[Show abstract][Hide abstract] ABSTRACT: Complete structural elucidation of natural products is often challenging due to structural complexity and limited availability. This is true for present-day secondary metabolites, but even more for exceptionally preserved secondary metabolites of ancient organisms that potentially provide insights into the evolutionary history of natural products. Here we report the full structure and absolute configuration of the borolithochromes, enigmatic boron-containing pigments from a Jurassic putative red alga, from samples of less than 50 µg using microcryoprobe NMR, CD spectroscopy, and DFT calculations, and reveal their polyke-tide origin. The pigments are identified as spiroborates with two pentacyclic sec-butyl-trihydroxy-methyl-benzo[gh]tetraphen-one ligands and less substituted deriva-tives. The configuration of the sec-butyl group is found to be (S). Because the exceptional benzo[gh]tetraphene scaffold is otherwise only observed in the recently discovered polyke-tide clostrubin from a present-day Clostridium bacterium, the Jurassic borolithochromes now can be unambiguously linked to the modern polyketide, providing evidence that the fossil pigments are almost originally preserved secondary metabolites and suggesting that the pigments in fact may have been produced by an ancient bacterium. The borolithochromes differ fundamentally from previously described boronated polyketides and represent the first boronated aromatic polyketides found so far. Our results demonstrate the potential of microcryoprobe NMR in the analysis of previously little-explored secondary metabolites from ancient organisms and reveal the evolutionary significance of clostrubin-type polyketides.
Journal of the American Chemical Society 10/2015; 137(42). DOI:10.1021/jacs.5b08191 · 12.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pathological tau aggregation leads to filamentous tau inclusions and characterizes neurodegenerative tauopathies such as Alzheimer's disease and frontotemporal dementia and parkinsonism linked to chromosome 17. Tau aggregation coincides with clinical symptoms and is thought to mediate neurodegeneration. Transgenic mice overexpressing mutant human P301S tau exhibit many neuropathological features of human tauopathies including behavioral deficits and increased mortality. Here, we show that the di-phenyl-pyrazole anle138b binds to aggregated tau and inhibits tau aggregation in vitro and in vivo. Furthermore, anle138b treatment effectively ameliorates disease symptoms, increases survival time and improves cognition of tau transgenic PS19 mice. In addition, we found decreased synapse and neuron loss accompanied by a decreased gliosis in the hippocampus. Our results suggest that reducing tau aggregates with anle138b may represent an effective and promising approach for the treatment of human tauopathies.
[Show abstract][Hide abstract] ABSTRACT: Together with NOE and J coupling, one-bond residual dipolar coupling (RDC), which reports on the three-dimensional orientation of an internuclear vector in the molecular frame, plays an important role in the conformation and configuration analysis of small molecules in solution by NMR spectroscopy. When the molecule has few CH bonds, or too many bonds are in parallel, the available RDCs may not be sufficient to obtain the alignment tensor used for structure elucidation. Long-range RDCs that connect nuclei over multiple bonds are normally not parallel to the single bonds and therefore complement one-bond RDCs. Herein we present a method for extracting the long-range RDC of a chosen proton or group of protons to all remotely connected carbon atoms, including non-protonated carbon atoms. Alignment tensors fitted directly to the total long-range couplings (T=J+D) enabled straightforward analysis of both the long-range and one-bond RDCs for strychnine.
[Show abstract][Hide abstract] ABSTRACT: Protein motions over various time scales are crucial for protein function. NMR relaxation dispersion experiments play a key role in explaining these motions. However, the study of slow conformational changes with lowly populated states remained elusive. The recently developed exchange-mediated saturation transfer experiments allow the detection and characterization of such motions, but require extensive measurement time. Here we show that, by making use of Fourier transform, the total acquisition time required to measure an exchange-mediated saturation transfer profile can be reduced by twofold in case that one applies linear prediction. In addition, we demonstrate that the analytical solution for R1ρ experiments can be used for fitting the exchange-mediated saturation transfer profile. Furthermore, we show that simultaneous analysis of exchange-mediated saturation transfer profiles with two different radio-frequency field strengths is required for accurate and precise characterization of the exchange process and the exchanging states.
[Show abstract][Hide abstract] ABSTRACT: Prion diseases are fatal neurodegenerative diseases characterized by accumulation of the pathogenic prion protein PrP in the brain. We established quantitative real-time quaking-induced conversion for the measurement of minute amounts of PrP in body fluids such as urine. Using this approach, we monitored the efficacy of antiprion therapy by quantifying the seeding activity of PrP from the brain and urine of mice after prion infection. We found that the aggregation inhibitor anle138b decreased the levels of PrP in the brain and urine. Importantly, variations of PrP levels in the urine closely corresponded to those in the brain. Our findings indicate that quantification of urinary PrP enables measurement of prion disease progression in body fluids and can substitute for immunodetection in brain tissue. We expect PrP quantification biologic fluids (such as urine and cerebrospinal fluid) with quantitative real-time quaking-induced conversion to emerge as a valuable noninvasive diagnostic tool for monitoring disease progression and the efficacy of therapeutic approaches in animal studies and human clinical trials of prion diseases. Moreover, highly sensitive methods for quantifying pathologic aggregate seeds might provide novel molecular biomarkers for other neurodegenerative diseases that may involve prion-like mechanisms (protein aggregation and spreading), such as Alzheimer disease and Parkinson disease.
[Show abstract][Hide abstract] ABSTRACT: INPHARMA (Internuclear NOEs for Pharmacophore Mapping) determines the relative orientation of two competitive ligands in the protein binding pocket. It is based on the observation of interligand transferred NOEs mediated by spin diffusion through protons of the protein and is, therefore, sensitive to the specific interactions of each of the two ligands with the protein. We show how this information can be directly included into a protein-ligand docking program to guide the prediction of the complex structures. Agreement between the experimental and back-calculated spectra based on the full relaxation matrix approach is translated into a score contribution that is combined with the scoring function ChemPLP of our docking tool PLANTS. This combined score is then used to predict the poses of 5 weakly bound cAMP-dependent Protein Kinase (PKA) ligands. After optimizing the setup, which finally also included trNOE data and optimized protonation states, very good success rates were obtained for all combinations of three ligands. For one additional ligand, no conclusive results could be obtained due to the ambiguous electron density of the ligand in the X-ray structure, which does not disprove alternative ligand poses. The failures of the remaining ligand are caused by suboptimal locations of specific protein side chains. Therefore, side-chain flexibility should be included in an improved INPHARMA-PLANTS version. This will reduce the strong dependence on the used protein input structure leading to improved scores not only for this last ligand.
Journal of Chemical Information and Modeling 07/2015; 55(9). DOI:10.1021/acs.jcim.5b00235 · 3.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Im Frühling 2013 versammelten sich NMR-Spektroskopiker am Weizmann-Institut in Israel, um neue Ansätze zur Verbesserung der Empfindlichkeit von NMR-Experimenten zu diskutieren, besonders im Hinblick auf Experimente an biomolekularen Systemen. Der vorliegende Aufsatz ist von vielen Autoren mit unterschiedlichem fachlichem Hintergrund verfasst worden; er beschreibt den Stand der Forschung auf diesem Gebiet, wie er im Rahmen des Treffens diskutiert wurde. Es werden Ansätze für Ultra-Hochfeld-NMR-Spektroskopie, für neuartige Techniken der NMR-Detektion, für Prinzipien und Methoden der Kernhyperpolarisation, aber auch für neue Probenpräparationsmethoden diskutiert. Diese Ansätze verbessern die Empfindlichkeit von NMR-Experimenten im Festkörper und in Lösung, sind voneinander unabhängig und können sich in ihren Auswirkungen multiplizieren. Auch wenn in allen genannten Bereichen bereits substanzielle Verbesserungen erzielt wurden, ist doch noch eine weite Strecke zu überwinden, um an die Empfindlichkeiten von optischen und elektronischen Spektroskopien heranzureichen. Diese Probleme und erste Lösungsansätze werden ebenfalls diskutiert.
[Show abstract][Hide abstract] ABSTRACT: Growing evidence supports a link between brain copper homeostasis, the formation of alpha-synuclein (AS)-copper complexes and the development of Parkinson´s disease (PD). Recently it was demonstrated that the physiological form of AS is N-terminally acetylated (AcAS). Here we used NMR spectroscopy to structurally characterize the interaction between Cu(I) and AcAS. We found that the formation of an AcAS-Cu(I) complex at the N-terminal re-gion stabilizes local conformations with α-helical secondary structure and restricted motility. Our work provides new evidence into the metallo-biology of PD and opens new lines of research as the formation of AcAS-Cu(I) complex might impact on AcAS membrane binding and aggregation.
Journal of the American Chemical Society 05/2015; 137(20). DOI:10.1021/jacs.5b01911 · 12.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lectins from different sources are known to interfere with HIV infection. The anti-viral activity is mediated by binding to high mannose sugars present on the viral envelope, thereby inhibiting cell entry. The lectin from Oscillatoria agardhii agglutinin (OAA) specifically recognizes a unique substructure of high mannose sugars and exhibits broad anti-HIV activity. Here we report the assignment of backbone and side-chain (1)H, (13)C and (15)N resonances of free OAA.
[Show abstract][Hide abstract] ABSTRACT: The use of NMR spectroscopy in anisotropic media, particularly the application of residual dipolar couplings (RDCs) for the structural analysis of small molecules is presented. Experimental procedures such as choice and preparation of alignment media, as well as the selection of the right pulse-sequences is discussed along with computational techniques for data analysis. Methods for the analysis of flexible molecules are also presented and illustrated with several examples from literature.
[Show abstract][Hide abstract] ABSTRACT: The eIF4E-binding protein 1 (4EBP1) has long been known to be completely unstructured without any secondary structures, which contributed significantly to the proposal of the induced fit mechanism for target binding of intrinsically disordered proteins. We show here that 4EBP1 is not completely unstructured, but contains a pre-structured helix.
[Show abstract][Hide abstract] ABSTRACT: The voltage-dependent anion channel (VDAC) is the most abundant protein of the outer mitochondrial membrane and constitutes the major pathway for the transport of ADP, ATP, and other metabolites. In this multidisciplinary study we combined solid-state NMR, electrophysiology, and molecular dynamics simulations, to study the structure of the human VDAC isoform 2 in a lipid bilayer environment. We find that the structure of hVDAC2 is similar to the structure of hVDAC1, in line with recent investigations on zfVDAC2. However, hVDAC2 appears to exhibit an increased conformational heterogeneity compared to hVDAC1 which is reflected in broader solid-state NMR spectra and less defined electrophysiological profiles.