FMP Publications

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

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Characterization of Membrane Proteins in Isolated Native Cellular Membranes by Dynamic Nuclear Polarization Solid-State NMR Spectroscopy without Purification and Reconstitution
Jacso, T., Franks, W. T., Rose, H., Fink, U., Broecker(*), J., Keller(*), S., Oschkinat, H.; Reif, B.
Angew Chem Int Edit, 51:432-435

Tags: Solid-State NMR Spectroscopy (Reif)

Structural properties of EGCG-induced, nontoxic Alzheimer's disease Abeta oligomers
Lopez del Amo, J. M., Fink, U., Dasari, M., Grelle(*), G., Wanker(*), E. E., Bieschke(*), J.; Reif, B.
J Mol Biol, 421:517-524

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: The green tea compound epigallocatechin-3-gallate (EGCG) inhibits Alzheimer's disease beta-amyloid peptide (Abeta) neurotoxicity. Solution-state NMR allows probing initial EGCG-Abeta interactions. We show that EGCG-induced Abeta oligomers adopt a well-defined structure and are amenable for magic angle spinning solid-state NMR investigations. We find that EGCG interferes with the aromatic hydrophobic core of Abeta. The C-terminal part of the Abeta peptide (residues 22-39) adopts a beta-sheet conformation, whereas the N-terminus (residues 1-20) is unstructured. The characteristic salt bridge involving residues D23 and K28 is present in the structure of these oligomeric Abeta aggregates as well. The structural analysis of small-molecule-induced amyloid aggregates will open new perspectives for Alzheimer's disease drug development.

An Asymmetric Dimer as the Basic Subunit in Alzheimer's Disease Amyloid beta Fibrils
Lopez del Amo, J. M., Schmidt(*), M., Fink, U., Dasari, M., Fändrich(*), M.; Reif, B.
Angew Chem Int Edit, 51:6136-6139

Tags: Solid-State NMR Spectroscopy (Reif)

Solid-state magic-angle spinning NMR of membrane proteins and protein-ligand interactions
Franks, W. T., Linden, A. H., Kunert, B., van Rossum, B. J.; Oschkinat, H.
Eur J Cell Biol, 91:340-348

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Structural biology is developing into a universal tool for visualizing biological processes in space and time at atomic resolution. The field has been built by established methodology like X-ray crystallography, electron microscopy and solution NMR and is now incorporating new techniques, such as small-angle X-ray scattering, electron tomography, magic-angle-spinning solid-state NMR and femtosecond X-ray protein nanocrystallography. These new techniques all seek to investigate non-crystalline, native-like biological material. Solid-state NMR is a relatively young technique that has just proven its capabilities for de novo structure determination of model proteins. Further developments promise great potential for investigations on functional biological systems such as membrane-integrated receptors and channels, and macromolecular complexes attached to cytoskeletal proteins. Here, we review the development and applications of solid-state NMR from the first proof-of-principle investigations to mature structure determination projects, including membrane proteins. We describe the development of the methodology by looking at examples in detail and provide an outlook towards future 'big' projects.

The effect of biradical concentration on the performance of DNP-MAS-NMR
Lange, S., Linden, A. H., Akbey, Ü., Franks, W. T., Loening(*), N. M., van Rossum, B. J.; Oschkinat, H.
J Magn Reson, 216:209-212

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: With the technique of dynamic nuclear polarization (DNP) signal intensity in solid-state MAS-NMR experiments can be enhanced by 2-3 orders of magnitude. DNP relies on the transfer of electron spin polarization from unpaired electrons to nuclear spins. For this reason, stable organic biradicals such as TOTAPOL are commonly added to samples used in DNP experiments. We investigated the effects of biradical concentration on the relaxation, enhancement, and intensity of NMR signals, employing a series of samples with various TOTAPOL concentrations and uniformly (13)C, (15)N labeled proline. A considerable decrease of the NMR relaxation times (T(1), T(2)( *), and T(1)(rho)) is observed with increasing amounts of biradical due to paramagnetic relaxation enhancement (PRE). For nuclei in close proximity to the radical, decreasing T(1)(rho) reduces cross-polarization efficiency and decreases in T(2)( *) broaden the signal. Additionally, paramagnetic shifts of (1)H signals can cause further line broadening by impairing decoupling. On average, the combination of these paramagnetic effects (PE; relaxation enhancement, paramagnetic shifts) quenches NMR-signals from nuclei closer than 10A to the biradical centers. On the other hand, shorter T(1) times allow the repetition rate of the experiment to be increased, which can partially compensate for intensity loss. Therefore, it is desirable to optimize the radical concentration to prevent additional line broadening and to maximize the signal-to-noise observed per unit time for the signals of interest.

Membrane-protein structure determination by solid-state NMR spectroscopy of microcrystals
Shahid, S. A., Bardiaux, B., Franks, W. T., Krabben, L., Habeck(*), M., van Rossum, B. J.; Linke(*), D.
Nature methods, 9:1212-1217

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Membrane proteins are largely underrepresented among available atomic-resolution structures. The use of detergents in protein purification procedures hinders the formation of well-ordered crystals for X-ray crystallography and leads to slower molecular tumbling, impeding the application of solution-state NMR. Solid-state magic-angle spinning NMR spectroscopy is an emerging method for membrane-protein structural biology that can overcome these technical problems. Here we present the solid-state NMR structure of the transmembrane domain of the Yersinia enterocolitica adhesin A (YadA). The sample was derived from crystallization trials that yielded only poorly diffracting microcrystals. We solved the structure using a single, uniformly (13)C- and (15)N-labeled sample. In addition, solid-state NMR allowed us to acquire information on the flexibility and mobility of parts of the structure, which, in combination with evolutionary conservation information, presents new insights into the autotransport mechanism of YadA.

Phosphatidylinositol 4-kinase II alpha function at endosomes is regulated by the ubiquitin ligase Itch
Mössinger(*), J., Wieffer, M., Krause, E., Freund, C., Gerth, F., Krauss, M.; Haucke, V.
Embo Rep, 13:1087-1094

Tags: Molecular Pharmacology and Cell Biology (Haucke), Protein Engineering (Freund), Mass Spectrometry (Krause, E.)

Abstract: Phosphatidylinositol (PI) 4-phosphate (PI(4) P) and its metabolizing enzymes serve important functions in cell signalling and membrane traffic. PI 4-kinase type II alpha (PI4KII alpha) regulates Wnt signalling, endosomal sorting of signalling receptors, and promotes adaptor protein recruitment to endosomes and the trans-Golgi network. Here we identify the E3 ubiquitin ligase Itch as binding partner and regulator of PI4KII alpha function. Itch directly associates with and ubiquitinates PI4KII alpha, and both proteins colocalize on endosomes containing Wnt-activated frizzled 4 (Fz4) receptor. Depletion of PI4KII alpha or Itch regulates Wnt signalling with corresponding changes in Fz4 internalization and degradative sorting. These findings unravel a new molecular link between phosphoinositide-regulated endosomal membrane traffic, ubiquitin and the modulation of Wnt signalling.

Improved two-dimensional reversed phase-reversed phase LC-MS/MS approach for identification of peptide-protein interactions
Stephanowitz, H., Lange, S., Lang, D., Freund, C.; Krause, E.
Journal of proteome research, 11:1175-1183

Tags: Mass Spectrometry (Krause, E.)

Abstract: Quantitative mass spectrometry (MS) in combination with affinity purification approaches allows for an unbiased study of protein-protein and peptide-protein interactions. In shotgun approaches that are based on proteolytic digestion of complex protein mixtures followed by two-dimensional liquid-phase chromatography, the separation effort prior to MS analysis is focused on tryptic peptides. Here we developed an improved offline 2-D liquid chromatography-MS/MS approach for the identification and quantification of binding proteins utilizing reversed-phase capillary columns with acidic acetonitrile-containing eluents in both chromatographic dimensions. A specific fractionation scheme was applied in order to obtain samples with evenly distributed peptides and to fully utilize the separation space in the second dimension nanoLC-MS/MS. We report peptide-protein interaction studies to identify phosphorylation-dependent binding partners of the T cell adapter protein ADAP. The results of the SILAC-based pull-down experiments show this approach is well suited for distinguishing phosphorylation-specific interactions from unspecific binding events. The data provide further evidence that phosphorylated Tyr 595 of ADAP may serve as a direct binding site for the SH2 domains of the T cell proteins SLP76 and NCK. From a technical point of view we provide a detailed protocol for an offline 2-D RP-RP LC-MS/MS method that offers a robust and time-saving alternative for quantitative interactome analysis.

Use of Kikume green-red fusions to study the influence of pharmacological chaperones on trafficking of G protein-coupled receptors
Ridelis, I., Schmidt, A., Teichmann, A., Furkert, J., Wiesner, B.; Schülein, R.
FEBS Lett, 586:784-791

Tags: Protein Trafficking (Schülein), Cellular Imaging (Wiesner)

Abstract: In this study we demonstrate that the photoconvertible monomeric Kikume green-red (mKikGR) protein is suitable to study trafficking of G protein-coupled receptors. Taking mKikGR-tagged mutants of the vasopressin V(2) receptor (V(2)R) as models, we analyzed whether the V(2)R-specific pharmacological chaperone SR121463B influences receptor folding on a co- or post-translational level. Misfolded mKikGR-tagged V(2)Rs were completely photoconverted in the early secretory pathway yielding a red receptor population (already synthesized receptors) and an arising green receptor population (newly synthesized receptors). Trafficking of both receptor populations could be rescued by treatment with SR121463B demonstrating that the substance can act co- and post-translationally.

The protease-activated receptor 1 possesses a functional and cleavable signal peptide which is necessary for receptor expression
Zampatis, D. E., Rutz, C., Furkert, J., Schmidt, A., Wüstenhagen(*), D., Kubick(*), S., Tsopanoglou(*), N. E.; Schülein, R.
FEBS Lett, 586:2351-2359

Tags: Protein Trafficking (Schülein)

Abstract: The protease-activated receptor 1 (PAR1) is activated by thrombin cleavage releasing the physiologically-relevant parstatin peptide (residues 1-41). However, the actual length of parstatin was unclear since the receptor may also possess a cleavable signal peptide (residues 1-21) according to prediction programs. Here, we show that this putative signal peptide is indeed functional and removed from the PAR1 resolving the question of parstatin length. Moreover, we show that the sequence encoding the signal peptide may surprisingly play a role in stabilization of the PAR1 mRNA, a function which would be novel for a G protein-coupled receptor.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
Robert-Roessle-Str. 10
13125 Berlin, Germany
+4930 94793 - 100 
+4930 94793 - 109 (Fax)

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