FhlA protein) domains (GAF1, GAF2 and GAF3). The respective full-length, truncated and cysteine point-mutated genes were expressed in Escherichia coli together with genes for chromophore biosynthesis. The resulting chromoproteins were analyzed by UV-visible absorption, fluorescence and circular dichroism spectroscopy as well as by mass spectrometry. RGS shows a red-green photochromism (λ(max) = 650 and 535 nm) that is assigned to the reversible 15Z/E isomerization of a single phycocyanobilin-chromophore (PCB) binding to Cys528 of GAF3. Of the three GAF domains, only GAF3 binds a chromophore and the binding is autocatalytic. RGS autophosphorylates in vitro; this reaction is photoregulated: the 535 nm state containing E-PCB was more active than the 650 nm state containing Z-PCB. AphC from Nostoc could be chromophorylated at two GAF domains, namely GAF1 and GAF3. PCB-GAF1 is photochromic, with the proposed 15E state (λ(max) = 685 nm) reverting slowly thermally to the thermostable 15Z state (λ(max)  = 635 nm). PCB-GAF3 showed a novel red-orange photochromism; the unstable state (putative 15E, λ(max) = 595 nm) reverts very rapidly (τ ~ 20 s) back to the thermostable Z state (λ(max) = 645 nm). The photochemistry of doubly chromophorylated AphC is accordingly complex, as is the autophosphorylation: E-GAF1/E-GAF3 shows the highest rate of autophosphorylation activity, while E-GAF1/Z-GAF3 has intermediate activity, and Z-GAF1/Z-GAF3 is the least active state.

Abstract: Phytochromes are well-known as photoactive red- and near IR-absorbing chromoproteins with cysteine-linked linear tetrapyrrole (bilin) prosthetic groups. Phytochrome photoswitching regulates adaptive responses to light in both photosynthetic and nonphotosynthetic organisms. Exclusively found in cyanobacteria, the related cyanobacteriochrome (CBCR) sensors extend the photosensory range of the phytochrome superfamily to shorter wavelengths of visible light. Blue/green light sensing by a well-studied subfamily of CBCRs proceeds via a photolabile thioether linkage to a second cysteine fully conserved in this subfamily. In the present study, we show that dual-cysteine photosensors have repeatedly evolved in cyanobacteria via insertion of a second cysteine at different positions within the bilin-binding GAF domain (cGMP-specific phosphodiesterases, cyanobacterial adenylate cyclases, and formate hydrogen lyase transcription activator FhlA) shared by CBCRs and phytochromes. Such sensors exhibit a diverse range of photocycles, yet all share ground-state absorbance of near-UV to blue light and a common mechanism of light perception: reversible photoisomerization of the bilin 15,16 double bond. Using site-directed mutagenesis, chemical modification and spectroscopy to characterize novel dual-cysteine photosensors from the cyanobacterium Nostoc punctiforme ATCC 29133, we establish that this spectral diversity can be tuned by varying the light-dependent stability of the second thioether linkage. We also show that such behavior can be engineered into the conventional phytochrome Cph1 from Synechocystis sp. PCC6803. Dual-cysteine photosensors thus allow the phytochrome superfamily in cyanobacteria to sense the full solar spectrum at the earth surface from near infrared to near ultraviolet.

Abstract: Light-mediated control of gene expression and thus of any protein function and metabolic process in living microbes is a rapidly developing field of research in the areas of functional genomics, systems biology, and biotechnology. The unique physical properties of the environmental factor light allow for an independent photocontrol of various microbial processes in a noninvasive and spatiotemporal fashion. This mini review describes recently developed strategies to generate photo-sensitive expression systems in bacteria and yeast. Naturally occurring and artificial photoswitches consisting of light-sensitive input domains derived from different photoreceptors and regulatory output domains are presented and individual properties of light-controlled expression systems are discussed.

Abstract: Signaling photoreceptors use the information contained in the absorption of a photon to modulate biological activity in plants and a wide range of organisms. The fundamental-and as yet imperfectly answered-question is, how is this achieved at the molecular level? We adopt the perspective of biophysicists interested in light-dependent signal transduction in nature and the three-dimensional structures that underpin signaling. Six classes of photoreceptors are known: light-oxygen-voltage (LOV) sensors, xanthopsins, phytochromes, blue-light sensors using flavin adenine dinucleotide (BLUF), cryptochromes, and rhodopsins. All are water-soluble proteins except rhodopsins, which are integral membrane proteins; all are based on a modular architecture except cryptochromes and rhodopsins; and each displays a distinct, light-dependent chemical process based on the photochemistry of their nonprotein chromophore, such as isomerization about a double bond (xanthopsins, phytochromes, and rhodopsins), formation or rupture of a covalent bond (LOV sensors), or electron transfer (BLUF sensors and cryptochromes).

Abstract: The spectroscopic identification of sensory rhodopsin I by Bogomolni and Spudich in 1982 provided a molecular link between the light environment and phototaxis in Halobacterium salinarum, and thus laid the foundation for the study of signal transducing photosensors in prokaryotes. In recent years, a number of new prokaryotic photosensory receptors have been discovered across a broad range of taxa, including dozens in chemotrophic species. Among these photoreceptors are new classes of rhodopsins, BLUF-domain proteins, bacteriophytochromes, cryptochromes, and LOV-family photosensors. Genetic and biochemical analyses of these receptors have demonstrated that they can regulate processes ranging from photosynthetic pigment biosynthesis to virulence.

Abstract: In the purple photosynthetic bacterium Rhodopseudomonas palustris, far-red illumination induces photosystem synthesis via the action of the bacteriophytochrome RpBphP1. This bacteriophytochrome antagonizes the repressive effect of the transcriptional regulator PpsR2 under aerobic condition. We show here that, in addition to photosystem synthesis, far-red light induces a significant growth rate limitation, compared to cells grown in the dark, linked to a decrease in the respiratory activity. The phenotypes of mutants inactivated in RpBphP1 and PpsR2 show their involvement in this regulation. Based on enzymatic and transcriptional studies, a 30% decrease in the expression of the alpha-ketoglutarate dehydrogenase complex, a central enzyme of the Krebs cycle, is observed under far-red light. We propose that this decrease is responsible for the down-regulation of respiration in this condition. This regulation mechanism at the Krebs cycle level still allows the formation of the photosynthetic apparatus via the synthesis of key biosynthesis precursors but lowers the production of NADH, i.e. the respiratory activity. Overall, the dual action of RpBphP1 on the regulation of both the photosynthesis genes and the Krebs cycle allows a fine adaptation of bacteria to environmental conditions by enhancement of the most favorable bioenergetic process in the light, photosynthesis versus respiration.