Stabilization of a flavoprotein for solid-state photo-CIDNP MAS NMR at room temperature by embedding in a glassy sugar matrix.

Hyperpolarization via the solid-state photochemically induced dynamic nuclear polarization (photo-CIDNP) effect can be detected in frozen solutions of electron transfer proteins generating a radical-pair upon illumination. The effect has been observed in various natural photosynthetic reaction centers and in light-oxygen-voltage (LOV) sensing domains incorporating a flavin mononucleotide (FMN) as chromophore. In LOV domains, where a highly conserved cysteine is mutated to a flavin to interrupt its natural photochemistry, a radical-pair is generated by electron transfer from a nearby tryptophan to the photoexcited triplet state of FMN.

Plastid Gene Transcription: An Update on Promoters and RNA Polymerases.

Chloroplasts, the sites of photosynthesis and sources of reducing power, are at the core of the success story that sets apart autotrophic plants from most other living organisms. Along with their fellow organelles (e.g.

Initializing 2 Pure 14-Qubit Entangled Nuclear Spin States in a Hyperpolarized Molecular Solid.

Quantum entanglement has been realized on a variety of physical platforms such as quantum dots, trapped atomic ions, and superconductors. Here we introduce specific molecular solids as promising alternative platforms. Our model system is triplet pentacene in a host single crystal at level anticrossing (LAC) conditions.

AtSIG6 and other members of the sigma gene family jointly but differentially determine plastid target gene expression in Arabidopsis thaliana.

Plants contain a nuclear gene family for plastid sigma factors, i.e., proteins that associate with the "bacterial-type" organellar RNA polymerase and confer the ability for correct promoter binding and transcription initiation.

Detecting a new source for photochemically induced dynamic nuclear polarization in the LOV2 domain of phototropin by magnetic-field dependent (13)C NMR spectroscopy.

Phototropin is a flavin mononucleotide (FMN) containing blue-light receptor, which regulates, governed by its two LOV domains, the phototropic response of higher plants. Upon photoexcitation, the FMN cofactor triplet state, (3)F, reacts with a nearby cysteine to form a covalent adduct. Cysteine-to-alanine mutants of LOV domains instead generate a flavin radical upon illumination.

Plastid gene transcription: promoters and RNA polymerases.

Chloroplasts, the sites of photosynthesis and sources of reducing power, are at the core of the success story that sets apart autotrophic plants from most other living organisms. Along with their fellow organelles (e.g.

Exploring the electron transfer pathways in photosystem I by high-time-resolution electron paramagnetic resonance: observation of the B-side radical pair P700(+)A1B(-) in whole cells of the deuterated green alga Chlamydomonas reinhardtii at cryogenic temperatures.

Crystallographic models of photosystem I (PS I) highlight a symmetrical arrangement of the electron transfer cofactors which are organized in two parallel branches (A, B) relative to a pseudo-C2 symmetry axis that is perpendicular to the membrane plane. Here, we explore the electron transfer pathways of PS I in whole cells of the deuterated green alga Chlamydomonas reinhardtii using high-time-resolution electron paramagnetic resonance (EPR) at cryogenic temperatures. Particular emphasis is given to quantum oscillations detectable in the tertiary radical pairs P700(+)A1A(-) and P700(+)A1B(-) of the electron transfer chain.

Phylogenetic and functional features of the plastid transcription kinase cpCK2 from Arabidopsis signify a role of cysteinyl SH-groups in regulatory phosphorylation of plastid sigma factors.

A plastidic serine/threonine protein kinase, initially named plastid transcription kinase (PTK) has been implicated in phosphorylation and redox control of chloroplast transcription. This kinase was later renamed as chloroplast casein kinase 2 (cpCK2) because of its physical and functional similarity to nucleocytosolic casein kinase 2 (ncCK2). It shares all four of its cysteine residues with ncCK2 from land plants, while only three of these residues are conserved in algal CK2-type sequences, and just two in animals.

Role and regulation of plastid sigma factors and their functional interactors during chloroplast transcription - recent lessons from Arabidopsis thaliana.

Chloroplasts and other plastids within plant cells together are responsible for autotrophic growth and biosynthesis of metabolic key components. Genetically, the plastids are hybrid organelles composed of proteins that are either products of their own organellar genes or are nucleus-encoded and imported from the cytosol. This dual genetic principle is evident in the case of the multi-subunit RNA polymerase, i.

Origin of light-induced spin-correlated radical pairs in cryptochrome.

Blue-light excitation of cryptochromes and homologues uniformly triggers electron transfer (ET) from the protein surface to the flavin adenine dinucleotide (FAD) cofactor. A cascade of three conserved tryptophan residues has been considered to be critically involved in this photoreaction. If the FAD is initially in its fully oxidized (diamagnetic) redox state, light-induced ET via the tryptophan triad generates a series of short-lived spin-correlated radical pairs comprising an FAD radical and a tryptophan radical.

Quantum oscillations and polarization of nuclear spins in photoexcited triplet states.

The unique physical properties of photoexcited triplet states have been explored in numerous spectroscopic studies employing electron paramagnetic resonance (EPR). So far, however, no quantum interference effects were found in these systems in the presence of a magnetic field. In this study, we report the successful EPR detection of nuclear quantum oscillations in an organic triplet state subject to an external magnetic field.

AtSIG6, a plastid sigma factor from Arabidopsis, reveals functional impact of cpCK2 phosphorylation.

Plastids contain sigma factors, i.e. gene-regulatory proteins for promoter binding and transcription initiation.

Arabidopsis mutants carrying chimeric sigma factor genes reveal regulatory determinants for plastid gene expression.

Like bacteria, plastids contain sigma factors for promoter binding and transcription initiation. Accumulating evidence suggests that members of the plant sigma factor family can have specialized non-redundant roles in terms of promoter preference in various developmental and environmental situations. To specify regulatory determinants, we have chosen pairwise exchange of portions of Arabidopsis sigma coding regions, followed by transformation of the chimeric constructs into a sigma 6 knockout line.

Flow-structure-acoustic interaction in a human voice model.

For the investigation of the physical processes of human phonation, inhomogeneous synthetic vocal folds were developed to represent the full fluid-structure-acoustic coupling. They consisted of polyurethane rubber with a stiffness in the range of human vocal folds and were mounted in a channel, shaped like the vocal tract in the supraglottal region. This test facility permitted extensive observations of flow-induced vocal fold vibrations, the periodic flow field, and the acoustic signals in the far field of the channel.

Structure of the charge separated state P865(+)Q(A)- in the photosynthetic reaction centers of Rhodobacter sphaeroides by quantum beat oscillations and high-field electron paramagnetic resonance: evidence for light-induced Q(A)- reorientation.

The structure of the secondary radical pair, P865(+)Q(A)-, in fully deuterated and Zn-substituted reaction centers (RCs) of the purple bacterium Rhodobacter sphaeroides R-26 has been determined by high-time resolution and high-field electron paramagnetic resonance (EPR). A computer analysis of quantum beat oscillations, observed in a two-dimensional Q-band (34 GHz) EPR experiment, provides the orientation of the various magnetic tensors of P865(+)Q(A)- with respect to a magnetic reference frame. The orientation of the g-tensor of P865(+) in an external reference system is adapted from a single-crystal W-band (95 GHz) EPR study [Klette, R.

Pulsed electron nuclear double resonance studies of the photoexcited triplet state of pentacene in p-terphenyl crystals at room temperature.

Pulsed electron nuclear double resonance (ENDOR) using a modified Davies-type [Phys. Lett. 47A, 1 (1974)] sequence is employed to study the hyperfine (HF) structure of the photoexcited triplet state of pentacene dispersed in protonated and deuterated p-terphenyl single crystals.

A promoter switch that can rescue a plant sigma factor mutant.

Chloroplasts sigma factors act in concert with PEP, the bacterial-type plastid RNA polymerase. Using a sigma knockout line from Arabidopsis thaliana, we investigated mutant-specific changes in plastid gene expression at RNA level. One characteristic feature was the appearance of a long transcript that spans the atpB-E operon and extends considerably into the far-upstream region of atpB.

Dual temporal role of plastid sigma factor 6 in Arabidopsis development.

Plants contain nuclear-coded sigma factors for initiation of chloroplast transcription. The in vivo function of individual members of the sigma gene family has become increasingly accessible by knockout and complementation strategies. Here we have investigated plastid gene expression in an Arabidopsis (Arabidopsis thaliana) mutant with a defective gene for sigma factor 6.

Structural organization in photosynthetic proteins as studied by high-field EPR of spin-correlated radical pair states.

We demonstrate the potential of high-field (HF) time-resolved electron paramagnetic resonance (EPR) spectroscopy to reveal unique information about electron transfer processes and the structure of photosynthetic systems. The lineshapes and electron spin polarization (ESP) of spin-correlated radical pair (SCRP) spectra recorded with HF-EPR are very sensitive to the magnetic parameters, interactions, and geometry of the radicals in the pair. This sensitivity facilitates an analysis of more sophisticated models and methods to reveal the important relationship between structural organization and light-induced electron transfer of the photosynthetic proteins.

Proteomics-based sequence analysis of plant gene expression--the chloroplast transcription apparatus.

The chloroplast transcription apparatus has turned out to be more complex than anticipated, with core polypeptides surrounded by multiple accessory proteins of diverse, and in part unexpected, functions. At least two different RNA-binding proteins and several redox-responsive proteins are components of the major chloroplast RNA polymerase termed PEP-A. One of the key-regulatory factors has been identified as a Ser/Thr-specific protein kinase that is sensitive to SH group modification by glutathione and by this means is able to modulate transcription.

DNA-binding and transcription characteristics of three cloned sigma factors from mustard (Sinapis alba L.) suggest overlapping and distinct roles in plastid gene expression.

We have isolated and studied the cloned sigma factors SASIG1-3 from mustard (Sinapis alba). In functional analyses using both promoter and factor mutants, the three recombinant proteins all had similar basic properties but also revealed differences in promoter preference and requirements for single nucleotide positions. Directed muta- genesis of SASIG1 identified critical residues within the conserved regions 2.

Redox regulation of chloroplast transcription.

Chloroplasts are the important plant cell organelles where photosynthesis takes place. Throughout this process, reaction center proteins are degraded and subsequently replenished by redox-responsive gene expression. In addition to well defined posttranscriptional mechanisms at the RNA and protein level, the transcription of chloroplast DNA into RNA precursors has been a focal point of studies in this area.

The plastid transcription kinase from mustard (Sinapis alba L.). A nuclear-encoded CK2-type chloroplast enzyme with redox-sensitive function.

The plastid transcription kinase (PTK), a component of the major RNA polymerase complex from mustard chloroplasts, has been implicated in redox-mediated regulation of plastid gene expression. A cloning strategy to define the PTK gene(s) resulted in the isolation of a full-length cDNA for a protein with overall high homology with the alpha subunit of cytosolic casein kinase (CK2) that contained an N-terminal extension for a putative plastid transit peptide. Using in organello chloroplast import studies, immunodetection and MS, we found that the corresponding protein, termed cpCK2alpha, is targeted to the chloroplast and is associated with the plastid RNA polymerase PEP-A.