Protein dynamics in the reductive activation of a B12-containing enzyme

B12-dependent proteins are involved in methyl transfer reactions ranging from the biosynthesis of methionine in humans to the formation of acetyl-CoA in anaerobic bacteria. During their catalytic cycle, they undergo large conformational changes to interact with various proteins. Recently, the crystal structure of the B12-containing corrinoid iron–sulfur protein (CoFeSP) in complex with its reductive activator (RACo) was determined, providing a first glimpse of how energy is transduced in the ATP-dependent reductive activation of corrinoid-containing methyltransferases. The thermodynamically uphill electron transfer from RACo to CoFeSP is accompanied by large movements of the cofactor-binding domains of CoFeSP. To refine the structure-based mechanism, we analyzed the conformational change of the B12-binding domain of CoFeSP by pulsed electron–electron double resonance and Förster resonance energy transfer spectroscopy. We show that the site-specific labels on the flexible B12-binding domain and the small subunit of CoFeSP move within 11 Å in the RACo:CoFeSP complex, consistent with the recent crystal structures. By analyzing the transient kinetics of formation and dissociation of the RACo:CoFeSP complex, we determined values of 0.75 μM–1 s–1 and 0.33 s–1 for rate constants kon and koff, respectively. Our results indicate that the large movement observed in crystals also occurs in solution and that neither the formation of the protein encounter complex nor the large movement of the B12-binding domain is rate-limiting for the ATP-dependent reductive activation of CoFeSP by RACo

Transient electrically detected magnetic resonance spectroscopy applied to organic solar cells

The influence of light-induced paramagnetic states on the photocurrent generated by polymer:fullerene solar cells is studied using spin-sensitive techniques in combination with laser-flash excitation. For this purpose, we developed a setup that allows for simultaneous detection of transient electron paramagnetic resonance as well as transient electrically detected magnetic resonance (trEDMR) signals from fully processed and encapsulated solar cells. Combining both techniques provides a direct link between photoinduced triplet excitons, charge transfer states, and free charge carriers as well as their influence on the photocurrent generated by organic photovoltaic devices. Our results obtained from solar cells based on poly(3-hexylthiophene) as electron donor and a fullerene-based electron acceptor show that the resonant signals observed in low-temperature (T = 80 K) trEDMR spectra can be attributed to positive polarons in the polymer as well as negative polarons in the fullerene phase, indicating that both centers are involved in spin-dependent processes that directly influence the photocurrent

A blue light receptor that mediates RNA binding and translational regulation

Sensory photoreceptor proteins underpin light-dependent adaptations in nature and enable the optogenetic control of organismal behavior and physiology. We identified the bacterial light-oxygen-voltage (LOV) photoreceptor PAL that sequence-specifically binds short RNA stem loops with around 20 nM affinity in blue light and weaker than 1 µM in darkness. A crystal structure rationalizes the unusual receptor architecture of PAL with C-terminal LOV photosensor and N-terminal effector units. The light-activated PAL–RNA interaction can be harnessed to regulate gene expression at the RNA level as a function of light in both bacteria and mammalian cells. The present results elucidate a new signal-transduction paradigm in LOV receptors and conjoin RNA biology with optogenetic regulation, thereby paving the way toward hitherto inaccessible optoribogenetic modalities

Site-selective measurement of coupled spin pairs in an organic semiconductor

From organic electronics to biological systems, understanding the role of intermolecular interactions between spin pairs is a key challenge. Here we show how such pairs can be selectively addressed with combined spin and optical sensitivity. We demonstrate this for bound pairs of spin-triplet excitations formed by singlet fission, with direct applicability across a wide range of synthetic and biological systems. We show that the site sensitivity of exchange coupling allows distinct triplet pairs to be resonantly addressed at different magnetic fields, tuning them between optically bright singlet (S=0) and dark triplet quintet (S=1,2) configurations: This induces narrow holes in a broad optical emission spectrum, uncovering exchange-specific luminescence. Using fields up to 60 T, we identify three distinct triplet-pair sites, with exchange couplings varying over an order of magnitude (0.3–5 meV), each with its own luminescence spectrum, coexisting in a single material. Our results reveal how site selectivity can be achieved for organic spin pairs in a broad range of systems

Blue-light reception through quaternary transitions

Sensory photoreceptors absorb light via their photosensor modules and trigger downstream physiological adaptations via their effector modules. Light reception accordingly depends on precisely orchestrated interactions between these modules, the molecular details of which often remain elusive. Using electron-electron double resonance (ELDOR) spectroscopy and site-directed spin labelling, we chart the structural transitions facilitating blue-light reception in the engineered light-oxygen-voltage (LOV) histidine kinase YF1 which represents a paradigm for numerous natural signal receptors. Structural modelling based on pair-wise distance constraints derived from ELDOR pinpoint light-induced rotation and splaying apart of the two LOV photosensors in the dimeric photoreceptor. Resultant molecular strain likely relaxes as left- handed supercoiling of the coiled-coil linker connecting sensor and effector units. ELDOR data on a photoreceptor variant with an inverted signal response indicate a drastically altered dimer interface but light-induced structural transitions in the linker that are similar to those in YF1. Taken together, we provide mechanistic insight into the signal trajectories of LOV photoreceptors and histidine kinases that inform molecular simulations and the engineering of novel receptors

Localization of dexamethasone within dendritic core-multishell (CMS) nanoparticles and skin penetration properties studied by multi-frequency electron paramagnetic resonance (EPR) spectroscopy

The skin and especially the stratum corneum (SC) act as a barrier and protect epidermal cells and thus the whole body against xenobiotica of the external environment. Topical skin treatment requires an efficient drug delivery system (DDS). Polymer-based nanocarriers represent novel transport vehicles for dermal application of drugs. In this study dendritic core-multishell (CMS) nanoparticles were investigated as promising candidates. CMS nanoparticles were loaded with a drug (analogue) and were applied to penetration studies of skin. We determined by dual-frequency electron paramagnetic resonance (EPR) how dexamethasone (Dx) labelled with 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PCA) is associated with the CMS. The micro-environment of the drug loaded to CMS nanoparticles was investigated by pulsed high-field EPR at cryogenic temperature, making use of the fact that magnetic parameters (g-, A-matrices, and spin-lattice relaxation time) represent specific probes for the micro-environment. Additionally, the rotational correlation time of spin-labelled Dx was probed by continuous wave EPR at ambient temperature, which provides independent information on the drug environment. Furthermore, the penetration depth of Dx into the stratum corneum of porcine skin after different topical applications was investigated. The location of Dx in the CMS nanoparticles is revealed and the function of CMS as penetration enhancers for topical application is shown

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. We show that our data are in agreement with the crystal structure of an engineered LOV-domain protein, YF1, that shows the N-terminus of the protein to be helical and to fold back in between the β-sheets of the two LOV domains, and argue for an identical arrangement in YtvA. While we could not detect any structural changes upon blue-light activation of the protein, this structural model now forms an ideal basis for identifying residues as targets for further spin labelling studies to detect potential conformational changes upon irradiation of the protein

a FD-FT THz-EPR study

A combined X-band and frequency-domain Fourier-transform THz electron paramagnetic resonance (FD-FT THz-EPR) approach has been employed to determine heme Fe(III) S = 5/2 zero-field splitting (ZFS) parameters of frozen metHb and metMb solutions, both with fluoro and aquo ligands. Frequency-domain EPR measurements have been carried out by an improved synchrotron-based FD-FT THz- EPR spectrometer. ZFS has been determined by field dependence of spin transitions within the mS = ±1/2 manifold, for all four protein systems, and by zero-field spin transitions between mS = ±1/2 and mS = ±3/2 levels, for metHb and metMb flouro-states. FD-FT THz-EPR data were simulated with a novel numerical routine based on Easyspin, which allows now for direct comparison of EPR spectra in field and frequency domain. We found purely axial ZFSs of D = 5.0(1) cm−1 (flouro-metMb), D = 9.2(4) cm−1 (aquo-metMb), D = 5.1(1) cm−1 (flouro-metHB) and D = 10.4(2) cm−1 (aquo-metHb)

Combined multifrequency EPR and DFT study of dangling bonds in a -Si:H

Multifrequency pulsed electron paramagnetic resonance (EPR) spectroscopy using S-, X-, Q-, and W-band frequencies (3.6, 9.7, 34, and 94 GHz, respectively) was employed to study paramagnetic coordination defects in undoped hydrogenated amorphous silicon (a-Si:H). The improved spectral resolution at high magnetic field reveals a rhombic splitting of the g tensor with the following principal values: g x=2.0079, g y=2.0061, and g z=2.0034, and shows pronounced g strain, i.e., the principal values are widely distributed. The multifrequency approach furthermore yields precise 29Si hyperfine data. Density functional theory (DFT) calculations on 26 computer-generated a-Si:H dangling-bond models yielded g values close to the experimental data but deviating hyperfine interaction values. We show that paramagnetic coordination defects in a-Si:H are more delocalized than computer-generated dangling-bond defects and discuss models to explain this discrepancy