FMP Publications

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

Year:  
All :: 2010, 2011, 2012, 2013, ... , 2017
Author:  
All :: (, A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T, U, V, W, X, Y, Z 
Preferences: 
References per page: Show keywords Show abstracts
References
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
(2010)

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
(2010)

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.

Quantification of protein backbone hydrogen-deuterium exchange rates by solid state NMR spectroscopy
del Amo, J. M., Fink, U.; Reif, B.
J Biomol NMR, 48:203-212
(2010)

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: We present the quantification of backbone amide hydrogen-deuterium exchange rates (HDX) for immobilized proteins. The experiments make use of the deuterium isotope effect on the amide nitrogen chemical shift, as well as on proton dilution by deuteration. We find that backbone amides in the microcrystalline alpha-spectrin SH3 domain exchange rather slowly with the solvent (with exchange rates negligible within the individual (15)N-T (1) timescales). We observed chemical exchange for 6 residues with HDX exchange rates in the range from 0.2 to 5 s(-1). Backbone amide (15)N longitudinal relaxation times that we determined previously are not significantly affected for most residues, yielding no systematic artifacts upon quantification of backbone dynamics (Chevelkov et al. 2008b). Significant exchange was observed for the backbone amides of R21, S36 and K60, as well as for the sidechain amides of N38, N35 and for W41epsilon. These residues could not be fit in our previous motional analysis, demonstrating that amide proton chemical exchange needs to be considered in the analysis of protein dynamics in the solid-state, in case D(2)O is employed as a solvent for sample preparation. Due to the intrinsically long (15)N relaxation times in the solid-state, the approach proposed here can expand the range of accessible HDX rates in the intermediate regime that is not accessible so far with exchange quench and MEXICO type experiments.

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
(2010)

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
(2010)

Tags: Protein Structure (Oschkinat)

Protein folding in membranes
Fiedler, S., Broecker, J.; Keller, S.
Cellular and molecular life sciences : CMLS, 67:1779-1798
(2010)

Tags: Biophysics of Membrane Proteins (Keller)

Abstract: Separation of cells and organelles by bilayer membranes is a fundamental principle of life. Cellular membranes contain a baffling variety of proteins, which fulfil vital functions as receptors and signal transducers, channels and transporters, motors and anchors. The vast majority of membrane-bound proteins contain bundles of alpha-helical transmembrane domains. Understanding how these proteins adopt their native, biologically active structures in the complex milieu of a membrane is therefore a major challenge in today's life sciences. Here, we review recent progress in the folding, unfolding and refolding of alpha-helical membrane proteins and compare the molecular interactions that stabilise proteins in lipid bilayers. We also provide a critical discussion of a detergent denaturation assay that is increasingly used to determine membrane-protein stability but is not devoid of conceptual difficulties.

Bidirectional binding of invariant chain peptides to an MHC class II molecule
Günther, S., Schlundt, A., Sticht, J., Roske(*), Y., Heinemann(*), U., Wiesmüller(*), K. H., Jung(*), G., Falk(*), K., Rötzschke(*), O.; Freund, C.
Proc Natl Acad Sci U S A, 107:22219-22224
(2010)

Tags: Protein Engineering (Freund)

Abstract: T-cell recognition of peptides bound to MHC class II (MHCII) molecules is a central event in cell-mediated adaptive immunity. The current paradigm holds that prebound class II-associated invariant chain peptides (CLIP) and all subsequent antigens maintain a canonical orientation in the MHCII binding groove. Here we provide evidence for MHCII-bound CLIP inversion. NMR spectroscopy demonstrates that the interconversion from the canonical to the inverse alignment is a dynamic process, and X-ray crystallography shows that conserved MHC residues form a hydrogen bond network with the peptide backbone in both orientations. The natural catalyst HLA-DM accelerates peptide reorientation and the exchange of either canonically or inversely bound CLIP against antigenic peptide. Thus, noncanonical MHC-CLIP displays the hallmarks of a structurally and functionally intact antigen-presenting complex.

Addressing protein-protein interactions with small molecules: a Pro-Pro dipeptide mimic with a PPII helix conformation as a module for the synthesis of PRD-binding ligands
Zaminer(*), J., Brockmann, C., Huy(*), P., Opitz, R., Reuter(*), C., Beyermann, M., Freund, C., Müller, M., Oschkinat, H., Kühne, R.; Schmalz(*), H. G.
Angew Chem Int Ed Engl, 49:7111-7115
(2010)

Tags: Computational Chemistry/Drug Design (Kühne), Protein Structure (Oschkinat), Peptide Synthesis (Beyermann)

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
(2010)

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.

Intermolecular protein-RNA interactions revealed by 2D 31P-15N magic angle spinning solid-state NMR spectroscopy
Jehle(*), S., Falb(*), M., Kirkpatrick(*), J. P., Oschkinat, H., van Rossum, B. J., Althoff(*), G.; Carlomagno(*), T.
J Am Chem Soc, 132:3842-3846
(2010)

Tags: Protein Structure (Oschkinat)

Abstract: The structural investigation of large RNP complexes by X-ray crystallography can be a difficult task due to the flexibility of the RNA and of the protein-RNA interfaces, which may hinder crystallization. In these cases, NMR spectroscopy is an attractive alternative to crystallography, although the large size of typical RNP complexes may limit the applicability of solution NMR. Solid-state NMR spectroscopy, however, is not subject to any intrinsic limitations with respect to the size of the object under investigation, with restrictions imposed solely by the sensitivity of the instrumentation. In addition, it does not require large, well-ordered crystals and can therefore be applied to flexible, partially disordered complexes. Here we show for the first time that solid-state NMR spectroscopy can be used to probe intermolecular interactions at the protein-RNA interface in RNP complexes. Distances between the (15)N nuclei of the protein backbone and the (31)P nuclei of the RNA backbone can be measured in TEDOR experiments and used as restraints in structure calculations. The distance measurement is accurate, as proven for the test case of the L7Ae-box C/D RNA complex, for which a crystal structure is available. The results presented here reveal the as yet unexplored potential of solid-state NMR spectroscopy in the investigation of large RNP complexes.

Page:  
Previous | 1, 2, 3 | Next
Export as:
BibTeX, XML

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)
info(at)fmp-berlin.de

Like many sites, we use cookies to optimize the user's browsing experience. Data Protection OK