FMP Publications

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

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Site-specific analysis of heteronuclear Overhauser effects in microcrystalline proteins
del Amo, J. M. L., Agarwal, V., Sarkar(*), R., Porter(*), J., Asami(*), S., Rubbelke(*), M., Fink, U., Xue(*), Y., Lange(*), O. F.; Reif, B.
J. Biomol. NMR, 59:241-249

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: Relaxation parameters such as longitudinal relaxation are susceptible to artifacts such as spin diffusion, and can be affected by paramagnetic impurities as e.g. oxygen, which make a quantitative interpretation difficult. We present here the site-specific measurement of [H-1]C-13 and [H-1]N-15 heteronuclear rates in an immobilized protein. For methyls, a strong effect is expected due to the three-fold rotation of the methyl group. Quantification of the [H-1]C-13 heteronuclear NOE in combination with C-13-R (1) can yield a more accurate analysis of side chain motional parameters. The observation of significant [H-1]N-15 heteronuclear NOEs for certain backbone amides, as well as for specific asparagine/glutamine sidechain amides is consistent with MD simulations. The measurement of site-specific heteronuclear NOEs is enabled by the use of highly deuterated microcrystalline protein samples in which spin diffusion is reduced in comparison to protonated samples.

Rapid proton-detected NMR assignment for proteins with fast magic angle spinning
Barbet-Massin(*), E., Pell(*), A. J., Retel, J. S., Andreas(*), L. B., Jaudzems(*), K., Franks, W. T., Nieuwkoop, A. J., Hiller, M., Higman(*), V., Guerry(*), P., Bertarello(*), A., Knight(*), M. J., Felletti(*), M., Le Marchand(*), T., Kotelovica(*), S., Akopjana(*), I., Tars(*), K., Stoppini(*), M., Bellotti(*), V., Bolognesi(*), M., Ricagno(*), S., Chou(*), J. J., Griffin(*), R. G., Oschkinat, H., Lesage(*), A., Emsley(*), L., Herrmann(*), T.; Pintacuda(*), G.
J Am Chem Soc, 136:12489-12497

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Using a set of six (1)H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5-30 kDa proteins. The approach relies on perdeuteration, amide (2)H/(1)H exchange, high magnetic fields, and high-spinning frequencies (omegar/2pi >/= 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems. In comparison to contemporary (13)C/(15)N-based methods, this approach facilitates and accelerates the MAS NMR assignment process, shortening the spectral acquisition times and enabling the use of unsupervised state-of-the-art computational data analysis protocols originally developed for solution NMR.

Cell tracking with caged xenon: using cryptophanes as MRI reporters upon cellular internalization
Klippel, S., Döpfert, J., Jayapaul, J., Kunth, M., Rossella, F., Schnurr, M., Witte, C., Freund, C.; Schröder, L.
Angew Chem Int Ed Engl, 53:493-496

Tags: Molecular Imaging (Schröder)

Abstract: Caged xenon has great potential in overcoming sensitivity limitations for solution-state NMR detection of dilute molecules. However, no application of such a system as a magnetic resonance imaging (MRI) contrast agent has yet been performed with live cells. We demonstrate MRI localization of cells labeled with caged xenon in a packed-bed bioreactor working under perfusion with hyperpolarized-xenon-saturated medium. Xenon hosts enable NMR/MRI experiments with switchable contrast and selectivity for cell-associated versus unbound cages. We present MR images with 10(3) -fold sensitivity enhancement for cell-internalized, dual-mode (fluorescence/MRI) xenon hosts at low micromolar concentrations. Our results illustrate the capability of functionalized xenon to act as a highly sensitive cell tracer for MRI detection even without signal averaging. The method will bridge the challenging gap for translation to in vivo studies for the optimization of targeted biosensors and their multiplexing applications.

The specific monomer/dimer equilibrium of the corticotropin-releasing factor receptor type 1 is established in the endoplasmic reticulum
Teichmann, A., Gibert, A., Lampe, A., Grzesik, P., Rutz, C., Furkert, J., Schmoranzer, J., Krause, G., Wiesner, B.; Schülein, R.
J Biol Chem, 289:24250-24262

Tags: Protein Trafficking (Schülein), Cellular Imaging (Wiesner), Structural Bioinformatics and Protein Design (Krause, G.), Molecular Pharmacology and Cell Biology (Haucke)

Abstract: G protein-coupled receptors (GPCRs) represent the most important drug targets. Although the smallest functional unit of a GPCR is a monomer, it became clear in the past decades that the vast majority of the receptors form dimers. Only very recently, however, data were presented that some receptors may in fact be expressed as a mixture of monomers and dimers and that the interaction of the receptor protomers is dynamic. To date, equilibrium measurements were restricted to the plasma membrane due to experimental limitations. We have addressed the question as to where this equilibrium is established for the corticotropin-releasing factor receptor type 1. By developing a novel approach to analyze single molecule fluorescence cross-correlation spectroscopy data for intracellular membrane compartments, we show that the corticotropin-releasing factor receptor type 1 has a specific monomer/dimer equilibrium that is already established in the endoplasmic reticulum (ER). It remains constant at the plasma membrane even following receptor activation. Moreover, we demonstrate for seven additional GPCRs that they are expressed in specific but substantially different monomer/dimer ratios. Although it is well known that proteins may dimerize in the ER in principle, our data show that the ER is also able to establish the specific monomer/dimer ratios of GPCRs, which sheds new light on the functions of this compartment.

AhR sensing of bacterial pigments regulates antibacterial defence
Moura-Alves(*), P., Fae(*), K., Houthuys(*), E., Dorhoi(*), A., Kreuchwig, A., Furkert, J., Barison(*), N., Diehl, A., Munder(*), A., Constant, P., Skrahina(*), T., Guhlich-Bornhof(*), U., Klemm(*), M., Koehler(*), A. B., Bandermann(*), S., Goosmann(*), C., Mollenkopf(*), H. J., Hurwitz(*), R., Brinkmann(*), V., Fillatreau(*), S., Daffe(*), M., Tummler, B., Kolbe(*), M., Oschkinat, H., Krause, G.; Kaufmann(*), S. H.
Nature, 512:387-392

Tags: Structural Bioinformatics and Protein Design (Krause, G.), NMR-Supported Structural Biology (Oschkinat), Protein Trafficking (Schülein)

Abstract: The aryl hydrocarbon receptor (AhR) is a highly conserved ligand-dependent transcription factor that senses environmental toxins and endogenous ligands, thereby inducing detoxifying enzymes and modulating immune cell differentiation and responses. We hypothesized that AhR evolved to sense not only environmental pollutants but also microbial insults. We characterized bacterial pigmented virulence factors, namely the phenazines from Pseudomonas aeruginosa and the naphthoquinone phthiocol from Mycobacterium tuberculosis, as ligands of AhR. Upon ligand binding, AhR activation leads to virulence factor degradation and regulated cytokine and chemokine production. The relevance of AhR to host defence is underlined by heightened susceptibility of AhR-deficient mice to both P. aeruginosa and M. tuberculosis. Thus, we demonstrate that AhR senses distinct bacterial virulence factors and controls antibacterial responses, supporting a previously unidentified role for AhR as an intracellular pattern recognition receptor, and identify bacterial pigments as a new class of pathogen-associated molecular patterns.

Differences between lutropin-mediated and choriogonadotropin-mediated receptor activation
Grzesik, P., Teichmann, A., Furkert, J., Rutz, C., Wiesner, B., Kleinau(*), G., Schülein, R., Gromoll(*), J.; Krause, G.
Febs J, 281:1479-1492

Tags: Structural Bioinformatics and Protein Design (Krause, G.), Protein Trafficking (Schülein), Cellular Imaging (Wiesner)

Abstract: The human lutropin/choriogonadotropin receptor (hLHR) for the gonadotropic hormones human luteinizing hormone (hLH; lutropin) and human choriogonadotropin (hCG) is crucial for normal sexual development and fertility. We aimed to unravel differences between the two hLHR hormones in molecular activation mechanisms at hLHR. We utilized a specific hLHR variant that lacks exon 10 (hLHR-delExon10), which maintains full cAMP signaling by hCG, but decreases hLH-induced receptor signaling, resulting in a pathogenic phenotype. Exon 10 encodes 27 amino acids within the hinge region, which is an extracellular segment that is important for signaling and hormone interaction. Initially, we assumed that the lack of exon 10 might disturb intermolecular trans-activation of hLH, a mechanism that has been reported for hCG at hLHR. Coexpression of signaling-deficient hLHR and binding-deficient hLHR can be used to examine the mechanisms of receptor signaling, in particular intermolecular cooperation and intramolecular cis-activation. Therefore, hLHR-delExon10 was combined with the hLHR Lys605-->Glu mutant, in which signaling is abolished, and the hLHR mutant Cys131-->Arg, in which binding is deficient. We found that hCG signaling was partially rescued, indicating trans-activation. However, the hLH signal could not be restored via forced trans-activation with any construct. Fluorescence cross-correlation spectroscopy detected oligomerization in all combinations, indicating that these functional differences cannot be explained by monomerization of hLHR-delExon10. Thus, our data demonstrate not only that the different behavior of hLH at hLHR-delExon10 is unlikely to be related to modified intermolecular receptor activation, but also that hLH may exclusively stimulate the targeted hLHR by cis-activation, whereas hCG is also capable of inducing trans-activation.

Development of an antibody-based, modular biosensor for 129Xe NMR molecular imaging of cells at nanomolar concentrations
Rose, H. M., Witte, C., Rossella, F., Klippel, S., Freund(*), C.; Schröder, L.
Proc Natl Acad Sci U S A, 111:11697-11702

Tags: Molecular Imaging (Schröder)

Abstract: Magnetic resonance imaging (MRI) is seriously limited when aiming for visualization of targeted contrast agents. Images are reconstructed from the weak diamagnetic properties of the sample and require an abundant molecule like water as the reporter. Micromolar to millimolar concentrations of conventional contrast agents are needed to generate image contrast, thus excluding many molecular markers as potential targets. To address this limitation, we developed and characterized a functional xenon NMR biosensor that can identify a specific cell surface marker by targeted (129)Xe MRI. Cells expressing the cell surface protein CD14 can be spatially distinguished from control cells with incorporation of as little as 20 nM of the xenon MRI readout unit, cryptophane-A. Cryptophane-A serves as a chemical host for hyperpolarized nuclei and facilitates the sensitivity enhancement achieved by xenon MRI. Although this paper describes the application of a CD14-specific biosensor, the construct has been designed in a versatile, modular fashion. This allows for quick and easy adaptation of the biosensor to any cell surface target for which there is a specific antibody. In addition, the modular design facilitates the creation of a multifunctional probe that incorporates readout modules for different detection methods, such as fluorescence, to complement the primary MRI readout. This modular antibody-based approach not only offers a practical technique with which to screen targets, but one which can be readily applied as the xenon MRI field moves closer to molecular imaging applications in vivo.

Multichannel MRI labeling of mammalian cells by switchable nanocarriers for hyperpolarized xenon
Klippel, S., Freund(*), C.; Schröder, L.
Nano Lett, 14:5721-5726

Tags: Molecular Imaging (Schröder)

Abstract: We demonstrate a concept for multichannel MRI cell-labeling using encapsulated laser-polarized xenon. Conceptually different Xe trapping properties of two nanocarriers, namely macrocyclic cages as individual hosts or compartmentalization into nanodroplets, ensure a large chemical shift separation for Xe bound in either of the carriers even after cellular internalization. Two differently labeled mammalian cell populations were imaged by frequency selective saturation transfer resulting in a switchable "two-color" xenon-MRI contrast at micro- to nanomolar Xe carrier concentrations.

Activation of Ligand Binding Domains of an AMPA-Type Glutamate Receptor
Baranovic, J., Chebli, M., Salazar, H. P., Faelber(*), K., Ghisi, V., Lau(*), A. Y., Daumke(*), O.; Plested, A. J. R.
Biophys. J., 106:29a-29a

Tags: Molecular Neuroscience and Biophysics (Plested)

Molecular characterization of ubiquitin-specific protease 18 reveals substrate specificity for interferon-stimulated gene 15
Basters(*), A., Geurink(*), P. P., El Oualid(*), F., Ketscher(*), L., Casutt(*), M. S., Krause, E., Ovaa(*), H., Knobeloch(*), K. P.; Fritz(*), G.
Febs J, 281:1918-1928

Tags: Mass Spectrometry (Krause, E.)

Abstract: UNLABELLED: Protein modification by interferon-stimulated gene 15 (ISG15), an ubiquitin-like modifier, affects multiple cellular functions and represents one of the major antiviral effector systems. Covalent linkage of ISG15 to proteins was previously reported to be counteracted by ubiquitin-specific protease 18 (USP18). To date, analysis of the molecular properties of USP18 was hampered by low expression yields and impaired solubility. We established high-yield expression of USP18 in insect cells and purified the protease to homogeneity. USP18 binds with high affinity to ISG15, as shown by microscale thermophoresis with a Kd of 1.3 +/- 0.2 mum. The catalytic properties of USP18 were characterized by a novel assay using ISG15 fused to a fluorophore via an isopeptide bond, giving a Km of 4.6 +/- 0.2 mum and a kcat of 0.23 +/- 0.004 s(-1) , respectively, at pH 7.5. Furthermore, the recombinant enzyme cleaves efficiently ISG15 but not ubiquitin from endogenous cellular substrates. In line with these data, USP18 exhibited neither cross-reactivity with an ubiquitin isopeptide fluorophore substrate, nor with a ubiquitin vinyl sulfone, showing that the enzyme is specific for ISG15. STRUCTURED DIGITAL ABSTRACT: ISG15 and USP18 bind by microscale thermophoresis (View interaction) USP18 cleaves ISG15 by enzymatic study (View interaction).

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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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