FMP Publications

Our publications are recorded in a searchable database since 2010, updates will be added regularly.

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References
Emerin self-assembly mechanism: role of the LEM domain
Samson(*), C., Celli(*), F., Hendriks, K., Zinke, M., Essawy(*), N., Herrada(*), I., Arteni(*), A. A., Theillet(*), F. X., Alpha-Bazin(*), B., Armengaud(*), J., Coirault(*), C., Lange, A.; Zinn-Justin(*), S.
Febs J, 284:338-352
(2017)

Tags: Molecular Biophysics (Lange, A.)

Abstract: At the nuclear envelope, the inner nuclear membrane protein emerin contributes to the interface between the nucleoskeleton and the chromatin. Emerin is an essential actor of the nuclear response to a mechanical signal. Genetic defects in emerin cause Emery-Dreifuss muscular dystrophy. It was proposed that emerin oligomerization regulates nucleoskeleton binding, and impaired oligomerization contributes to the loss of function of emerin disease-causing mutants. We here report the first structural characterization of emerin oligomers. We identified an N-terminal emerin region from amino acid 1 to amino acid 132 that is necessary and sufficient for formation of long curvilinear filaments. In emerin monomer, this region contains a globular LEM domain and a fragment that is intrinsically disordered. Solid-state nuclear magnetic resonance analysis identifies the LEM beta-fragment as part of the oligomeric structural core. However, the LEM domain alone does not self-assemble into filaments. Additional residues forming a beta-structure are observed within the filaments that could correspond to the unstructured region in emerin monomer. We show that the delK37 mutation causing muscular dystrophy triggers LEM domain unfolding and increases emerin self-assembly rate. Similarly, inserting a disulfide bridge that stabilizes the LEM folded state impairs emerin N-terminal region self-assembly, whereas reducing this disulfide bridge triggers self-assembly. We conclude that the LEM domain, responsible for binding to the chromatin protein BAF, undergoes a conformational change during self-assembly of emerin N-terminal region. The consequences of these structural rearrangement and self-assembly events on emerin binding properties are discussed.

Bacteriophage Tail-Tube Assembly Studied by Proton-Detected 4D Solid-State NMR
Zinke, M., Fricke, P., Samson(*), C., Hwang, S., Wall(*), J. S., Lange, S., Zinn-Justin(*), S.; Lange, A.
Angew Chem Int Ed Engl,
(2017)

Tags: Molecular Biophysics (Lange, A.)

Abstract: Obtaining unambiguous resonance assignments remains a major bottleneck in solid-state NMR studies of protein structure and dynamics. Particularly for supramolecular assemblies with large subunits (>150 residues), the analysis of crowded spectral data presents a challenge, even if three-dimensional (3D) spectra are used. Here, we present a proton-detected 4D solid-state NMR assignment procedure that is tailored for large assemblies. The key to recording 4D spectra with three indirect carbon or nitrogen dimensions with their inherently large chemical shift dispersion lies in the use of sparse non-uniform sampling (as low as 2 %). As a proof of principle, we acquired 4D (H)COCANH, (H)CACONH, and (H)CBCANH spectra of the 20 kDa bacteriophage tail-tube protein gp17.1 in a total time of two and a half weeks. These spectra were sufficient to obtain complete resonance assignments in a straightforward manner without use of previous solution NMR data.

Backbone assignment of perdeuterated proteins by solid-state NMR using proton detection and ultrafast magic-angle spinning
Fricke, P., Chevelkov, V., Zinke, M., Giller(*), K., Becker(*), S.; Lange, A.
Nat Protoc, 12:764-782
(2017)

Tags: Molecular Biophysics (Lange, A.)

Abstract: Solid-state NMR (ssNMR) is a technique that allows the study of protein structure and dynamics at atomic detail. In contrast to X-ray crystallography and cryo-electron microscopy, proteins can be studied under physiological conditions-for example, in a lipid bilayer and at room temperature (0-35 degrees C). However, ssNMR requires considerable amounts (milligram quantities) of isotopically labeled samples. In recent years, 1H-detection of perdeuterated protein samples has been proposed as a method of alleviating the sensitivity issue. Such methods are, however, substantially more demanding to the spectroscopist, as compared with traditional 13C-detected approaches. As a guide, this protocol describes a procedure for the chemical shift assignment of the backbone atoms of proteins in the solid state by 1H-detected ssNMR. It requires a perdeuterated, uniformly 13C- and 15N-labeled protein sample with subsequent proton back-exchange to the labile sites. The sample needs to be spun at a minimum of 40 kHz in the NMR spectrometer. With a minimal set of five 3D NMR spectra, the protein backbone and some of the side-chain atoms can be completely assigned. These spectra correlate resonances within one amino acid residue and between neighboring residues; taken together, these correlations allow for complete chemical shift assignment via a 'backbone walk'. This results in a backbone chemical shift table, which is the basis for further analysis of the protein structure and/or dynamics by ssNMR. Depending on the spectral quality and complexity of the protein, data acquisition and analysis are possible within 2 months.

Helical Polyisocyanopeptides as Lyotropic Liquid Crystals for Measuring Residual Dipolar Couplings
Li(*), G. W., Cao(*), J. M., Zong(*), W., Hu(*), L., Hu(*), M. L., Lei(*), X., Sun, H.; Tan(*), R. X.
Chemistry, 23:7653-7656
(2017)

Tags: Computational Chemistry and Protein Design (Kühne)

Abstract: Residual dipolar couplings (RDC) emerged to be an important structural parameter for organic and biomolecules. Herein, a new helical polyisocyanopeptide (l,l-PIAF-OBn) that forms lyotropic liquid crystals (LLC) in CDCl3 is proposed as a novel weakly orienting medium for acquiring residual dipolar couplings (RDCs) of organic molecules. We demonstrate its application for the structural elucidation of strychnine and triptolide.

NMR Hyperpolarization Techniques of Gases
Barskiy(*), D. A., Coffey(*), A. M., Nikolaou(*), P., Mikhaylov(*), D. M., Goodson(*), B. M., Branca(*), R. T., Lu(*), G. J., Shapiro(*), M. G., Telkki(*), V. V., Zhivonitko(*), V. V., Koptyug(*), I. V., Salnikov(*), O. G., Kovtunov(*), K. V., Bukhtiyarov(*), V. I., Rosen(*), M. S., Barlow(*), M. J., Safavi(*), S., Hall(*), I. P., Schroeder, L.; Chekmenev(*), E. Y.
Chemistry, 23:725-751
(2017)

Tags: Molecular Imaging (Schröder)

Abstract: Nuclear spin polarization can be significantly increased through the process of hyperpolarization, leading to an increase in the sensitivity of nuclear magnetic resonance (NMR) experiments by 4-8 orders of magnitude. Hyperpolarized gases, unlike liquids and solids, can often be readily separated and purified from the compounds used to mediate the hyperpolarization processes. These pure hyperpolarized gases enabled many novel MRI applications including the visualization of void spaces, imaging of lung function, and remote detection. Additionally, hyperpolarized gases can be dissolved in liquids and can be used as sensitive molecular probes and reporters. This Minireview covers the fundamentals of the preparation of hyperpolarized gases and focuses on selected applications of interest to biomedicine and materials science.

Human iPSC-Derived Neural Progenitors Are an Effective Drug Discovery Model for Neurological mtDNA Disorders
Lorenz(*), C., Lesimple(*), P., Bukowiecki(*), R., Zink(*), A., Inak(*), G., Mlody(*), B., Singh(*), M., Semtner(*), M., Mah(*), N., Aure(*), K., Leong(*), M., Zabiegalov(*), O., Lyras(*), E. M., Pfiffer(*), V., Fauler(*), B., Eichhorst, J., Wiesner, B., Huebner(*), N., Priller(*), J., Mielke(*), T., Meierhofer(*), D., Izsvak(*), Z., Meier(*), J. C., Bouillaud(*), F., Adjaye(*), J., Schuelke(*), M., Wanker(*), E. E., Lombes(*), A.; Prigione(*), A.
Cell stem cell,
(2017)

Tags: Cellular Imaging (Wiesner)

Abstract: Mitochondrial DNA (mtDNA) mutations frequently cause neurological diseases. Modeling of these defects has been difficult because of the challenges associated with engineering mtDNA. We show here that neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) retain the parental mtDNA profile and exhibit a metabolic switch toward oxidative phosphorylation. NPCs derived in this way from patients carrying a deleterious homoplasmic mutation in the mitochondrial gene MT-ATP6 (m.9185T>C) showed defective ATP production and abnormally high mitochondrial membrane potential (MMP), plus altered calcium homeostasis, which represents a potential cause of neural impairment. High-content screening of FDA-approved drugs using the MMP phenotype highlighted avanafil, which we found was able to partially rescue the calcium defect in patient NPCs and differentiated neurons. Overall, our results show that iPSC-derived NPCs provide an effective model for drug screening to target mtDNA disorders that affect the nervous system.

Phosphatidylinositol 4,5-bisphosphate optical uncaging potentiates exocytosis
Walter(*), A. M., Müller(*), R., Tawfik(*), B., Wierda(*), K. D., Pinheiro(*), P. S., Nadler(*), A., McCarthy(*), A. W., Ziomkiewicz(*), I., Kruse(*), M., Reither(*), G., Rettig(*), J., Lehmann, M., Haucke, V., Hille(*), B., Schultz(*), C.; Sorensen(*), J. B.
Elife, 6
(2017)

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is essential for exocytosis. Classical ways of manipulating PI(4,5)P2 levels are slower than metabolism, making it difficult to distinguish effects of PI(4,5)P2 from those of its metabolites. We developed a membrane-permeant, photoactivatable PI(4,5)P2, which is loaded into cells in an inactive form and activated by light, allowing sub-second increases in PI(4,5)P2 levels. By combining this compound with electrophysiological measurements in mouse adrenal chromaffin cells, we show that PI(4,5)P2 uncaging potentiates exocytosis and identify synaptotagmin-1 (the Ca2+ sensor for exocytosis) and Munc13-2 (a vesicle priming protein) as the relevant effector proteins. PI(4,5)P2 activation of exocytosis did not depend on the PI(4,5)P2-binding CAPS-proteins, suggesting that PI(4,5)P2 uncaging bypasses CAPS-function. Finally, PI(4,5)P2 uncaging triggered the rapid fusion of a subset of readily-releasable vesicles, revealing a rapid role of PI(4,5)P2 in fusion triggering. Thus, optical uncaging of signaling lipids can uncover their rapid effects on cellular processes and identify lipid effectors.

Unitary Properties of AMPA Receptors with Reduced Desensitization
Zhang(*), W., Eibl, C., Weeks(*), A. M., Riva, I., Li(*), Y. J., Plested, A. J. R.; Howe(*), J. R.
Biophys J,
(2017)

Tags: Molecular Neuroscience and Biophysics (Plested)

Abstract: Wild-type AMPA receptors display a characteristic rapidly desensitizing phenotype. Many studies point to the dimer interface between pairs of extracellular ligand binding domains as the key region controlling the rate at which the receptors desensitize. However, mutations at the extracellular end of the pore-forming regions (near the putative ion channel gate) have also been shown to alter desensitization. Here we report the behavior of single GluA4 receptors carrying one of two mutations that greatly reduce desensitization at the level of ensemble currents: the dimer interface mutation L484Y and the Lurcher mutation (A623T, GluA4-Lc) in the extracellular end of M3 (the second true transmembrane helix). Analysis of unitary currents in patches with just one active receptor showed that each mutation greatly prolongs bursts of openings without prolonging the apparent duration of individual openings. Each mutation decreases the frequency with which individual receptors visit desensitized states, but both mutant receptors still desensitize multiple times per second. Cyclothiazide (CTZ) reduced desensitization of wild-type receptors and both types of mutant receptor. Analysis of shut-time distributions revealed a form of short-lived desensitization that was resistant to CTZ and was especially prominent for GluA4-Lc receptors. Despite reducing desensitization of GluA4 L484Y receptors, CTZ decreased the amplitude of ensemble currents through GluA2 and GluA4 LY receptor mutants. Single-channel analysis and comparison of the GluA2 L483Y ligand binding domain dimer in complex with glutamate with and without CTZ is consistent with the conclusion that CTZ binding to the dimer interface prevents effects of the LY mutation to modulate receptor activation, resulting in a reduction in the prevalence of large-conductance substates that accounts for the decrease in ensemble current amplitudes. Together, the results show that similar nondesensitizing AMPA-receptor phenotypes of population currents can arise from distinct underlying molecular mechanisms that produce different types of unitary activity.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
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13125 Berlin, Germany
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info(at)fmp-berlin.de

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