QuasAr Odyssey: the origin of fluorescence and its voltage sensitivity in microbial rhodopsins.

Rhodopsins had long been considered non-fluorescent until a peculiar voltage-sensitive fluorescence was reported for archaerhodopsin-3 (Arch3) derivatives. These proteins named QuasArs have been used for imaging membrane voltage changes in cell cultures and small animals, but they could not be applied in living rodents. To develop the next generation of sensors, it is indispensable to first understand the molecular basis of the fluorescence and its modulation by the membrane voltage.

Ultrafast proton-coupled isomerization in the phototransformation of phytochrome.

The biological function of phytochromes is triggered by an ultrafast photoisomerization of the tetrapyrrole chromophore biliverdin between two rings denoted C and D. The mechanism by which this process induces extended structural changes of the protein is unclear. Here we report ultrafast proton-coupled photoisomerization upon excitation of the parent state (Pfr) of bacteriophytochrome Agp2.

Photoinduced reaction mechanisms in prototypical and bathy phytochromes.

Phytochromes, found in plants, fungi, and bacteria, exploit light as a source of information to control physiological processes photoswitching between two states of different physiological activity, a red-absorbing Pr and a far-red-absorbing Pfr state. Depending on the relative stability in the dark, bacterial phytochromes are divided into prototypical and bathy phytochromes, where the stable state is Pr and Pfr, respectively. In this work we studied representatives of these groups (prototypical Agp1 and bathy Agp2 from ) together with the bathy-like phytochrome BphP from by resonance Raman and IR difference spectroscopy.

Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome.

Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance.

Light- and temperature-dependent dynamics of chromophore and protein structural changes in bathy phytochrome Agp2.

Bacterial phytochromes are sensoric photoreceptors that transform light absorbed by the photosensor core module (PCM) to protein structural changes that eventually lead to the activation of the enzymatic output module. The underlying photoinduced reaction cascade in the PCM starts with the isomerization of the tetrapyrrole chromophore, followed by conformational relaxations, proton transfer steps, and a secondary structure transition of a peptide segment (tongue) that is essential for communicating the signal to the output module. In this work, we employed various static and time-resolved IR and resonance Raman spectroscopic techniques to study the structural and reaction dynamics of the Meta-F intermediate of both the PCM and the full-length (PCM and output module) variant of the bathy phytochrome Agp2 from Agrobacterium fabrum.

The Bacterial and Fungal Microbiota of the Mexican Rubiaceae Family Medicinal Plant Bouvardia ternifolia.

Bouvardia ternifolia is a medicinal plant considered a source of therapeutic compounds, like the antitumoral cyclohexapeptide bouvardin. It is known that large number of secondary metabolites produced by plants results from the interaction of the host and adjacent or embedded microorganisms. Using high-throughput DNA sequencing of V3-16S and V5-18S ribosomal gene libraries, we characterized the endophytic, endophytic + epiphyte bacterial, and fungal communities associated to flowers, leaves, stems, and roots, as well as the rhizosphere.

Comparison of the Forward and Reverse Photocycle Dynamics of Two Highly Similar Canonical Red/Green Cyanobacteriochromes Reveals Unexpected Differences.

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors that exhibit photochromism between two states: a thermally stable dark-adapted state and a metastable light-adapted state with bound linear tetrapyrrole (bilin) chromophores possessing 15 and 15 configurations, respectively. The photodynamics of canonical red/green CBCRs have been extensively studied; however, the time scales of their excited-state lifetimes and subsequent ground-state evolution rates widely differ and, at present, remain difficult to predict. Here, we compare the photodynamics of two closely related red/green CBCRs that have substantial sequence identity (∼68%) and similar chromophore environments: AnPixJg2 from sp.

Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome.

Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance.

The Lumi-R Intermediates of Prototypical Phytochromes.

Phytochromes are photoreceptors that upon light absorption initiate a physiological reaction cascade. The starting point is the photoisomerization of the tetrapyrrole cofactor in the parent Pr state, followed by thermal relaxation steps culminating in activation of the physiological signal. Here we have employed resonance Raman (RR) spectroscopy to study the chromophore structure in the primary photoproduct Lumi-R, trapped between 130 and 200 K.

Intramolecular Proton Transfer Controls Protein Structural Changes in Phytochrome.

Phytochromes are biological photoswitches that interconvert between two parent states (Pr and Pfr). The transformation is initiated by photoisomerization of the tetrapyrrole chromophore, followed by a sequence of chromophore and protein structural changes. In the last step, a phytochrome-specific peptide segment (tongue) undergoes a secondary structure change, which in prokaryotic phytochromes is associated with the (de)activation of the output module.

Role of the Propionic Side Chains for the Photoconversion of Bacterial Phytochromes.

Bacteriophytochromes harboring a biliverdin IXα (BV) chromophore undergo photoinduced reaction cascades to switch between physiologically inactive and active states. Employing vibrational spectroscopic and computational methods, we analyzed the role of propionic substituents of BV in the transformations between parent states Pr and Pfr in prototypical (Agp1) and bathy (Agp2) phytochromes from . Both proteins form adducts with BV monoesters (BVM), esterified at propionic side chain (Ps) or (Ps), but in each case, only one monoester adduct is reactive.

Photoreactions of the Histidine Kinase Rhodopsin Ot-HKR from the Marine Picoalga Ostreococcus tauri.

The tiny picoalga, Ostreococcus tauri, originating from the Thau Lagoon is a member of the marine phytoplankton. Because of its highly reduced genome and small cell size, while retaining the fundamental requirements of a eukaryotic photosynthetic cell, it became a popular model organism for studying photosynthesis or circadian clock-related processes. We analyzed the spectroscopic properties of the photoreceptor domain of the histidine kinase rhodopsin Ot-HKR that is suggested to be involved in the light-induced entrainment of the Ostreococcus circadian clock.

An Assessment of the Bioactivity of Coffee Silverskin Melanoidins.

Melanoidins present in coffee silverskin, the only by-product of the roasting process, are formed via the Maillard reaction. The exact structure, biological properties, and mechanism of action of coffee silverskin melanoidins, remain unknown. This research work aimed to contribute to this novel knowledge.

Chromophore binding to two cysteines increases quantum yield of near-infrared fluorescent proteins.

Phytochromes are red/far-red light sensing photoreceptors employing linear tetrapyrroles as chromophores, which are covalently bound to a cysteine (Cys) residue in the chromophore-binding domain (CBD, composed of a PAS and a GAF domain). Recently, near-infrared (NIR) fluorescent proteins (FPs) engineered from bacterial phytochromes binding biliverdin IXα (BV), such as the iRFP series, have become invaluable probes for multicolor fluorescence microscopy and in vivo imaging. However, all current NIR FPs suffer from relatively low brightness.

Structural and Vibrational Characterization of the Chromophore Binding Site of Bacterial Phytochrome Agp1.

Agp1 is a prototypical bacterial phytochrome from Agrobacterium fabrum harboring a biliverdin cofactor which reversibly photoconverts between a red-light-absorbing (Pr) and a far-red-light-absorbing (Pfr) states. The reaction mechanism involves the isomerization of the bilin-chromophore followed by large structural changes of the protein matrix that are coupled to protonation dynamics at the chromophore binding site. Histidines His250 and His280 participate in this process.

Common Structural Elements in the Chromophore Binding Pocket of the Pfr State of Bathy Phytochromes.

Phytochromes are bimodal photoreceptors which, upon light absorption by the tetrapyrrole chromophore, can be converted between a red-absorbing state (Pr) and far-red-absorbing state (Pfr). In bacterial phytochromes, either Pr or Pfr are the thermally stable states, thereby constituting the classes of prototypical and bathy phytochromes, respectively. In this work, we have employed vibrational spectroscopies to elucidate the origin of the thermal stability of the Pfr states in bathy phytochromes.

Structural communication between the chromophore-binding pocket and the N-terminal extension in plant phytochrome phyB.

The N-terminal extension (NTE) of plant phytochromes has been suggested to play a functional role in signaling photoinduced structural changes. Here, we use resonance Raman spectroscopy to study the effect of the NTE on the chromophore structure of B-type phytochromes from two evolutionarily distant plants. NTE deletion seems to have no effect on the chromophore in the inactive Pr state, but alters the torsion of the C-D ring methine bridge and the surrounding hydrogen bonding network in the physiologically active Pfr state.

Protonation-Dependent Structural Heterogeneity in the Chromophore Binding Site of Cyanobacterial Phytochrome Cph1.

Phytochromes are biological red/far-red light sensors found in many organisms. Photoisomerization of the linear methine-bridged tetrapyrrole triggers transient proton translocation events in the chromophore binding pocket (CBP) leading to major conformational changes of the protein matrix that are in turn associated with signaling. By combining pH-dependent resonance Raman and UV-visible absorption spectroscopy, we analyzed protonation-dependent equilibria in the CBP of Cph1 involving the proposed Pr-I and Pr-II substates that prevail below and above pH 7.

The role of local and remote amino acid substitutions for optimizing fluorescence in bacteriophytochromes: A case study on iRFP.

Bacteriophytochromes are promising tools for tissue microscopy and imaging due to their fluorescence in the near-infrared region. These applications require optimization of the originally low fluorescence quantum yields via genetic engineering. Factors that favour fluorescence over other non-radiative excited state decay channels are yet poorly understood.

Structure of the Full-Length Bacteriophytochrome from the Plant Pathogen Xanthomonas campestris Provides Clues to its Long-Range Signaling Mechanism.

Phytochromes constitute a major superfamily of light-sensing proteins that are reversibly photoconverted between a red-absorbing (Pr) and a far-red-absorbing (Pfr) state. Bacteriophytochromes (BphPs) are found among photosynthetic and non-photosynthetic bacteria, including pathogens. To date, several BphPs have been biophysically characterized.

A Red/Green Cyanobacteriochrome Sustains Its Color Despite a Change in the Bilin Chromophore's Protonation State.

The second GAF domain of AnPixJ, AnPixJg2, a bilin-binding protein from the cyanobacterium Anabaena PCC 7120, undergoes a photoinduced interconversion between a red-absorbing state, Pr, and a green-absorbing state, Pg. Combining ultraviolet-vis (UV-vis), infrared, resonance Raman (RR), and magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, we have studied this cyanobacteriochrome (CBCR) assembled with phycocyanobilin (PCB) either in vivo or in vitro. In both assembly routes, the spectroscopic data of the Pr state reveal nearly identical chromophore structures with a protonated (cationic) bilin.

Conformational heterogeneity of the Pfr chromophore in plant and cyanobacterial phytochromes.

Phytochromes are biological photoreceptors that can be reversibly photoconverted between a dark and photoactivated state. The underlying reaction sequences are initiated by the photoisomerization of the tetrapyrrole cofactor, which in plant and cyanobacterial phytochromes are a phytochromobilin (PΦB) and a phycocyanobilin (PCB), respectively. The transition between the two states represents an on/off-switch of the output module activating or deactivating downstream physiological processes.

A protonation-coupled feedback mechanism controls the signalling process in bathy phytochromes.

Phytochromes are bimodal photoswitches composed of a photosensor and an output module. Photoactivation of the sensor is initiated by a double bond isomerization of the tetrapyrrole chromophore and eventually leads to protein conformational changes. Recently determined structural models of phytochromes identify differences between the inactive and the signalling state but do not reveal the mechanism of photosensor activation or deactivation.