FMP Publications

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

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References
NMR spectroscopy reveals unexpected structural variation at the protein-protein interface in MHC class I molecules
Beerbaum, M., Ballaschk, M., Erdmann, N., Schnick(*), C., Diehl, A., Uchanska-Ziegler(*), B., Ziegler(*), A.; Schmieder, P.
J Biomol NMR, 57:167-178
(2013)

Tags: Solution NMR (Schmieder)

Abstract: beta2-Microglobulin (beta2m) is a small, monomorphic protein non-covalently bound to the heavy chain (HC) in polymorphic major histocompatibility complex (MHC) class I molecules. Given the high evolutionary conservation of structural features of beta2m in various MHC molecules as shown by X-ray crystallography, beta2m is often considered as a mere scaffolding protein. Using nuclear magnetic resonance (NMR) spectroscopy, we investigate here whether beta2m residues at the interface to the HC exhibit changes depending on HC polymorphisms and the peptides bound to the complex in solution. First we show that human beta2m can effectively be produced in deuterated form using high-cell-density-fermentation and we employ the NMR resonance assignments obtained for triple-labeled beta2m bound to the HLA-B*27:09 HC to examine the beta2m-HC interface. We then proceed to compare the resonances of beta2m in two minimally distinct subtypes, HLA-B*27:09 and HLA-B*27:05, that are differentially associated with the spondyloarthropathy Ankylosing Spondylitis. Each of these subtypes is complexed with four distinct peptides for which structural information is already available. We find that only the resonances at the beta2m-HC interface show a variation of their chemical shifts between the different complexes. This indicates the existence of an unexpected plasticity that enables beta2m to accommodate changes that depend on HC polymorphism as well as on the bound peptide through subtle structural variations of the protein-protein interface.

Structural and biochemical characterization of Rv2140c, a phosphatidylethanolamine-binding protein from Mycobacterium tuberculosis
Eulenburg(*), G., Higman, V. A., Diehl, A., Wilmanns(*), M.; Holton(*), S. J.
Febs Letters, 587:2936-2942
(2013)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Rv2140c is one of many conserved Mycobacterium tuberculosis proteins for which no molecular function has been identified. We have determined a high-resolution crystal structure of the Rv2140c gene product, which reveals a dimeric complex that shares strong structural homology with the phosphatidylethanolamine-binding family of proteins. Rv2140c forms low-millimolar interactions with a selection of soluble phosphatidylethanolamine analogs, indicating that it has a role in lipid metabolism. Furthermore, the small molecule locostatin binds to the Rv2140c ligand-binding site and also inhibits the growth of the model organism Mycobacterium smegmatis. Structured digital abstract: Rv2140c and Rv2140c bind by molecular sieving (View interaction) v2140c and Rv2140c bind by cosedimentation in solution (View interaction) Rv2140c and Rv2140c bind by x-ray crystallography (View interaction) (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Hydrogen bonding involving side chain exchangeable groups stabilizes amyloid quarternary structure
Agarwal, V., Linser, R., Dasari, M., Fink, U., del Amo, J. M.; Reif, B.
Phys Chem Chem Phys, 15:12551-12557
(2013)

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: The amyloid beta-peptide (Abeta) is the major structural component of amyloid fibrils in the plaques of brains of Alzheimer's disease patients. Numerous studies have addressed important aspects of secondary and tertiary structure of fibrils. In electron microscopic images, fibrils often bundle together. The mechanisms which drive the association of protofilaments into bundles of fibrils are not known. We show here that amino acid side chain exchangeable groups like e.g. histidines can provide useful restraints to determine the quarternary assembly of an amyloid fibril. Exchangeable protons are only observable if a side chain hydrogen bond is formed and the respective protons are protected from exchange. The method relies on deuteration of the Abeta peptide. Exchangeable deuterons are substituted with protons, before fibril formation is initiated.

Functional properties of cell-free expressed human endothelin A and endothelin B receptors in artificial membrane environments
Proverbio(*), D., Roos(*), C., Beyermann, M., Orban(*), E., Dötsch(*), V.; Bernhard(*), F.
Bba-Biomembranes, 1828:2182-2192
(2013)

Tags: Peptide Chemistry (Beyermann)

Abstract: The human endothelin receptors are members of the rhodopsin class A of G-protein coupled receptors and key modulators of blood pressure regulation. Their functional in vitro characterization has widely been limited by the availability of high quality samples. We have optimized cell-free expression protocols for the human endothelin A and endothelin B receptors by implementing co-translational association approaches of the synthesized proteins with supplied liposomes or nanodiscs. Efficiency of membrane association and ligand binding properties of the receptors have systematically been studied in correlation to different membrane environments and lipid types. Ligand binding was analyzed by a number of complementary assays including radioassays, surface plasmon resonance and fluorescence measurements. High affinity binding of the peptide ligand ET-I to both endothelin receptors could be obtained with several conditions and the highest Bmax values were measured In association with nanodiscs. We could further obtain the characteristic differential binding pattern of the two endothelin receptors with a panel of selected agonists and antagonists. Two intrinsic properties of the functionally folded endothelin B receptor, the proteolytic processing based on conformational recognition as well as the formation of SDS-resistant complexes with the peptide ligand ET-1, were observed with samples obtained from several cell-free expression conditions. High affinity and specific binding of ligands could furthermore be obtained with non-purified receptor samples in crude cell-free reaction mixtures, thus providing new perspectives for fast in vitro screening applications. (C) 2013 Elsevier B.V. All rights reserved.

Oligomerization of Dynamin Superfamily Proteins in Health and Disease
Faelber(*), K., Gao(*), S., Held(*), M., Posor, Y., Haucke, V., Noe(*), F.; Daumke(*), O.
Prog Mol Biol Transl, 117:411-443
(2013)

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Proteins of the dynamin superfamily are mechanochemical GTPases, which mediate nucleotide-dependent membrane remodeling events. The founding member dynamin is recruited to the neck of clathrin-coated endocytic vesicles where it oligomerizes into helical filaments. Nucleotide-hydrolysis-induced conformational changes in the oligomer catalyze scission of the vesicle neck. Here, we review recent insights into structure, function, and oligomerization of dynamin superfamily proteins and their roles in human diseases. We describe in detail the molecular mechanisms how dynamin oligomerizes at membranes and introduce a model how oligomerization is linked to membrane fission. Finally, we discuss molecular mechanisms how mutations in dynamin could lead to the congenital diseases, Centronuclear Myopathy and Charcot-Marie Tooth disease.

Structural insights into the mechanism of GTPase activation in the GIMAP family
Schwefel(*), D., Arasu(*), B. S., Marino(*), S. F., Lamprecht(*), B., Kochert(*), K., Rosenbaum(*), E., Eichhorst, J., Wiesner, B., Behlke(*), J., Rocks(*), O., Mathas(*), S.; Daumke(*), O.
Structure (London, England : 1993), 21:550-559
(2013)

Tags: Cellular Imaging (Wiesner)

Abstract: GTPases of immunity-associated proteins (GIMAPs) are regulators of lymphocyte survival and homeostasis. We previously determined the structural basis of GTP-dependent GIMAP2 scaffold formation on lipid droplets. To understand how its GTP hydrolysis is activated, we screened for other GIMAPs on lipid droplets and identified GIMAP7. In contrast to GIMAP2, GIMAP7 displayed dimerization-stimulated GTP hydrolysis. The crystal structure of GTP-bound GIMAP7 showed a homodimer that assembled via the G domains, with the helical extensions protruding in opposite directions. We identified a catalytic arginine that is supplied to the opposing monomer to stimulate GTP hydrolysis. GIMAP7 also stimulated GTP hydrolysis by GIMAP2 via an analogous mechanism. Finally, we found GIMAP2 and GIMAP7 expression differentially regulated in several human T cell lymphoma lines. Our findings suggest that GTPase activity in the GIMAP family is controlled by homo- and heterodimerization. This may have implications for the differential roles of some GIMAPs in lymphocyte survival.

Characterization of Cell-Penetrating Lipopeptide Micelles by Spectroscopic Methods
Gehne(*), S., Sydow, K., Dathe, M.; Kumke(*), M. U.
J Phys Chem B, 117:14215-14225
(2013)

Tags: Peptide-Lipid-Interaction/ Peptide Transport (Dathe)

Abstract: The transport of bioactive compounds to the site of action is a great challenge. A promising approach to overcome application-related problems is the development of targeting colloidal transport systems, such as micelles which are equipped with uptake mediating moieties. Here, we investigated a set of novel lipopeptides which exhibit a surfactant-like structure due to attachment of two palmitoyl chains to the Nterminus of cationic or anionic amino acid sequences. We analyzed the association behavior of these lipopeptides by using 5(6)-carboxyfluorescein (CF)-labeled derivatives as a fluorescent probe and different spectroscopic methods such as fluorescence anisotropy and fluorescence correlation spectroscopy (FCS). The photophysical properties as well as the diffusion and rotational movements of the CF-labeled lipopeptides were exploited to determine the cmc and the size of the micelles consisting of lipopeptides. We could distinguish cationic and anionic lipopeptides by their association behavior and by studying the interactions with mouse brain capillary endothelial cells (b.end3). The cationic derivatives turned out to be very strong surfactants with a very low cmc in the micromolar range (0.5-14 mu M). The unique combination of micelle-forming property and cell-penetrating ability can pave the road for the development of a novel class of efficient drug carrier systems.

What Goes around Comes around-A Comparative Study of the Influence of Chemical Modifications on the Antimicrobial Properties of Small Cyclic Peptides
Scheinpflug, K., Nikolenko, H., Komarov(*), I. V., Rautenbach(*), M.; Dathe, M.
Pharmaceuticals (Basel), 6:1130-1144
(2013)

Tags: Peptide-Lipid-Interaction/ Peptide Transport (Dathe)

Abstract: Tryptophan and arginine-rich cyclic hexapeptides of the type cyclo-RRRWFW combine high antibacterial activity with rapid cell killing kinetics, but show low toxicity in human cell lines. The peptides fulfil the structural requirements for membrane interaction such as high amphipathicity and cationic charge, but membrane permeabilisation, which is the most common mode of action of antimicrobial peptides (AMPs), could not be observed. Our current studies focus on elucidating a putative membrane translocation mechanism whereupon the peptides might interfere with intracellular processes. These investigations require particular analytical tools: fluorescent analogues and peptides bearing appropriate reactive groups were synthesized and characterized in order to be used in confocal laser scanning microscopy and HPLC analysis. We found that minimal changes in both the cationic and hydrophobic domain of the peptides in most cases led to significant reduction of antimicrobial activity and/or changes in the mode of action. However, we were able to identify two modified peptides which exhibited properties similar to those of the cyclic parent hexapeptide and are suitable for subsequent studies on membrane translocation and uptake into bacterial cells.

Visualizing Brain Inflammation with a Shingled-Leg Radio-Frequency Head Probe for F-19/H-1 MRI
Waiczies(*), H., Lepore(*), S., Drechsler(*), S., Qadri(*), F., Purfurst(*), B., Sydow, K., Dathe, M., Kühne(*), A., Lindel(*), T., Hoffmann(*), W., Pohlmann(*), A., Niendorf(*), T.; Waiczies(*), S.
Sci Rep-Uk, 3
(2013)

Tags: Peptide-Lipid-Interaction/ Peptide Transport (Dathe)

Abstract: Magnetic resonance imaging (MRI) provides the opportunity of tracking cells in vivo. Major challenges in dissecting cells from the recipient tissue and signal sensitivity constraints albeit exist. In this study, we aimed to tackle these limitations in order to study inflammation in autoimmune encephalomyelitis. We constructed a very small dual-tunable radio frequency (RF) birdcage probe tailored for F-19 (fluorine) and H-1 (proton) MR mouse neuroimaging. The novel design eliminated the need for extra electrical components on the probe structure and afforded a uniform B-1(+)-field as well as good SNR. We employed fluorescently-tagged F-19 nanoparticles and could study the dynamics of inflammatory cells between CNS and lymphatic system during development of encephalomyelitis, even within regions of the brain that are otherwise not easily visualized by conventional probes. F-19/H-1 MR Neuroimaging will allow us to study the nature of immune cell infiltration during brain inflammation over an extensive period of time.

Cryogenic solid state NMR studies of fibrils of the Alzheimer's disease amyloid-beta peptide: perspectives for DNP
del Amo, J. M. L., Schneider(*), D., Loquet(*), A., Lange(*), A.; Reif, B.
J. Biomol. NMR, 56:359-363
(2013)

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: Dynamic Nuclear Polarization solid-state NMR holds the potential to enable a dramatic increase in sensitivity by exploiting the large magnetic moment of the electron. However, applications to biological solids are hampered in uniformly isotopically enriched biomacromolecules due to line broadening which yields a limited spectral resolution at cryogenic temperatures. We show here that high magnetic fields allow to overcome the broadening of resonance lines often experienced at liquid nitrogen temperatures. For a fibril sample of the Alzheimer's disease beta-amyloid peptide, we find similar line widths at low temperature and at room temperature. The presented results open new perspectives for structural investigations in the solid-state.

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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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