Balance between photoreduction efficiency, cofactor affinity, and allosteric coupling of halogenase flavoenzymes.

Flavin-dependent halogenases (FDHs) are promising candidates for the sustainable production of halogenated organic molecules by biocatalysis. FDHs require only oxygen, halide and a fully reduced flavin adenine dinucleotide (FADH) cofactor to generate the reactive HOX that diffuses 10 Å to the substrate binding pocket and enables regioselective oxidative halogenation. A key challenge for the application of FDHs is the regeneration of the FADH.

The NADH-dependent flavin reductase ThdF follows an ordered sequential mechanism though crystal structures reveal two FAD molecules in the active site.

Two-component flavin-dependent monooxygenases are of great interest as biocatalysts for the production of pharmaceuticals and other relevant molecules, as they catalyze chemically important reactions such as hydroxylation, epoxidation and halogenation. The monooxygenase components require a separate flavin reductase, which provides the necessary reduced flavin cofactor. The tryptophan halogenase Thal from Streptomyces albogriseolus is a well-characterized two-component flavin-dependent halogenase.

Coupling and regulation mechanisms of the flavin-dependent halogenase PyrH observed by infrared difference spectroscopy.

Flavin-dependent halogenases are central enzymes in the production of halogenated secondary metabolites in various organisms and they constitute highly promising biocatalysts for regioselective halogenation. The mechanism of these monooxygenases includes formation of hypohalous acid from a reaction of fully reduced flavin with oxygen and halide. The hypohalous acid then diffuses via a tunnel to the substrate-binding site for halogenation of tryptophan and other substrates.

Core-shell microgels synthesized in continuous flow: deep insight into shell growth using temperature-dependent FTIR.

While core-shell microgels have been intensively studied in their fully synthesized state, the formation mechanism of the shell has not been completely understood. Such insight is decisive for a customization of microgel properties for applications. In this work, microgels based on a -isopropylmethacrylamide (NiPMAM) core and a -propylacrylamide (NnPAM) shell are synthesized in a continuous flow reactor.

Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization Rates.

pH-sensitive fluorescent proteins as genetically encoded pH sensors are promising tools for monitoring intra- and extracellular pH. However, there is a lack of ratiometric pH sensors, which offer a good dynamic range and can be purified and applied extracellularly to investigate uptake. In our study, the bright fluorescent protein CoGFP_V0 was C-terminally fused to the ligand epidermal growth factor (EGF) and retained its dual-excitation and dual-emission properties as a purified protein.

Plant Cryptochromes Illuminated: A Spectroscopic Perspective on the Mechanism.

Plant cryptochromes are central blue light receptors for the control of land plant and algal development including the circadian clock and the cell cycle. Cryptochromes share a photolyase homology region with about 500 amino acids and bind the chromophore flavin adenine dinucleotide. Characteristic for plant cryptochromes is a conserved aspartic acid close to flavin and an exceptionally long C-terminal extension.

The World of Algae Reveals a Broad Variety of Cryptochrome Properties and Functions.

Algae are photosynthetic eukaryotic (micro-)organisms, lacking roots, leaves, and other organs that are typical for land plants. They live in freshwater, marine, or terrestrial habitats. Together with the cyanobacteria they contribute to about half of global carbon fixation.

DASH cryptochrome 1, a UV-A receptor, balances the photosynthetic machinery of Chlamydomonas reinhardtii.

Drosophila, Arabidopsis, Synechocystis, Homo (DASH) cryptochromes belong to the cryptochrome/photolyase family and can act as DNA repair enzymes. In bacteria and fungi, they also can play regulatory roles, but in plants their biological functions remain elusive. Here, we characterize CRY-DASH1 from the green alga Chlamydomonas reinhardtii.

C-Terminal Extension of a Plant Cryptochrome Dissociates from the β-Sheet of the Flavin-Binding Domain.

Plant cryptochromes are central blue light receptors in land plants and algae. Photoreduction of the flavin bound to the photolyase homology region (PHR) causes a dissociation of the C-terminal extension (CCT) as effector via an unclear pathway. We applied the recently developed in-cell infrared difference (ICIRD) spectroscopy to study the response of the full-length pCRY from in living bacterial cells, because the receptor degraded upon isolation.

Peripheral Methionine Residues Impact Flavin Photoreduction and Protonation in an Engineered LOV Domain Light Sensor.

Proton-coupled electron transfer reactions play critical roles in many aspects of sensory phototransduction. In the case of flavoprotein light sensors, reductive quenching of flavin excited states initiates chemical and conformational changes that ultimately transmit light signals to downstream targets. These reactions generally require neighboring aromatic residues and proton-donating side chains for rapid and coordinated electron and proton transfer to flavin.

Resolving Structural Changes of Photoreceptors in Living via In-cell Infrared Difference Spectroscopy.

Several in-cell spectroscopic techniques have been developed recently to investigate the structure and mechanism of proteins in their native environment. Conditions differ dramatically from those selected for experiments. Accordingly, the cellular environment can affect the protein mechanism for example by molecular crowding or binding of small molecules.

Site-by-site tracking of signal transduction in an azidophenylalanine-labeled bacteriophytochrome with step-scan FTIR spectroscopy.

Signal propagation in photosensory proteins is a complex and multidimensional event. Unraveling such mechanisms site-specifically in real time is an eligible but a challenging goal. Here, we elucidate the site-specific events in a red-light sensing phytochrome using the unnatural amino acid azidophenylalanine, vibrationally distinguishable from all other protein signals.

Smart membranes by electron beam cross-linking of copolymer microgels.

Poly(N-isopropylacrylamide) (pNIPAM) based copolymer microgels were used to create free-standing, transferable, thermoresponsive membranes. The microgels were synthesized by copolymerization of NIPAM with N-benzylhydrylacrylamide (NBHAM). Monolayers of these colloidal gels were subsequently cross-linked using an electron gun leading to the formation of a connected monolayer.

A quantum cascade laser setup for studying irreversible photoreactions in HO with nanosecond resolution and microlitre consumption.

Time-resolved infrared spectroscopy on irreversible reactions requires in general an exchange of sample for thousands of acquisitions leading to high sample consumption. Here, we present a setup employing a modern quantum cascade laser (QCL) as a probe light source to record time-resolved difference spectra of irreversible photoreactions in H2O. The combination of the focused QCL with a pressure-tolerant flow cell and a micrometre stage orthogonal to the flow allowed us to drastically reduce the sample consumption.

Tailored flavoproteins acting as light-driven spin machines pump nuclear hyperpolarization.

The solid-state photo-chemically induced dynamic nuclear polarization (photo-CIDNP) effect generates non-Boltzmann nuclear spin magnetization, referred to as hyperpolarization, allowing for high gain of sensitivity in nuclear magnetic resonance (NMR). Well known to occur in photosynthetic reaction centers, the effect was also observed in a light-oxygen-voltage (LOV) domain of the blue-light receptor phototropin, in which the functional cysteine was removed to prevent photo-chemical reactions with the cofactor, a flavin mononucleotide (FMN). Upon illumination, the FMN abstracts an electron from a tryptophan to form a transient spin-correlated radical pair (SCRP) generating the photo-CIDNP effect.

In-cell infrared difference spectroscopy of LOV photoreceptors reveals structural responses to light altered in living cells.

Proteins are usually studied in well-defined buffer conditions, which differ substantially from those within a host cell. In some cases, the intracellular environment has an impact on the mechanism, which might be missed by experiments. IR difference spectroscopy previously has been applied to study the light-induced response of photoreceptors and photoenzymes Here, we established the in-cell IR difference (ICIRD) spectroscopy in the transmission and attenuated total reflection configuration to investigate the light-induced response of soluble proteins in living bacterial cells.

Core-shell microgels as thermoresponsive carriers for catalytic palladium nanoparticles.

Responsive core-shell microgels are promising systems for a stabilization of Pd nanoparticles and control of their catalytic activity. Here, poly-N-n-propylacrylamide (PNNPAM) was copolymerized with methacrylic acid to yield microgel core particles, which were subsequently coated with an additional, acid-free poly-N-isopropylmethacrylamide (PNIPMAM) shell. Both core and core-shell systems were used as pH- and temperature-responsive carrier systems for the incorporation of palladium nanoparticles.

Tongue Refolding in the Knotless Cyanobacterial Phytochrome All2699.

Phytochromes regulate central responses of plants and microorganisms such as shade avoidance and photosystem synthesis. Canonical phytochromes comprise a photosensory module of three domains. The C-terminal phytochrome-specific (PHY) domain interacts via a tongue element with the bilin chromophore in the central GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA) domain.

Calculation of the Geometries and Infrared Spectra of the Stacked Cofactor Flavin Adenine Dinucleotide (FAD) as the Prerequisite for Studies of Light-Triggered Proton and Electron Transfer.

Flavin cofactors, like flavin adenine dinucleotide (FAD), are important electron shuttles in living systems. They catalyze a wide range of one- or two-electron redox reactions. Experimental investigations include UV-vis as well as infrared spectroscopy.

Nuclear spin-hyperpolarization generated in a flavoprotein under illumination: experimental field-dependence and theoretical level crossing analysis.

The solid-state photo-chemically induced dynamic nuclear polarization (photo-CIDNP) effect generates non-equilibrium nuclear spin polarization in frozen electron-transfer proteins upon illumination and radical-pair formation. The effect can be observed in various natural photosynthetic reaction center proteins using magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, and in a flavin-binding light-oxygen-voltage (LOV) domain of the blue-light receptor phototropin. In the latter system, a functionally instrumental cysteine has been mutated to interrupt the natural cysteine-involving photochemistry allowing for an electron transfer from a more distant tryptophan to the excited flavin mononucleotide chromophore.

Time-Resolved Infrared and Visible Spectroscopy on Cryptochrome aCRY: Basis for Red Light Reception.

Cryptochromes function as flavin-binding photoreceptors in bacteria, fungi, algae, land plants, and insects. The discovery of an animal-like cryptochrome in the green alga Chlamydomonas reinhardtii has expanded the spectral range of sensitivity of these receptors from ultraviolet A/blue light to almost the complete visible spectrum. The broadened light response has been explained by the presence of the flavin neutral radical as a chromophore in the dark.

Arguments for an additional long-lived intermediate in the photocycle of the full-length aureochrome 1c receptor: A time-resolved small-angle X-ray scattering study.

Aureochromes (AUREO) act as blue-light photoreceptors in algae. They consist of a light-, oxygen-, voltage-sensitive (LOV) domain and a DNA-binding basic region/leucine zipper. Illumination of the flavin cofactor in LOV leads to the formation of an adduct, followed by global structural changes.

Synthesis of smart dual-responsive microgels: correlation between applied surfactants and obtained particle morphology.

Thermo- and pH-responsive copolymer microgels were obtained by surfactant-assisted precipitation polymerization of N-isopropylacrylamide (NIPAM) and acrylic acid (AAc). The surfactants used were sodium dodecylsulfate (SDS), dodecyltrimethylammonium bromide (DTAB) and the nonionic n-octyl-β-d-glucopyranoside (C8G1). We investigate the influence of the surfactants on the acrylic acid incorporation rate, the particle size, particle morphology, and the swelling behaviour at pH 4 and pH 7, at which AAc is neutral or charged, respectively.

Biphasic Formation of 2D Nanomembranes by Photopolymerization of Diacetylene Lipids as Revealed by Infrared Difference Spectroscopy.

Two-dimensional nanomembranes are promising materials for filtration or separation by providing the basis for controlled and rapid transport between two compartments. The polymerization by UV light of diacetylene-containing lipids at an interface produces free-standing 2D nanomembranes. Here, we analyzed the nanomembrane formation of 1,2-bis(10,12-tricosadiynoyl)--glycero-3-phosphocholine (DiynePC) and 1-palmitoyl-2-(10,12-tricosadiynoyl)--glycero-3-phosphoethanolamine (PTPE) on germanium using light-induced infrared difference spectroscopy with attenuated total reflection to obtain insights into the kinetics and mechanism of the polymerization process.