FMP Publications

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

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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

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.

Unraveling the existence of dynamic water channels in light-harvesting proteins: alpha-C-phycocyanobilin in vitro
Elgabarty(*), H., Schmieder, P.; Sebastiani(*), D.
Chem Sci, 4:755-763

Tags: Solution NMR (Schmieder)

Abstract: We present hybrid ab initio QM/MM MD simulations and theoretical NMR chemical shift calculations of the bilin chromophore phycocyanobilin (PCB) in the binding pocket of the alpha-subunit of C-phycocyanin (alpha-C-PC). The good overall agreement between the computed NMR chemical shifts and the experimental values confirm the overall structural picture. A particular discrepancy is observed for the pyrrole nitrogen and hydrogen on ring A, which points to a disagreement between the reported X-ray structure and the experimental solution-state NMR spectrum. Our results suggest that in the solution-state, the binding pocket of alpha-C-PC slightly opens up allowing one water molecule to form a stable bridge between ring A in PCB and the protein backbone at the ASN73 residue. With this modified solution-state structure, the computed NMR chemical shifts are in excellent agreement with experimental values. For proteins still lacking a fully-resolved solution-state NMR-based structure, this approach of combining ab initio MD/NMR provides a very sensitive probe for local geometries at the sub-angstrom ngstrom range that can be utilized to compare/reconcile simple experimental one-and two-dimensional NMR data with X-ray structures.

The Clip-Segment of the von Willebrand Domain 1 of the BMP Modulator Protein Crossveinless 2 Is Preformed
Fiebig(*), J. E., Weidauer(*), S. E., Qiu(*), L. Y., Bauer(*), M., Schmieder, P., Beerbaum, M., Zhang(*), J. L., Oschkinat, H., Sebald(*), W.; Mueller(*), T. D.
Molecules, 18:11658-11682

Tags: NMR-Supported Structural Biology (Oschkinat), Solution NMR (Schmieder)

Abstract: Bone Morphogenetic Proteins (BMPs) are secreted protein hormones that act as morphogens and exert essential roles during embryonic development of tissues and organs. Signaling by BMPs occurs via hetero-oligomerization of two types of serine/threonine kinase transmembrane receptors. Due to the small number of available receptors for a large number of BMP ligands ligand-receptor promiscuity presents an evident problem requiring additional regulatory mechanisms for ligand-specific signaling. Such additional regulation is achieved through a plethora of extracellular antagonists, among them members of the Chordin superfamily, that modulate BMP signaling activity by binding. The key-element in Chordin-related antagonists for interacting with BMPs is the von Willebrand type C (VWC) module, which is a small domain of about 50 to 60 residues occurring in many different proteins. Although a structure of the VWC domain of the Chordin-member Crossveinless 2 (CV2) bound to BMP-2 has been determined by X-ray crystallography, the molecular mechanism by which the VWC domain binds BMPs has remained unclear. Here we present the NMR structure of the Danio rerio CV2 VWC1 domain in its unbound state showing that the key features for high affinity binding to BMP-2 is a pre-oriented peptide loop.

Efficient alpha-helix induction in a linear peptide chain by N-capping with a bridged-tricyclic diproline analogue
Hack(*), V., Reuter(*), C., Opitz, R., Schmieder, P., Beyermann, M., Neudörfl(*), J. M., Kühne, R.; Schmalz(*), H. G.
Angew Chem Int Ed Engl, 52:9539-9543

Tags: Solution NMR (Schmieder), Peptide Synthesis (Beyermann), Computational Chemistry/Drug Design (Kühne)

Highly functionalized terpyridines as competitive inhibitors of AKAP-PKA interactions
Schäfer(*), G., Milic(*), J., Eldahshan, A., Götz(*), F., Zühlke(*), K., Schillinger, C., Kreuchwig, A., Elkins(*), J. M., Abdul Azeez(*), K. R., Oder(*), A., Moutty(*), M. C., Masada(*), N., Beerbaum, M., Schlegel, B., Niquet(*), S., Schmieder, P., Krause, G., von Kries, J. P., Cooper(*), D. M., Knapp(*), S., Rademann, J., Rosenthal(*), W.; Klussmann(*), E.
Angew Chem Int Ed Engl, 52:12187-12191

Tags: Medicinal Chemistry (Rademann), Screening Unit (von Kries), Solution NMR (Schmieder)

Determination of glucan phosphorylation using heteronuclear 1H, 13C double and 1H, 13C, 31P triple-resonance NMR spectra
Schmieder, P., Nitschke(*), F., Steup(*), M., Mallow, K.; Specker, E.
Magn Reson Chem, 51:655-661

Tags: Solution NMR (Schmieder), Screening Unit (von Kries)

Abstract: Phosphorylation and dephosphorylation of starch and glycogen are important for their physicochemical properties and also their physiological functions. It is therefore desirable to reliably determine the phosphorylation sites. Heteronuclear multidimensional NMR-spectroscopy is in principle a straightforward analytical approach even for complex carbohydrate molecules. With heterogeneous samples from natural sources, however, the task becomes more difficult because a full assignment of the resonances of the carbohydrates is impossible to obtain. Here, we show that the combination of heteronuclear (1) H,(13) C and (1) H,(13) C,(31) P techniques and information derived from spectra of a set of reference compounds can lead to an unambiguous determination of the phosphorylation sites even in heterogeneous samples.

What's in a name? Why these proteins are intrinsically disordered: Why these proteins are intrinsically disordered
Dunker(*), A. K., Babu(*), M. M., Barbar(*), E., Blackledge(*), M., Bondos(*), S. E., Dosztanyi(*), Z., Dyson(*), H. J., Forman-Kay(*), J., Fuxreiter(*), M., Gsponer(*), J., Han(*), K. H., Jones(*), D. T., Longhi(*), S., Metallo(*), S. J., Nishikawa(*), K., Nussinov(*), R., Obradovic(*), Z., Pappu(*), R. V., Rost(*), B., Selenko, P., Subramaniam(*), V., Sussman(*), J. L., Tompa(*), P.; Uversky(*), V. N.
Intrinsically disordered proteins, 1:e24157

Tags: In-Cell NMR (Selenko)

Abstract: "What's in a name? That which we call a rose By any other name would smell as sweet." From "Romeo and Juliet", William Shakespeare (1594) This article opens a series of publications on disambiguation of the basic terms used in the field of intrinsically disordered proteins. We start from the beginning, namely from the explanation of what the expression "intrinsically disordered protein" actually means and why this particular term has been chosen as the common denominator for this class of proteins characterized by broad structural, dynamic and functional characteristics.

Quantitative NMR analysis of Erk activity and inhibition by U0126 in a panel of patient-derived colorectal cancer cell lines
Rose, H. M., Stuiver, M., Thongwichian, R., Theillet, F. X., Feller(*), S. M.; Selenko, P.
Biochim Biophys Acta, 1834:1396-1401

Tags: In-Cell NMR (Selenko)

Abstract: We comparatively analyzed the basal activity of extra-cellular signal-regulated kinase (Erk1/2) in lysates of 10 human colorectal cancer cell lines by semi-quantitative Western blotting and time-resolved NMR spectroscopy. Both methods revealed heterogeneous levels of endogenous Erk1/2 activities in a highly consistent manner. Upon treatment with U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK) acting upstream of Erk1/2, Western-blotting and NMR congruently reported specific modulations of cellular phospho-Erk levels that translated into reduced kinase activities. Results obtained in this study highlight the complementary nature of antibody- and NMR-based phospho-detection techniques. They further exemplify the usefulness of time-resolved NMR measurements in providing fast and quantitative readouts of kinase activities and kinase inhibitor efficacies in native cellular environments. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).

The alphabet of intrinsic disorder: I. Act like a Pro: On the abundance and roles of proline residues in intrinsically disordered proteins
Theillet, F. X., Kalmar(*), L., Tompa(*), P., Han(*), K. H., Selenko, P., Dunker(*), A. K., Daughdrill(*), G. W.; Uversky(*), V. N.
Intrinsically disordered proteins, 1:e24360

Tags: In-Cell NMR (Selenko)

Abstract: A significant fraction of every proteome is occupied by biologically active proteins that do not form unique three-dimensional structures. These intrinsically disordered proteins (IDPs) and IDP regions (IDPRs) have essential biological functions and are characterized by extensive structural plasticity. Such structural and functional behavior is encoded in the amino acid sequences of IDPs/IDPRs, which are enriched in disorder-promoting residues and depleted in order-promoting residues. In fact, amino acid residues can be arranged according to their disorder-promoting tendency to form an alphabet of intrinsic disorder that defines the structural complexity and diversity of IDPs/IDPRs. This review is the first in a series of publications dedicated to the roles that different amino acid residues play in defining the phenomenon of protein intrinsic disorder. We start with proline because data suggests that of the 20 common amino acid residues, this one is the most disorder-promoting.

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.

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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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