FMP Publications

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

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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

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.

NMR Spectroscopy of Soluble Protein Complexes at One Mega-Dalton and Beyond
Mainz, A., Religa, T. L., Sprangers, R., Linser, R., Kay, L. E.; Reif, B.
Angew Chem Int Edit, 52:8746-8751

Tags: Solid-State NMR Spectroscopy (Reif)

Dynamic nuclear polarization of spherical nanoparticles
Akbey, Ü., Altin(*), B., Linden, A., Ozcelik(*), S., Gradzielski(*), M.; Oschkinat, H.
Phys Chem Chem Phys, 15:20706-20716

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Spherical silica nanoparticles of various particle sizes (~10 to 100 nm), produced by a modified Stoeber method employing amino acids as catalysts, are investigated using Dynamic Nuclear Polarization (DNP) enhanced Nuclear Magnetic Resonance (NMR) spectroscopy. This study includes ultra-sensitive detection of surface-bound amino acids and their supramolecular organization in trace amounts, exploiting the increase in NMR sensitivity of up to three orders of magnitude via DNP. Moreover, the nature of the silicon nuclei on the surface and the bulk silicon nuclei in the core (sub-surface) is characterized at atomic resolution. Thereby, we obtain unique insights into the surface chemistry of these nanoparticles, which might result in improving their rational design as required for promising applications, e.g. as catalysts or imaging contrast agents. The non-covalent binding of amino acids to surfaces was determined which shows that the amino acids not just function as catalysts but become incorporated into the nanoparticles during the formation process. As a result only three distinct Q-types of silica signals were observed from surface and core regions. We observed dramatic changes of DNP enhancements as a function of particle size, and very small particles (which suit in vivo applications better) were hyperpolarized with the best efficiency. Nearly one order of magnitude larger DNP enhancement was observed for nanoparticles with 13 nm size compared to particles with 100 nm size. We determined an approximate DNP penetration-depth (~4.2 or ~5.7 nm) for the polarization transfer from electrons to the nuclei of the spherical nanoparticles. Faster DNP polarization buildup was observed for larger nanoparticles. Efficient hyperpolarization of such nanoparticles, as achieved in this work, can be utilized in applications such as magnetic resonance imaging (MRI).

Site-specific NMR mapping and time-resolved monitoring of serine and threonine phosphorylation in reconstituted kinase reactions and mammalian cell extracts
Theillet, F. X., Rose, H. M., Liokatis, S., Binolfi, A., Thongwichian, R., Stuiver, M.; Selenko, P.
Nat Protoc, 8:1416-1432

Tags: In-Cell NMR (Selenko)

Abstract: We outline NMR protocols for site-specific mapping and time-resolved monitoring of protein phosphorylation reactions using purified kinases and mammalian cell extracts. These approaches are particularly amenable to intrinsically disordered proteins and unfolded, regulatory protein domains. We present examples for the (1)(5)N isotope-labeled N-terminal transactivation domain of human p53, which is either sequentially reacted with recombinant enzymes or directly added to mammalian cell extracts and phosphorylated by endogenous kinases. Phosphorylation reactions with purified enzymes are set up in minutes, whereas NMR samples in cell extracts are prepared within 1 h. Time-resolved NMR measurements are performed over minutes to hours depending on the activities of the probed kinases. Phosphorylation is quantitatively monitored with consecutive 2D (1)H-(1)(5)N band-selective optimized-flip-angle short-transient (SOFAST)-heteronuclear multiple-quantum (HMQC) NMR experiments, which provide atomic-resolution insights into the phosphorylation levels of individual substrate residues and time-dependent changes thereof, thereby offering unique advantages over western blotting and mass spectrometry.

Alzheimer's Disease: Identification and Development of -Secretase (BACE-1) Binding Fragments and Inhibitors by Dynamic Ligation Screening (DLS)
Fernandez-Bachiller, M. I., Horatscheck, A., Lisurek, M.; Rademann, J.
Chemmedchem, 8:1041-1056

Tags: Medicinal Chemistry (Rademann)

Abstract: The application of dynamic ligation screening (DLS), a methodology for fragment-based drug discovery (FBDD), to the aspartic protease -secretase (BACE-1) is reported. For this purpose, three new fluorescence resonance energy transfer (FRET) substrates were designed and synthesized. Their kinetic parameters (Vmax, KM, and kcat) were determined and compared with a commercial substrate. Secondly, a peptide aldehyde was designed as a chemically reactive inhibitor (CRI) based on the Swedish mutation substrate sequence. Incubation of this CRI with the protease, a FRET substrate, and one amine per well taken from an amine library, which was assembled by a maximum common substructure (MCS) approach, revealed the fragment 3-(3-aminophenyl)-2H-chromen-2-one (1) to be a competitive BACE-1 inhibitor that enhanced the activity of the CRI. Irreversibly formed fragment combination products of 1 with the initial peptide sequence were active and confirmed the targeting of the active site through the ethane-1,2-diamine isostere. Finally, structure-assisted combination of fragment 1 with secondary fragments that target the S1 site in hit optimization yielded novel, entirely fragment-based BACE-1 inhibitors with up to 30-fold improved binding affinity. Interactions with the protein were explained by molecular modeling studies, which indicate that the new fragment combinations interact with the catalytic aspartic acid dyad, as well as with the adjacent binding sites required for potency.

Common gating of both CLC transporter subunits underlies voltage-dependent activation of the 2Cl-/1H+ exchanger ClC-7/Ostm1
Ludwig, C. F., Ullrich, F., Leisle, L., Stauber, T.; Jentsch, T. J.
J Biol Chem, 288:28611-28619

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: CLC anion transporters form dimers that function either as Cl(-) channels or as electrogenic Cl(-)/H(+) exchangers. CLC channels display two different types of "gates," "protopore" gates that open and close the two pores of a CLC dimer independently of each other and common gates that act on both pores simultaneously. ClC-7/Ostm1 is a lysosomal 2Cl(-)/1H(+) exchanger that is slowly activated by depolarization. This gating process is drastically accelerated by many CLCN7 mutations underlying human osteopetrosis. Making use of some of these mutants, we now investigate whether slow voltage activation of plasma membrane-targeted ClC-7/Ostm1 involves protopore or common gates. Voltage activation of wild-type ClC-7 subunits was accelerated by co-expressing an excess of ClC-7 subunits carrying an accelerating mutation together with a point mutation rendering these subunits transport-deficient. Conversely, voltage activation of a fast ClC-7 mutant could be slowed by co-expressing an excess of a transport-deficient mutant. These effects did not depend on whether the accelerating mutation localized to the transmembrane part or to cytoplasmic cystathionine-beta-synthase (CBS) domains of ClC-7. Combining accelerating mutations in the same subunit did not speed up gating further. No currents were observed when ClC-7 was truncated after the last intramembrane helix. Currents and slow gating were restored when the C terminus was co-expressed by itself or fused to the C terminus of the beta-subunit Ostm1. We conclude that common gating underlies the slow voltage activation of ClC-7. It depends on the CBS domain-containing C terminus that does not require covalent binding to the membrane domain of ClC-7.

Human mast cell line-1 (HMC-1) cells exhibit a membrane capacitance increase when dialysed with high free-Ca(2+) and GTPgammaS containing intracellular solution
Balletta(*), A., Lorenz, D., Rummel(*), A., Gerhard(*), R., Bigalke(*), H.; Wegner(*), F.
Eur J Pharmacol, 720:227-236

Tags: Cellular Imaging (Wiesner)

Abstract: An increase in cytosolic free calcium concentration [Ca(2+)]i initiates the exocytotic activity in various types of secretory cells. The guanosine 5'-O-[3-thio]triphosphate (GTPgammaS), a non-hydrolysable analogue of GTP (guanosine 5'-triphosphate), is an effective secretagogue for different cell types of different species, like mast cells, neutrophils or eosinophils. Consequently, the internal administration of GTPgammaS causes degranulation of mouse and rat mast cells. Regarding rat mast cells, it is proved that Ca(2+) can cooperate with GTP or GTPgammaS in accelerating and increasing amplitude of the secretory response. All the previous studies with respect to capacitance recordings and mast cells were performed using mouse or rat mast cells, usually derived from peritoneum or the rat basophilic leukaemia cell line RBL. In this study, we applied the capacitance measurement technique to the human mast cell line-1 (HMC-1) cells, an immature cell line established from a patient with mast cell leukaemia. Patch-clamp dialysis experiments revealed that high intracellular free Ca(2+) and GTPgammaS concentrations are both required for considerable capacitance increases in HMC-1 cells. During degranulation of HMC-1 cells, the total membrane capacitance (Cm) increase appeared continuously and, in some cases, as a discrete capacitance change, developing in a stepwise manner. Then, we tested the effect of latrunculin B upon HMC-1 cell capacitance increase as well as of some classic mast cell stimulators like PMA, A23187 and IL-1beta in hexosaminidase release. Finally, we could conclude that the HMC-1 cell line represents a suitable model for the study of human mast cell degranulation.

A conformational intermediate in glutamate receptor activation
Lau(*), A. Y., Salazar, H., Blachowicz(*), L., Ghisi, V., Plested, A. J.; Roux(*), B.
Neuron, 79:492-503

Tags: Molecular Neuroscience and Biophysics (Plested)

Abstract: Ionotropic glutamate receptors (iGluRs) transduce the chemical signal of neurotransmitter release into membrane depolarization at excitatory synapses in the brain. The opening of the transmembrane ion channel of these ligand-gated receptors is driven by conformational transitions that are induced by the association of glutamate molecules to the ligand-binding domains (LBDs). Here, we describe the crystal structure of a GluA2 LBD tetramer in a configuration that involves an approximately 30 degrees rotation of the LBD dimers relative to the crystal structure of the full-length receptor. The configuration is stabilized by an engineered disulfide crosslink. Biochemical and electrophysiological studies on full-length receptors incorporating either this crosslink or an engineered metal bridge show that this LBD configuration corresponds to an intermediate state of receptor activation. GluA2 activation therefore involves a combination of both intra-LBD (cleft closure) and inter-LBD dimer conformational transitions. Overall, these results provide a comprehensive structural characterization of an iGluR intermediate state.

A Well-Defined Pd Hybrid Material for the Z-Selective Semihydrogenation of Alkynes Characterized at the Molecular Level by DNP SENS
Conley(*), M. P., Drost(*), R. M., Baffert(*), M., Gajan(*), D., Elsevier(*), C., Franks, W. T., Oschkinat, H., Veyre(*), L., Zagdoun(*), A., Rossini(*), A., Lelli(*), M., Lesage(*), A., Casano(*), G., Ouari(*), O., Tordo(*), P., Emsley(*), L., Coperet(*), C.; Thieuleux(*), C.
Chem-Eur J, 19:12234-12238

Tags: NMR-Supported Structural Biology (Oschkinat)

Improved Dynamic Nuclear Polarization Surface-Enhanced NMR Spectroscopy through Controlled Incorporation of Deuterated Functional Groups
Zagdoun(*), A., Rossini(*), A. J., Conley(*), M. P., Grüning(*), W. R., Schwarzwälder(*), M., Lelli(*), M., Franks, W. T., Oschkinat, H., Coperet(*), C., Emsley(*), L.; Lesage(*), A.
Angew Chem Int Edit, 52:1222-1225

Tags: NMR-Supported Structural Biology (Oschkinat)

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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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