Time-resolved photoacoustics of channelrhodopsins: early energetics and light-driven volume changes.

In biological photoreceptors, the energy stored in early transient species is a key feature to drive the photocycle or a chain of reactions. Time-resolved photoacoustics (PA) can explore the energy landscape of transient species formed within few ns after photoexcitation, as well as volumetric changes (ΔV) of these intermediates with respect to the parental state. In this work, PA identified these important parameters for several channelrhodopsins, namely CaChR1 from Chlamydomonas augustae and CrChR2 from Chlamydomonas reinhardtii and various variants.

A red-green photochromic bacterial protein as a new contrast agent for improved photoacoustic imaging.

The GAF3 domain of the cyanobacteriochrome Slr1393 from . PCC6803, binding phycocyanobilin as a chromophore, shows photochromicity between two stable, green- and red-absorbing states, characterized by relatively high photoconversion yields. Using nanosecond-pulsed excitation by red or green light, respectively, and suitable cw photoconversion beams, we demonstrate that the light-modulatable photoacoustic waveforms arising from GAF3 can be easily distinguished from background signals originating from non-modulatable competitive absorbers and scattering media.

Oxygen diffusion pathways in mutated forms of a LOV photoreceptor from : A molecular dynamics study.

4511 from is a photoreceptor of the light, oxygen voltage (LOV) family, binding flavin mononucleotide (FMN) as a chromophore. It exhibits the prototypical LOV photocycle, with the reversible formation of an FMN-Cys71 adduct via fast decay of the FMN triplet state. 4511 has high potential as a photosensitiser for singlet oxygen (SO) upon mutation of C71.

Mapping the role of aromatic amino acids within a blue-light sensing LOV domain.

Photosensing LOV (Light, Oxygen, Voltage) domains detect and respond to UVA/Blue (BL) light by forming a covalent adduct between the flavin chromophore and a nearby cysteine, via the decay of the flavin triplet excited state. LOV domains where the reactive cysteine has been mutated are valuable fluorescent tools for microscopy and as genetically encoded photosensitisers for reactive oxygen species. Besides being convenient tools for applications, LOV domains without the reactive cysteine (naturally occurring or engineered) can still be functionally photoactivated via formation of a neutral flavin radical.

A light life together: photosensing in the plant microbiota.

Bacteria and fungi of the plant microbiota can be phytopathogens, parasites or symbionts that establish mutually advantageous relationships with plants. They are often rich in photoreceptors for UVA-Visible light, and in many cases, they exhibit light regulation of growth patterns, infectivity or virulence, reproductive traits, and production of pigments and of metabolites. In addition to the light-driven effects, often demonstrated via the generation of photoreceptor gene knock-outs, microbial photoreceptors can exert effects also in the dark.

Interplay among the "flipping" glutamine, a conserved phenylalanine, water and hydrogen bonds within a blue-light sensing LOV domain.

In this work we exploited time-resolved photoacoustics (PA) and molecular dynamics (MD) simulations to investigate the function of a conserved phenylalanine residue in blue sensing (BL) LOV domains. The LOV photocycle involves reversible formation of a photoproduct (LOV390) where the flavin mononucleotide (FMN) chromophore is covalently bound to a cysteine. LOV390 thermally returns to the dark adapted state (LOV447) with a lifetime τrec (s-to-h).

The first molecular characterisation of blue- and red-light photoreceptors from Methylobacterium radiotolerans.

Methylobacteria are facultative methylotrophic phytosymbionts of great industrial and agronomical interest, and they are considered as opportunistic pathogens posing a health threat to humans. So far only a few reports mention photoreceptor coding sequences in Methylobacteria genomes, but no investigation at the molecular level has been performed yet. We here present comprehensive in silico research into potential photoreceptors in this bacterial phylum and report the photophysical and photochemical characterisation of two representatives of the most widespread photoreceptor classes, a blue-light sensing LOV (light, oxygen, voltage) protein and a red/far red light sensing BphP (biliverdin-binding bacterial phytochrome) from M.

Single mutation in a novel bacterial LOV protein yields a singlet oxygen generator.

Mr4511 from Methylobacterium radiotolerans is a 164 amino acid protein built of a flavin mononucleotide (FMN) binding, blue-light responsive LOV (Light, Oxygen, Voltage) core domain plus flanking regions. In contrast to the majority of LOV domains, Mr4511 lacks a tryptophan residue that was previously identified as a major quencher for the FMN triplet state in photosensitizers for singlet oxygen (SO) engineered from these photoreceptors. Here we show that for Mr4511 it is sufficient to only mutate the reactive cysteine responsible for the photocycle (Cys71) in the native protein to generate an efficient SO photosensitizer: both C71S and C71G variants exhibit SO quantum yields of formation, Φ, around 0.

Dynamics and efficiency of photoswitching in biliverdin-binding phytochromes.

The light-driven conversions between the dark-adapted and the photoproduct state were recorded for bacteriophytochromes (BphP) carrying biliverdin IXα (BV) as chromophore by time-resolved absorption spectroscopy. BphPs can be photoswitched between a red absorbing (Pr, maximum at ca. 700 nm) and a far-red/near-infrared (Pfr, maximum at ca.

MAS NMR on a Red/Far-Red Photochromic Cyanobacteriochrome All2699 from .

Unlike canonical phytochromes, the GAF domain of cyanobacteriochromes (CBCRs) can bind bilins autonomously and is sufficient for functional photocycles. Despite the astonishing spectral diversity of CBCRs, the GAF1 domain of the three-GAF-domain photoreceptor all2699 from the cyanobacterium 7120 is the only CBCR-GAF known that converts from a red-absorbing (Pr) dark state to a far-red-absorbing (Pfr) photoproduct, analogous to the more conservative phytochromes. Here we report a solid-state NMR spectroscopic study of all2699g1 in its Pr state.

Far-red acclimating cyanobacterium as versatile source for bright fluorescent biomarkers.

Far-red and near-infrared emitting chromophores extend applications of fluorescent proteins to regions of maximal transmission of most tissues, but present considerable engineering challenges. Far-red adapting cyanobacteria generate a novel set of biliproteins. One of them, ApcF2, from a thermophilic cyanobacterium was subjected to structure-guided, site-directed random and specific mutagenesis, and was screened for bright far-red emission.

Blue-Light Receptors for Optogenetics.

Sensory photoreceptors underpin light-dependent adaptations of organismal physiology, development, and behavior in nature. Adapted for optogenetics, sensory photoreceptors become genetically encoded actuators and reporters to enable the noninvasive, spatiotemporally accurate and reversible control by light of cellular processes. Rooted in a mechanistic understanding of natural photoreceptors, artificial photoreceptors with customized light-gated function have been engineered that greatly expand the scope of optogenetics beyond the original application of light-controlled ion flow.

Time-Resolved Energetics of Photoprocesses in Prokaryotic Phytochrome-Related Photoreceptors.

Time-resolved photoacoustics (PA) is uniquely able to explore the energy landscape of photoactive proteins and concomitantly detects light-induced volumetric changes (ΔV) accompanying the formation and decay of transient species in a time window between ca. 20 ns and 5 μs. Here, we report PA measurements on diverse photochromic bilin-binding photoreceptors of prokaryotic origin: (1) the chromophore-binding GAF3 domain of the red (R)/green (G) switching cyanobacteriochrome 1393 (Slr1393g3) from Synechocystis; (2) the red/far red (R/FR) Synechocystis Cph1 phytochrome; (3) full-length and truncated constructs of Xanthomonas campestris bacteriophytochrome (XccBphP), absorbing up to the NIR spectral region.

FRET in a Synthetic Flavin- and Bilin-binding Protein.

The last decade has seen development and application of a large number of novel fluorescence-based techniques that have revolutionized fluorescence microscopy in life sciences. Preferred tags for such applications are genetically encoded fluorescent proteins (FP), mostly derivatives of the green fluorescent protein (GFP). Combinations of FPs with wavelength-separated absorption/fluorescence properties serve as excellent tools for molecular interaction studies, for example, protein-protein complexes or enzyme-substrate interactions, based on the FRET phenomenon (Förster resonance energy transfer).

Solving Blue Light Riddles: New Lessons from Flavin-binding LOV Photoreceptors.

Detection of blue light (BL) via flavin-binding photoreceptors (Fl-Blues) has evolved throughout all three domains of life. Although the main BL players, that is light, oxygen and voltage (LOV), blue light sensing using flavins (BLUF) and Cry (cryptochrome) proteins, have been characterized in great detail with respect to structure and function, still several unresolved issues at different levels of complexity remain and novel unexpected findings were reported. Here, we review the most prevailing riddles of LOV-based photoreceptors, for example: the relevance of water and/or small metabolites for the dynamics of the photocycle; molecular details of light-to-signal transduction events; the interplay of BL sensing by LOV domains with other environmental stimuli, such as BL plus oxygen-mediating photodamage and its impact on microbial lifestyles; the importance of the cell or chromophore redox state in determining the fate of BL-driven reactions; the evolutionary pathways of LOV-based BL sensing and associated functions through the diverse phyla.

Functional Characterization of a LOV-Histidine Kinase Photoreceptor from Xanthomonas citri subsp. citri.

The blue-light (BL) absorbing protein Xcc-LOV from Xanthomonas citri subsp. citri is composed of a LOV-domain, a histidine kinase (HK) and a response regulator. Spectroscopic characterization of Xcc-LOV identified intermediates and kinetics of the protein's photocycle.

Visualizing the relevance of bacterial blue- and red-light receptors during plant-pathogen interaction.

The foliar pathogen Pseudomonas syringae pv. tomato DC3000 (Pst) leads to consistent losses in tomato crops, urging to multiply investigations on the physiological bases for its infectiveness. As other P.

The Evolution and Functional Role of Flavin-based Prokaryotic Photoreceptors.

Flavin-based photoreceptor proteins of the LOV (light, oxygen and voltage) superfamily are ubiquitous and appear to be essential blue-light sensing systems not only in plants, algae and fungi, but also in prokaryotes, where they are represented in more than 10% of known species. Despite their broad occurrence, only in few cases LOV proteins have been correlated with important phenomena such as bacterial infectivity, selective growth patterns or/and stress responses; nevertheless these few known roles are helping us understand the multiple ways by which prokaryotes can exploit these soluble blue-light photoreceptors. Given the large number of sequences now deposited in databases, it becomes meaningful to define a signature for bona fide LOV domains, a procedure that facilitates identification of proteins with new properties and phylogenetic analysis.

Photochromic conversion in a red/green cyanobacteriochrome from Synechocystis PCC6803: quantum yields in solution and photoswitching dynamics in living E. coli cells.

The protein encoded by the gene slr1393 from the cyanobacterium Synechocystis sp. PCC6803 (Slr1393) is composed of three GAF domains, a PAS domain, and a histidine kinase motif. The third GAF domain (referred to as GAF3) was previously characterized as the sole domain in this protein, being able to carry phycocyanobilin (PCB) as the chromophore and to accomplish photochemistry.

The dark recovery rate in the photocycle of the bacterial photoreceptor YtvA is affected by the cellular environment and by hydration.

We report thermal recovery kinetics of the lit state into the parental dark state, measured for the blue light-sensing photoreceptor YtvA inside overexpressing E. coli and B. subtilis bacterial cells, performed for the wild type and several mutated proteins.

A cyanobacterial light activated adenylyl cyclase partially restores development of a Dictyostelium discoideum, adenylyl cyclase a null mutant.

A light-regulated adenylyl cyclase, mPAC, was previously identified from the cyanobacterium Microcoleus chthonoplastes PCC7420. MPAC consists of a flavin-based blue light-sensing LOV domain and a catalytic domain. In this work, we expressed mPAC in an adenylate cyclase A null mutant (aca-) of the eukaryote Dictyostelium discoideum and tested to what extent light activation of mPAC could restore the cAMP-dependent developmental programme of this organism.

From Plant Infectivity to Growth Patterns: The Role of Blue-Light Sensing in the Prokaryotic World.

Flavin-based photoreceptor proteins of the LOV (Light, Oxygen, and Voltage) and BLUF (Blue Light sensing Using Flavins) superfamilies are ubiquitous among the three life domains and are essential blue-light sensing systems, not only in plants and algae, but also in prokaryotes. Here we review their biological roles in the prokaryotic world and their evolution pathways. An unexpected large number of bacterial species possess flavin-based photosensors, amongst which are important human and plant pathogens.

A LOV-domain-mediated blue-light-activated adenylate (adenylyl) cyclase from the cyanobacterium Microcoleus chthonoplastes PCC 7420.

Genome screening of the cyanobacterium Microcoleus chthonoplastes PCC 7420 identified a gene encoding a protein (483 amino acids, 54.2 kDa in size) characteristic of a BL (blue light)-regulated adenylate (adenylyl) cyclase function. The photoreceptive part showed signatures of a LOV (light, oxygen, voltage) domain.

A structural model for the full-length blue light-sensing protein YtvA from Bacillus subtilis, based on EPR spectroscopy.

A model for the full-length structure of the blue light-sensing protein YtvA from Bacillus subtilis has been determined by EPR spectroscopy, performed on spin labels selectively inserted at amino acid positions 54, 80, 117 and 179. Our data indicate that YtvA forms a dimer in solution and enable us, based on the known structures of the individual domains and modelling, to propose a three-dimensional model for the full length protein. Most importantly, this includes the YtvA N-terminus that has so far not been identified in any structural model.