FMP Publications

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

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Narrow carbonyl resonances in proton-diluted proteins facilitate NMR assignments in the solid-state
Linser, R., Fink, U.; Reif, B.
J. Biomol. NMR, 47:1-6

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: HNCO/HNCACO type correlation experiments are an alternative for assignment of backbone resonances in extensively deuterated proteins in the solid-state, given the fact that line widths on the order of 14-17 Hz are achieved in the carbonyl dimension without the need of high power decoupling. The achieved resolution demonstrates that MAS solid-state NMR on extensively deuterated proteins is able to compete with solution-state NMR spectroscopy if proteins are investigated with correlation times tau (c) that exceed 25 ns.

Assignment of dynamic regions in biological solids enabled by spin-state selective NMR experiments
Linser, R., Fink, U.; Reif, B.
J Am Chem Soc, 132:8891-8893

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: Structural investigations are a prerequisite to understand protein function. Intermediate time scale motional processes (ns-micros) are deleterious for NMR of biological solids and obscure the detection of amide moieties in traditional CP based solid-state NMR approaches as well as in regular scalar coupling based experiments. We show that this obstacle can be overcome by using TROSY type techniques in triple resonance experiments, which enable the assignment of resonances in loop regions of a microcrystalline protein. The presented approach provides an exemplified solution for the analysis of secondary structure elements undergoing slow dynamics that might be particularly crucial for understanding protein function.

Comparison of solid-state dipolar couplings and solution relaxation data provides insight into protein backbone dynamics
Chevelkov, V., Xue(*), Y., Linser, R., Skrynnikov(*), N. R.; Reif, B.
J Am Chem Soc, 132:5015-5017

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: Analyses of solution (15)N relaxation data and solid-state (1)H(N)-(15)N dipolar couplings from a small globular protein, alpha-spectrin SH3 domain, produce a surprisingly similar pattern of order parameters. This result suggests that there is little or no ns-mus dynamics throughout most of the sequence and, in particular, in the structured portion of the backbone. At the same time, evidence of ns-mus motions is found in the flexible loops and termini. These findings, corroborated by the MD simulations of alpha-spectrin SH3 in a hydrated crystalline environment and in solution, are consistent with the picture of protein dynamics that has recently emerged from the solution studies employing residual dipolar couplings.

Optimum levels of exchangeable protons in perdeuterated proteins for proton detection in MAS solid-state NMR spectroscopy
Akbey, Ü., Lange, S., Trent Franks, W., Linser, R., Rehbein, K., Diehl, A., van Rossum, B. J., Reif, B.; Oschkinat, H.
J Biomol NMR, 46:67-73

Tags: Protein Structure (Oschkinat), Solid-State NMR Spectroscopy (Reif)

Abstract: We present a systematic study of the effect of the level of exchangeable protons on the observed amide proton linewidth obtained in perdeuterated proteins. Decreasing the amount of D(2)O employed in the crystallization buffer from 90 to 0%, we observe a fourfold increase in linewidth for both (1)H and (15)N resonances. At the same time, we find a gradual increase in the signal-to-noise ratio (SNR) for (1)H-(15)N correlations in dipolar coupling based experiments for H(2)O concentrations of up to 40%. Beyond 40%, a significant reduction in SNR is observed. Scalar-coupling based (1)H-(15)N correlation experiments yield a nearly constant SNR for samples prepared with < or =30% H(2)O. Samples in which more H(2)O is employed for crystallization show a significantly reduced NMR intensity. Calculation of the SNR by taking into account the reduction in (1)H T (1) in samples containing more protons (SNR per unit time), yields a maximum SNR for samples crystallized using 30 and 40% H(2)O for scalar and dipolar coupling based experiments, respectively. A sensitivity gain of 3.8 is obtained by increasing the H(2)O concentration from 10 to 40% in the CP based experiment, whereas the linewidth only becomes 1.5 times broader. In general, we find that CP is more favorable compared to INEPT based transfer when the number of possible (1)H,(1)H interactions increases. At low levels of deuteration (> or =60% H(2)O in the crystallization buffer), resonances from rigid residues are broadened beyond detection. All experiments are carried out at MAS frequency of 24 kHz employing perdeuterated samples of the chicken alpha-spectrin SH3 domain.

Identification of hydroxyl protons, determination of their exchange dynamics, and characterization of hydrogen bonding in a microcrystallin protein
Agarwal, V., Linser, R., Fink, U., Faelber, K.; Reif, B.
J Am Chem Soc, 132:3187-3195

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: Heteronuclear correlation experiments employing perdeuterated proteins enable the observation of all hydroxyl protons in a microcrystalline protein by MAS solid-state NMR. Dipolar-based sequences allow magnetization transfers that are >50 times faster compared to scalar-coupling-based sequences, which significantly facilitates their assignment. Hydroxyl exchange rates were measured using EXSY-type experiments. We find a biexponential decay behavior for those hydroxyl groups that are involved in side chain-side chain C-O-H...O horizontal lineC hydrogen bonds. The quantification of the distances between the hydroxyl proton and the carbon atoms in the hydrogen-bonding donor as well as acceptor group is achieved via a REDOR experiment. In combination with X-ray data and isotropic proton chemical shifts, availability of (1)H,(13)C distance information can aid in the quantitative description of the geometry of these hydrogen bonds. Similarly, correlations between backbone amide proton and carbonyl atoms are observed, which will be useful in the analysis of the registry of beta-strand arrangement in amyloid fibrils.

Dynamic nuclear polarization of deuterated proteins
Akbey, Ü., Franks, W. T., Linden, A., Lange, S., Griffin(*), R. G., van Rossum, B. J.; Oschkinat, H.
Angew Chem Int Ed Engl, 49:7803-7806

Tags: Protein Structure (Oschkinat)

Adhesion and degranulation promoting adapter protein (ADAP) is a central hub for phosphotyrosine-mediated interactions in T cells
Sylvester, M., Kliche(*), S., Lange, S., Geithner(*), S., Klemm, C., Schlosser(*), A., Grossmann(*), A., Stelzl(*), U., Schraven(*), B., Krause, E.; Freund, C.
Plos One, 5:e11708

Tags: Protein Engeneering (Freund), Mass Spectrometry (Krause, E.)

Abstract: TCR stimulation leads to an increase in cellular adhesion among other outcomes. The adhesion and degranulation promoting adapter protein (ADAP) is known to be rapidly phosphorylated after T cell stimulation and relays the TCR signal to adhesion molecules of the integrin family. While three tyrosine phosphorylation sites have been characterized biochemically, the binding capabilities and associated functions of several other potential phosphotyrosine motifs remain unclear. Here, we utilize in vitro phosphorylation and mass spectrometry to map novel phosphotyrosine sites in the C-terminal part of human ADAP (486-783). Individual tyrosines were then mutated to phenylalanine and their relevance for cellular adhesion and migration was tested experimentally. Functionally important tyrosine residues include two sites within the folded hSH3 domains of ADAP and two at the C-terminus. Furthermore, using a peptide pulldown approach in combination with stable isotope labeling in cell culture (SILAC) we identified SLP-76, PLCgamma, PIK3R1, Nck, CRK, Gads, and RasGAP as phospho-dependent binding partners of a central YDDV motif of ADAP. The phosphorylation-dependent interaction between ADAP and Nck was confirmed by yeast two-hybrid analysis, immunoprecipitation and binary pulldown experiments, indicating that ADAP directly links integrins to modulators of the cytoskeleton independent of SLP-76.

The use of small molecule high-throughput screening to identify inhibitors of the proteinase 3-NB1 interaction
Choi(*), M., Eulenberg(*), C., Rolle(*), S., von Kries, J. P., Luft(*), F. C.; Kettritz(*), R.
Clin Exp Immunol, 161:389-396

Tags: Screening Unit (von Kries)

Abstract: P>Anti-neutrophil cytoplasmic antibodies (ANCA) to proteinase 3 (PR3) are found in patients with small-vessel vasculitis. PR3-ANCA bind strongly to membrane PR3 (mPR3) that is presented by the NB1 receptor. We performed high-throughput screening using a small molecule library to identify compounds that inhibit PR3-NB1 binding. We established a human embryonic kidney (HEK293) cell-based system, where approximately 95 +/- 2% of the NB1-transfected cells expressed the NB1 receptor on the cell surface. Addition of 0 center dot 1 mu g/ml human PR3 to 104 NB1-expressing HEK293 cells resulted in PR3 binding that was detected by immunofluorescence using a fluorescence plate reader assay. We identified 13 of 20 000 molecules that inhibited PR3 binding by > 70%. Seven of 13 substances showed reproducible inhibition in four additional validation experiments. Two selected compounds (27519 and 27549) demonstrated a dose-dependent inhibition over a range from 6 center dot 25 to 100 mu M as measured by the plate reader assay. We used flow cytometry as a second assay, and found that both compounds reproducibly inhibited PR3 binding to NB1-transfected HEK293 cells at 50 mu M (inhibition to 42 +/- 4% with compound 27519 and to 47 +/- 6% with compound 27549 compared to the dimethylsulphoxide control). Furthermore, compounds 27519 and 27549 also inhibited binding of exogenous PR3 to human neutrophils. In contrast, the compounds did not decrease mPR3 expression on resting neutrophils, but reduced the tumour necrosis factor-alpha-mediated mPR3 increase on NB1pos neutrophils when present continuously during the assay. The findings suggest that small inhibitory compounds provide a potential therapeutic tool to reduce mPR3 by preventing its binding to NB1.

Modulation of G-protein coupled receptor sample quality by modified cell-free expression protocols: A case study of the human endothelin A receptor
Junge(*), F., Luh(*), L. M., Proverbio(*), D., Schäfer(*), B., Abele(*), R., Beyermann, M., Dötsch(*), V.; Bernhard(*), F.
J Struct Biol, 172:94-106

Tags: Peptide Synthesis (Beyermann)

Abstract: G-protein coupled receptors still represent one of the most challenging targets in membrane protein research. Here we present a strategic approach for the cell-free synthesis of these complex membrane proteins exemplified by the preparative scale production of the human endothelin A receptor. The versatility of the cell-free expression system was used to modulate sample quality by alteration of detergents hence presenting different solubilization environments to the synthesized protein at different stages of the production process. Sample properties after co-translational and post-translational solubilization have been analysed by evaluation of homogeneity, protein stability and receptor ligand binding competence. This is a first quality evaluation of a membrane protein obtained in two different cell-free expression modes and we demonstrate that both can be used for the production of ligand-binding competent endothelin A receptor in quantities sufficient for structural approaches. The presented strategy of cell-free expression protocol development could serve as basic guideline for the production of related receptors in similar systems. (C) 2010 Elsevier Inc. All rights reserved.

A MAS NMR Study of the Bacterial ABC Transporter ArtMP
Lange, V., Becker-Baldus, J., Kunert, B., van Rossum, B. J., Casagrande(*), F., Engel(*), A., Roske(*), Y., Scheffel(*), F. M., Schneider(*), E.; Oschkinat, H.
Chembiochem, 11:547-555

Tags: Protein Structure (Oschkinat)

Abstract: ATP-binding cassette (ABC) transport systems facilitate the translocation of substances, like amino acids, across cell membranes energised by ATP hydrolysis. This work describes first structural studies on the ABC transporter ArtMP from Geobacillus stearothermophilus in native lipid environment by magic-angle spinning NMR spectroscopy. The 2D crystals of ArtMP and 3D crystals of isolated ArtP were prepared in different nucleotide-bound or -unbound states. From selectively C-13,N-15-labelled ArtP, several sequence-specific assignments were obtained, most of which could be transferred to spectra of ArtMP. Residues Tyr133 and Pro134 protrude directly into the ATP-binding pocket at the interface of the ArtP subunits, and hence, are sensitive monitors for structural changes during nucleotide binding and hydrolysis. Distinct sets of NMR shifts were obtained for ArtP with different phosphorylation states of the ligand. Indications were found for an asymmetric or inhomogeneous state of the ArtP dimer bound with triphosphorylated nucleotides. With this investigation, a model system was established for screening all functional states occurring in one ABC transporter in native lipid environment.

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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 
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