Metabolic Profiling of Intact Arabidopsis thaliana Leaves during Circadian Cycle Using 1H High Resolution Magic Angle Spinning NMR.

Arabidopsis thaliana is the most widely used model organism for research in plant biology. While significant advances in understanding plant growth and development have been made by focusing on the molecular genetics of Arabidopsis, extracting and understanding the functional framework of metabolism is challenging, both from a technical perspective due to losses and modification during extraction of metabolites from the leaves, and from the biological perspective, due to random variation obscuring how well the function is performed. The purpose of this work is to establish the in vivo metabolic profile directly from the Arabidopsis thaliana leaves without metabolite extraction, to reduce the complexity of the results by multivariate analysis, and to unravel the mitigation of cellular complexity by predominant functional periodicity. To achieve this, we use the circadian cycle that strongly influences metabolic and physiological processes and exerts control over the photosynthetic machinery. High resolution-magic angle spinning nuclear magnetic resonance (HR-MAS NMR) was applied to obtain the metabolic profile directly from intact Arabidopsis leaves. Combining one- and two-dimensional 1H HR-MAS NMR allowed the identification of several metabolites including sugars and amino acids in intact leaves. Multivariate analysis on HR-MAS NMR spectra of leaves throughout the circadian cycle revealed modules of primary metabolites with significant and consistent variations of their molecular components at different time points of the circadian cycle. Since robust photosynthetic performance in plants relies on the functional periodicity of the circadian rhythm, our results show that HR-MAS NMR promises to be an important non-invasive method that can be used for metabolomics of the Arabidopsis thaliana mutants with altered physiology and photosynthetic efficiency.

In Vivo Longitudinal Monitoring of Changes in the Corpus Callosum Integrity During Disease Progression in a Mouse Model of Alzheimer's Disease.

The corpus callosum is the largest commissural fiber connecting left and right hemisphere of the brain. Emerging evidence suggests that a variety of abnormalities detected in the microstructure of this white matter fiber can be an early event in Alzheimer's disease (AD) pathology. However, little is known about tissue characteristics of these abnormalities and how these abnormalities evolve during AD progression. In this study, we measured in vivo magnetic resonance transverse relaxation times (T2) to longitudinally monitor changes in tissue integrity and abnormalities related to myelination and demyelination processes in corpus callosum of AD mouse models. The most striking finding of our study was a significant elongation of T2 values in the corpus callosum at 10, 14, 16 and 18 months of age compared to age-matched wild-type mice. In contrast, the gray matter regions surrounding the corpus callosum, such as the cortex and hippocampus, showed a significant T2 decrease compared to wild-type mice. Histological analyses clearly revealed demyelination, gliosis and amyloid-plaque deposition in the corpus callosum. Our results suggest that demyelinating and inflammatory pathology may result in prolonged relaxation time during AD progression. To our knowledge, this is the first in vivo T2 study assessing the microstructural changes with age in the corpus callosum of the Tg2576 mouse model and it demonstrates the application of T2 measurement to noninvasively detect tissue integrity of the corpus callosum, which can be an early event in disease progression.

Analysis of electron donors in photosystems in oxygenic photosynthesis by photo-CIDNP MAS NMR.

Both photosystem I and photosystem II are considerably similar in molecular architecture but they operate at very different electrochemical potentials. The origin of the different redox properties of these RCs is not yet clear. In recent years, insight was gained into the electronic structure of photosynthetic cofactors through the application of photochemically induced dynamic nuclear polarization (photo-CIDNP) with magic-angle spinning NMR (MAS NMR). Non-Boltzmann populated nuclear spin states of the radical pair lead to strongly enhanced signal intensities that allow one to observe the solid-state photo-CIDNP effect from both photosystem I and II from isolated reaction center of spinach (Spinacia oleracea) and duckweed (Spirodela oligorrhiza) and from the intact cells of the cyanobacterium Synechocystis by (13)C and (15)N MAS NMR. This review provides an overview on the photo-CIDNP MAS NMR studies performed on PSI and PSII that provide important ingredients toward reconstruction of the electronic structures of the donors in PSI and PSII.

Biosolar cells: global artificial photosynthesis needs responsive matrices with quantum coherent kinetic control for high yield.

This contribution discusses why we should consider developing artificial photosynthesis with the tandem approach followed by the Dutch BioSolar Cells consortium, a current operational paradigm for a global artificial photosynthesis project. We weigh the advantages and disadvantages of a tandem converter against other approaches, including biomass. Owing to the low density of solar energy per unit area, artificial photosynthetic systems must operate at high efficiency to minimize the land (or sea) area required. In particular, tandem converters are a much better option than biomass for densely populated countries and use two photons per electron extracted from water as the raw material into chemical conversion to hydrogen, or carbon-based fuel when CO2 is also used. For the average total light sum of 40 mol m(-2) d(-1) for The Netherlands, the upper limits are many tons of hydrogen or carbon-based fuel per hectare per year. A principal challenge is to forge materials for quantitative conversion of photons to chemical products within the physical limitation of an internal potential of ca 2.9 V. When going from electric charge in the tandem to hydrogen and back to electricity, only the energy equivalent to 1.23 V can be stored in the fuel and regained. A critical step is then to learn from nature how to use the remaining difference of ca 1.7 V effectively by triple use of one overpotential for preventing recombination, kinetic stabilization of catalytic intermediates and finally generating targeted heat for the release of oxygen. Probably the only way to achieve this is by using bioinspired responsive matrices that have quantum-classical pathways for a coherent conversion of photons to fuels, similar to what has been achieved by natural selection in evolution. In appendix A for the expert, we derive a propagator that describes how catalytic reactions can proceed coherently by a convergence of time scales of quantum electron dynamics and classical nuclear dynamics. We propose that synergy gains by such processes form a basis for further progress towards high efficiency and yield for a global project on artificial photosynthesis. Finally, we look at artificial photosynthesis research in The Netherlands and use this as an example of how an interdisciplinary approach is beneficial to artificial photosynthesis research. We conclude with some of the potential societal consequences of a large-scale roll out of artificial photosynthesis.

Structural rearrangements and reaction intermediates in a di-Mn water oxidation catalyst.

By using first-principles molecular dynamics simulations combined with metadynamics to simulate rare events we analyse competing reaction coordinates for a di-Mn water oxidation catalyst ([(bis(imino)pyridine)(H(2)O)Mn(IV)(µ-O)(2)Mn(V)(O)(bis(imino)pyridine)](3+)). The catalytic water oxidation cycle of the complex is examined by addressing the thermodynamic accessibility of the hydroperoxo species that is considered a critical and rate-limiting intermediate. To achieve this, hybrid quantum-mechanics/molecular-mechanics (QM/MM) and full QM simulations have been performed for an explicit treatment of the water environment that plays an active role in the reaction processes. Starting from a likely active species for the O-O bond formation, we observe that during the water approach to the oxo ligand a facile structural rearrangement of the complex takes place, leading to the opening of one µ-O bridge and the release of a water ligand, and resulting in two pentacoordinated Mn centers. This complex appears weakly active in the water oxidation process, since a concerted reaction is required to establish a Mn-OOH hydroperoxo intermediate. The slow kinetics of a concerted reaction can allow other processes, including linear degradation of the catalyst, to take precedence over catalytic water oxidation.

Monitoring blood flow alterations in the Tg2576 mouse model of Alzheimer's disease by in vivo magnetic resonance angiography at 17.6 T.

Many neurodegenerative diseases including Alzheimer's disease are linked to abnormalities in the vascular system. In AD, the deposition of amyloid ß (Aß) peptide in the cerebral vessel walls, known as cerebral amyloid angiopathy (CAA) is frequently observed, leading to blood flow abnormalities. Visualization of the changes in vascular structure is important for early diagnosis and treatment. Blood vessels can be imaged non-invasively by magnetic resonance angiography (MRA). In this study we optimized high resolution MRA at 17.6 T to longitudinally monitor morphological changes in cerebral arteries in a Tg2576 mouse model, a widely used model of AD. Our results at 17.6 T show that MRA significantly benefits from the ultra-high magnetic field strength especially to visualize smaller vessels. Visual and quantitative analysis of MRA results revealed severe blood flow defects in large and medium sized arteries in Tg2576 mice. In particular blood flow defects were observed in the middle cerebral artery (MCA) and in the anterior communicating artery (AComA) in Tg2576 mice. Histological data show that Aß levels in the vessel wall may be responsible for impaired cerebral blood flow, thereby contributing to the early progression of AD. To our knowledge this is the first ultra-high field MRA study monitoring blood flow alterations longitudinally in living Tg2576 mice, consequently providing a powerful tool to test new therapeutic intervention related to CAA in a mouse model of AD.

Acetyl group orientation modulates the electronic ground-state asymmetry of the special pair in purple bacterial reaction centers.

Recent experimental data point to an asymmetric ground-state electronic distribution in the special pair (P) of purple bacterial reaction centers, which acts as the primary electron donor in photosynthesis. We have performed a density functional theory investigation on an extended model including the bacteriochlorophyll dimer and a few relevant surrounding residues to explore the origin of this asymmetry. We find strong evidence that the ground-state electron density in P is intrinsically asymmetric due to protein-induced distortions of the porphyrin rings, with excess electron charge on the P(M) bacteriochlorophyll cofactor. Moreover, the electron charge asymmetry is strongly modulated by the specific orientation of the C3(1) acetyl group, which is hydrogen bonded to His168. The electronic excitation has a significant charge transfer character inducing a displacement of electron charge from P(L) to P(M), in agreement with experimental data in the excited state. These results are relevant for the understanding of the unidirectional electron transfer path in photosynthesis.

Prospects for early detection of Alzheimer's disease from serial MR images in transgenic mice.

The existing literature on the magnetic resonance imaging of murine models of Alzheimer's disease is reviewed. Particular attention is paid to the possibilities for the early detection of the disease. To this effect, not only are relaxometric and volumetric approaches discussed, but also mathematical models for plaque distribution and aggregation. Image analysis plays a prominent role in this line of research, as stochastic image models and texture analysis have shown some success in the classification of subjects affected by Alzheimer's disease. It is concluded that relaxometric approaches seem to be a promising candidate for the task at hand, especially when combined with sophisticated image analysis, and when data from more than one time-point is available. There have been few longitudinal studies of mice models so far, so this direction of research warrants future efforts.

Residual backbone and side-chain 13C and 15N resonance assignments of the intrinsic transmembrane light-harvesting 2 protein complex by solid-state Magic Angle Spinning NMR spectroscopy.

This study reports the sequence specific chemical shifts assignments for 76 residues of the 94 residues containing monomeric unit of the photosynthetic light-harvesting 2 transmembrane protein complex from Rhodopseudomonas acidophila strain 10050, using Magic Angle Spinning (MAS) NMR in combination with extensive and selective biosynthetic isotope labeling methods. The sequence specific chemical shifts assignment is an essential step for structure determination by MAS NMR. Assignments have been performed on the basis of 2-dimensional proton-driven spin diffusion (13)C-(13)C correlation experiments with mixing times of 20 and 500 ms and band selective (13)C-(15)N correlation spectroscopy on a series of site-specific biosynthetically labeled samples. The decreased line width and the reduced number of correlation signals of the selectively labeled samples with respect to the uniformly labeled samples enable to resolve the narrowly distributed correlation signals of the backbone carbons and nitrogens involved in the long alpha-helical transmembrane segments. Inter-space correlations between nearby residues and between residues and the labeled BChl a cofactors, provided by the (13)C-(13)C correlation experiments using a 500 ms spin diffusion period, are used to arrive at sequence specific chemical shift assignments for many residues in the protein complex. In this way it is demonstrated that MAS NMR methods combined with site-specific biosynthetic isotope labeling can be used for sequence specific assignment of the NMR response of transmembrane proteins.

Biosynthetic site-specific (13) C labeling of the light-harvesting 2 protein complex: a model for solid state NMR structure determination of transmembrane proteins.

Partly biosynthetic site-directed isotopically (13)C enriched photosynthetic light-harvesting 2(LH2) complexes have been prepared from Rhodopseudomonas acidophila strain 10050 by using chemically labeled [1,2,3,4-(13)C], [1,4-(13)C] and [2,3-(13)C] succinic acid as a precursor in the growth medium. Two-dimensional proton driven spin diffusion (PDSD) solid state NMR correlation spectroscopy has been used to trace each individual (13)C isotope from the labeled succinic acid precursor to its destination into the protein and into the embedded major light-absorbing bacteriochlorophyll cofactors. For both the residues of the protein and for the cofactors distinct labeling patterns have been deduced, for protein complexes prepared from [1,4-(13)C]-succinic acid or [2,3-(13)C]-succinic labeled media. All residues, except isoleucine and leucine, have been labeled almost homogeneously by the succinic acid precursor. Carbonyl carbons in the protein backbone were labeled by [1,4-(13)C]-succinic acid, while the Calpha and Cbeta carbons of the residues were labeled by [2,3 (13)C]-succinic acid. Leucine and isoleucine residues were labeled using a uniformly labeled amino acid mixture in the medium. The pattern labeling yields an increase of the resolution and less spectral crowding. The partial labeling technique in combination with conventional solid state NMR methods at ultra high magnetic fields provides an attractive route to resolve chemical shifts for alpha-helical transmembrane protein structures.

Analysis of histamine and modeling of ligand-receptor interactions in the histamine H(1) receptor for Magic Angle Spinning NMR studies.

OBJECTIVE AND DESIGN: Investigation of the principles of ligand-receptor interaction in histamine receptors can help to provide a solid foundation for structure-based drug design. Stable isotope labelling of the ligand 'Histamine' has been performed and 1D (13)C CP MAS and 2D Radio Frequency Dipolar Recoupling (RFDR) spectra for the ligand are presented. Hyperfine signals were well spread and did not suffer from any sizable line broadening. The production of H(1) receptor for Magic Angle Spinning NMR studies is currently in progress. TREATMENT: An agonist binding domain is proposed using homology modeling, database searches and mutagenesis data for the H(1) receptor. METHODS: Homology modeling, Database searches for Expressed sequence Tag (ESTs), Magic Angle Spinning Nuclear Magnetic Resonance analysis of the ligand histamine. RESULTS: The three-dimensional receptor model and mutagenesis studies suggest that the amine of the agonist histamine may form an ion pair with the TM III Asp, whereas the imidazole ring of histamine may associate with TM V Asp and Thr. CONCLUSIONS: Homology modeling studies confirms the absence of TM VIII in the H(1) receptor. According to the model the histamine in particular interacts with the transmembrane (TM) regions of the H(1) receptor structure, in particular TM helix III and V. This is in line with recent mutagenesis studies. Database search methods for ESTs have been used for electronic prediction of tissue distribution of H(1) receptor expression. The results indicate that the H(1) expression is highest in heart and skeletal muscle, which may be of importance for drug targeting.