FMP Publications

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

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References
Semisynthesis and optimization of G protein-coupled receptor mimics
Abel, S., Geltinger, B., Heinrich, N., Michl, D., Klose, A., Beyermann, M.; Schwarzer(*), D.
J Pept Sci, 20:831-836
(2014)

Tags: Peptide Chemistry (Beyermann)

Abstract: We have recently developed a soluble mimic of the corticotropin-releasing factor receptor type 1 (CRF1), a membrane-spanning G protein-coupled receptor, which allowed investigations on receptor-ligand interactions. The CRF1 mimic consists of the receptor N-terminus and three synthetic extracellular loops (ECL1-3), which constitute the extracellular receptor domains (ECDs) of CRF1, coupled to a linear peptide template. Here, we report the synthesis of a modified CRF1 mimic, which is more similar to the native receptor possessing a cyclic template that displays the ECDs in a more physiological conformation compared with the initial linear design. In order to facilitate detailed biophysical investigations on CRF1 mimics, we have further established a cost-efficient access to the CRF1 mimic, which is suitable for isotopic labeling for NMR spectroscopy. To this end, the loop-mimicking cyclic peptide of the ECL2 of CRF1 was produced recombinantly and cyclized by expressed protein ligation. Cyclic ECL2 was obtained in milligram scale, and CRF1 mimics synthesized from this material displayed the same binding properties as synthetic CRF1 constructs.

Quadruple-resonance magic-angle spinning NMR spectroscopy of deuterated solid proteins
Akbey, Ü., Nieuwkoop, A. J., Wegner, S., Voreck, A., Kunert, B., Bandara, P., Engelke, F., Nielsen, N. C.; Oschkinat, H.
Angew Chem Int Ed Engl, 53:2438-2442
(2014)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: (1)H-detected magic-angle spinning NMR experiments facilitate structural biology of solid proteins, which requires using deuterated proteins. However, often amide protons cannot be back-exchanged sufficiently, because of a possible lack of solvent exposure. For such systems, using (2)H excitation instead of (1)H excitation can be beneficial because of the larger abundance and shorter longitudinal relaxation time, T1, of deuterium. A new structure determination approach, "quadruple-resonance NMR spectroscopy", is presented which relies on an efficient (2)H-excitation and (2)H-(13)C cross-polarization (CP) step, combined with (1)H detection. We show that by using (2)H-excited experiments better sensitivity is possible on an SH3 sample recrystallized from 30 % H2O. For a membrane protein, the ABC transporter ArtMP in native lipid bilayers, different sets of signals can be observed from different initial polarization pathways, which can be evaluated further to extract structural properties.

Interrelation between protein synthesis, proteostasis and life span
Arnsburg, K.; Kirstein-Miles, J.
Current genomics, 15:66-75
(2014)

Tags: Proteostasis in Aging and Disease (Kirstein)

Abstract: The production of newly synthesized proteins is a key process of protein homeostasis that initiates the biosynthetic flux of proteins and thereby determines the composition, stability and functionality of the proteome. Protein synthesis is highly regulated on multiple levels to adapt the proteome to environmental and physiological challenges such as aging and proteotoxic conditions. Imbalances of protein folding conditions are sensed by the cell that then trigger a cascade of signaling pathways aiming to restore the protein folding equilibrium. One regulatory node to rebalance proteostasis upon stress is the control of protein synthesis itself. Translation is reduced as an immediate response to perturbations of the protein folding equilibrium that can be observed in the cytosol as well as in the organelles such as the endoplasmatic reticulum and mitochondria. As reduction of protein synthesis is linked to life span increase, the signaling pathways regu-lating protein synthesis might be putative targets for treatments of age-related diseases. Eukaryotic cells have evolved a complex system for protein synthesis regulation and this review will summarize cellular strategies to regulate mRNA translation upon stress and its impact on longevity.

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
(2014)

Tags: Molecular Neuroscience and Biophysics (Plested)

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
(2014)

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.

CLCN7 and TCIRG1 Mutations Differentially Affect Bone Matrix Mineralization in Osteopetrotic Individuals
Barvencik(*), F., Kurth(*), I., Koehne(*), T., Stauber, T., Zustin(*), J., Tsiakas, K., Ludwig, C. F., Beil(*), F. T., Pestka(*), J. M., Hahn(*), M., Santer(*), R., Supanchart(*), C., Kornak(*9, U., Del Fattore(*), A., Jentsch, T. J., Teti(*), A., Schulz(*), A., Schinke(*), T.; Amling(*), M.
J Bone Miner Res, 29:982-991
(2014)

Tags: Physiology and Pathology of Ion Transport (Jentsch)

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
(2014)

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).

Chemical protein synthesis
Becker(*), C. F., Brik(*), A., Dawson(*), P.; Hackenberger, C. P.
J Pept Sci, 20:63
(2014)

Tags: Chemical Biology II (Hackenberger)

Highly conserved cysteines are involved in the oligomerization of occludin-redox dependency of the second extracellular loop
Bellmann, C., Schreivogel, S., Günther, R., Dabrowski, S., Schümann, M., Wolburg(*), H.; Blasig, I. E.
Antioxid Redox Signal, 20:855-867
(2014)

Tags: Molecular Cell Physiology (Blasig, I.E.), Mass Spectrometry (Krause, E.)

Abstract: UNLABELLED: The tight junction (TJ) marker occludin is a 4-transmembrane domain (TMD) protein with unclear physiological and pathological functions, interacting with other TJ proteins. It oligomerizes and is redox sensitive. However, oligomerization sites and mechanisms are unknown. AIMS: To identify hypoxia-sensitive binding sites, we investigated the consequences of amino-acid substitutions of highly conserved cysteines in human occludin, under normal and hypoxic incubations. RESULTS: (i) The extracellular loop 2 (ECL2) showed homophilic trans- and cis-association between opposing cells and along the cell membrane, respectively, caused by a loop properly folded via an intraloop disulfide bridge between the shielded C216 and C237. Hypoxia and reductants prevented the associations. (ii) C82 in TMD1 directly cis-associated without disulfide formation. (iii) C76 in TMD1 and C148 in TMD2 limited the trans-interaction; C76 also limited occludin-related paracellular tightness and changed the strand morphology of claudin-1. (iv) The diminished binding strength found after substituting C82, C216, or C237 was accompanied by increased occludin mobility in the cell membrane. INNOVATION: The data enable the first experimentally proven structural model of occludin and its homophilic interaction sites, in which the ECL2, via intraloop disulfide formation, has a central role in occludin's hypoxia-sensitive oligomerization and to regulate the structure of TJs. CONCLUSION: Our findings support the new concept that occludin acts as a hypoxiasensor and contributes toward regulating the TJ assembly redox dependently. This is of pathogenic relevance for tissue barrier injury with reducing conditions. The ECL2 disulfide might be a model for four TMD proteins in TJs with two conserved cysteines in an ECL.

Site-specifically phosphorylated lysine peptides
Bertran-Vicente, J., Serwa(*), R. A., Schümann, M., Schmieder, P., Krause, E.; Hackenberger, C. P.
J Am Chem Soc, 136:13622-13628
(2014)

Tags: Chemical Biology II (Hackenberger), Mass Spectrometry (Krause, E.), Solution NMR (Schmieder)

Abstract: Protein phosphorylation controls major processes in cells. Although phosphorylation of serine, threonine, and tyrosine and also recently histidine and arginine are well-established, the extent and biological significance of lysine phosphorylation has remained elusive. Research in this area has been particularly limited by the inaccessibility of peptides and proteins that are phosphorylated at specific lysine residues, which are incompatible with solid-phase peptide synthesis (SPPS) due to the intrinsic acid lability of the P( horizontal lineO)-N phosphoramidate bond. To address this issue, we have developed a new synthetic route for the synthesis of site-specifically phospholysine (pLys)-containing peptides by employing the chemoselectivity of the Staudinger-phosphite reaction. Our synthetic approach relies on the SPPS of unprotected epsilon-azido lysine-containing peptides and their subsequent reaction to phosphoramidates with phosphite esters before they are converted into the natural modification via UV irradiation or basic deprotection. With these peptides in hand, we demonstrate that electron-transfer dissociation tandem mass spectrometry can be used for unambiguous assignment of phosphorylated-lysine residues within histone peptides and that these peptides can be detected in cell lysates using a bottom-up proteomic approach. This new tagging method is expected to be an essential tool for evaluating the biological relevance of lysine phosphorylation.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
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