FMP Publications

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

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References
Antimicrobial peptide cWFW kills by combining lipid phase separation with autolysis
Scheinpflug, K., Wenzel(*), M., Krylova, O., Bandow(*), J. E., Dathe, M.; Strahl(*), H.
Sci Rep, 7:44332
(2017)

Tags: Peptide-Lipid-Interaction/ Peptide Transport (Dathe)

Abstract: The synthetic cyclic hexapeptide cWFW (cyclo(RRRWFW)) has a rapid bactericidal activity against both Gram-positive and Gram-negative bacteria. Its detailed mode of action has, however, remained elusive. In contrast to most antimicrobial peptides, cWFW neither permeabilizes the membrane nor translocates to the cytoplasm. Using a combination of proteome analysis, fluorescence microscopy, and membrane analysis we show that cWFW instead triggers a rapid reduction of membrane fluidity both in live Bacillus subtilis cells and in model membranes. This immediate activity is accompanied by formation of distinct membrane domains which differ in local membrane fluidity, and which severely disrupts membrane protein organisation by segregating peripheral and integral proteins into domains of different rigidity. These major membrane disturbances cause specific inhibition of cell wall synthesis, and trigger autolysis. This novel antibacterial mode of action holds a low risk to induce bacterial resistance, and provides valuable information for the design of new synthetic antimicrobial peptides.

Fluorescent labelling in living cells
Schneider, A. F.; Hackenberger, C. P.
Current opinion in biotechnology, 48:61-68
(2017)

Tags: Chemical Biology II (Hackenberger)

Abstract: The labelling of proteins with green fluorescent protein enabled the visualization of proteins in living cells for the first time. Since then, much progress has been made in the field. Modern strategies allow the labelling of proteins in live cells through a range of specialized methods with sophisticated chemical probes that show enhanced photophysical properties compared to fluorescent proteins. This review briefly summarizes recent advances in the field of fluorescent chemical protein labelling inside living cells and illustrates key aspects on the requirements and advantages of each given method.

A straightforward approach to N-substituted-2H-indazol-2-amines through reductive cyclization
Schöne, J., Abed, H. B., Christmann(*), M.; Nazare, M.
Tetrahedron Lett, 58:1633-1635
(2017)

Tags: Medicinal Chemistry (Nazare)

Abstract: A versatile two-step, one-pot reaction to access N-substituted-2H-indazol-2-amine derivatives has been elaborated. A diverse set of analogues was obtained by a sequential hydrazone formation and reductive cyclization in moderate to good yields from readily available starting materials. The strategy tolerates a broad range of substitutions pattern and functional groups allowing further derivatizations. (C) 2017 Elsevier Ltd. All rights reserved.

Lipid-mediated PX-BAR domain recruitment couples local membrane constriction to endocytic vesicle fission
Schöneberg(*), J., Lehmann, M., Ullrich(*), A., Posor, Y., Lo, W. T., Lichtner, G., Schmoranzer, J., Haucke, V.; Noe(*), F.
Nat Commun, 8:15873
(2017)

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Clathrin-mediated endocytosis (CME) involves membrane-associated scaffolds of the bin-amphiphysin-rvs (BAR) domain protein family as well as the GTPase dynamin, and is accompanied and perhaps triggered by changes in local lipid composition. How protein recruitment, scaffold assembly and membrane deformation is spatiotemporally controlled and coupled to fission is poorly understood. We show by computational modelling and super-resolution imaging that phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] synthesis within the clathrin-coated area of endocytic intermediates triggers selective recruitment of the PX-BAR domain protein SNX9, as a result of complex interactions of endocytic proteins competing for phospholipids. The specific architecture induces positioning of SNX9 at the invagination neck where its self-assembly regulates membrane constriction, thereby providing a template for dynamin fission. These data explain how lipid conversion at endocytic pits couples local membrane constriction to fission. Our work demonstrates how computational modelling and super-resolution imaging can be combined to unravel function and mechanisms of complex cellular processes.

Recombinant expression of porcine spermadhesin AWN and its phospholipid interaction: Indication for a novel lipid binding property
Schröter(*), F., Müller(*), K., Müller(*), P., Krause, E.; Braun(*), B. C.
Reprod Domest Anim, 52:585-595
(2017)

Tags: Mass Spectrometry (Krause, E.)

Abstract: AWN is a porcine (Sus scrofa domestica) seminal plasma protein and has been linked to a variety of processes related to fertilization. To acquire the protein in sufficient amount and purity for functional studies, we established its recombinant expression in E. coli and a three-step purification protocol based on different chromatographies. The test for AWN-phospholipid interaction revealed phosphatidic acid and cardiolipin as potential binding partners. As phosphatidic acid is surmised to play a role in cation-induced membrane destabilization and fusion events, we propose a membrane protective function of the presented binding affinity. Further studies with recombinant AWN will allow new insights into the mechanism of sperm-spermadhesin interaction and might provide new approaches for artificial reproduction techniques.

Functional Significance of the Signal Peptides of Corticotropin-Releasing Factor Receptors
Schülein, R., Gibert, A.; Rutz, C.
Current molecular pharmacology,
(2017)

Tags: Protein Trafficking (Schülein)

Abstract: The corticotropin releasing factor (CRF) receptors belong to the large family of G protein-coupled receptors (GPCRs) and must be transported to the plasma membrane to function properly. The first step of the intracellular transport of GPCRs is their insertion into the membrane of the endoplasmic reticulum (ER). This process is mediated by the translocon complex of the ER membrane and the signal sequences of the receptors. Most GPCRs possess signal sequences which form part of the mature proteins, the so called signal anchor sequences (usually transmembrane domain 1). The CRF receptors possess instead signal sequences at their extreme N tails which were thought to be cleaved off following integration of the receptors into the ER membrane (signal peptides, SPs, also called cleaved signal sequences). Recent work, however, showed that not all subtypes of CRF receptors stick to this rule. Whereas the corticotropin-releasing factor receptor type 1 (CRF1R) and the corticotropin-releasing factor receptor type 2b (CRF2(b)R) possess conventional SPs which are indeed cleaved off following ER insertion, the SP of the cortictropin-releasing factor receptor type 2a (CRF2(a)R) remains uncleaved. It forms a unique N-terminal domain (pseudo signal peptide, PSP) which has surprising functions beyond the ER level. Its presence not only influences expression levels at the plasma membrane but also receptor homodimerisation and, as a consequence, G protein selectivity. In this mini-review, we summarize the progress in understanding the functions of SPs of CRF receptors. Recent data also allow deriving hypotheses for a physiological significance of these sequences.

Chemical functionalization strategies and intracellular applications of nanobodies
Schumacher, D., Helma(*), J., Schneider, A. F. L., Leonhardt(*), H.; Hackenberger, C.
Angew Chem Int Ed Engl,
(2017)

Tags: Chemical Biology II (Hackenberger)

Abstract: Nanobodies can be considered as next-generation life science tools for the recognition and modulation of antigens that are inaccessible to conventional antibodies. Due to their compact structure and high stability, nanobodies see frequent usage in basic research. Their chemical functionalization facilitates powerful diagnostic tools and opens the way towards promising therapeutic applications. In this review, central aspects of nanobody functionalization are given together with selected applications in molecular cell biology. While first-generation conjugation strategies rely on random modification of natural amino acids, more recent studies focus on a site-specific attachment of functional groups. Such techniques include chemoenzymatic approaches, expressed protein ligation and amber suppression in combination with bioorthogonal modification strategies. With an ever growing toolkit of protein synthesis and conjugation, functional applications are on the rise as well. Such recent applications range from sophisticated imaging and mass spectrometry to the delivery of nanobodies into living cells, enabling visualization and manipulation of intracellular antigens.

Broad substrate tolerance of tubulin tyrosine ligase enables one-step site-specific enzymatic protein labeling
Schumacher, D., Lemke(*), O., Helma(*), J., Gerszonowicz(*), L., Waller(*), V., Stoschek(*), T., Durkin(*), P. M., Budisa(*), N., Leonhardt(*), H., Keller(*), B. G.; Hackenberger, C. P. R.
Chem Sci, 8:3471-3478
(2017)

Tags: Chemical Biology II (Hackenberger)

Abstract: The broad substrate tolerance of tubulin tyrosine ligase is the basic rationale behind its wide applicability for chemoenzymatic protein functionalization. In this context, we report that the wild-type enzyme enables ligation of various unnatural amino acids that are substantially bigger than and structurally unrelated to the natural substrate, tyrosine, without the need for extensive protein engineering. This unusual substrate flexibility is due to the fact that the enzyme's catalytic pocket forms an extended cavity during ligation, as confirmed by docking experiments and all-atom molecular dynamics simulations. This feature enabled one-step C-terminal biotinylation and fluorescent coumarin labeling of various functional proteins as demonstrated with ubiquitin, an antigen binding nanobody, and the apoptosis marker Annexin V. Its broad substrate tolerance establishes tubulin tyrosine ligase as a powerful tool for in vitro enzyme-mediated protein modification with single functional amino acids in a specific structural context.

Synaptic Vesicle Endocytosis Occurs on Multiple Timescales and Is Mediated by Formin-Dependent Actin Assembly
Soykan, T., Kaempf, N., Sakaba(*), T., Vollweiter, D., Goerdeler, F., Puchkov, D., Kononenko, N. L.; Haucke, V.
Neuron, 93:854-866.e854
(2017)

Tags: Molecular Pharmacology and Cell Biology (Haucke), Cellular Imaging (Wiesner, Puchkov)

Abstract: Neurotransmission is based on the exocytic fusion of synaptic vesicles (SVs) followed by endocytic membrane retrieval and the reformation of SVs. Recent data suggest that at physiological temperature SVs are internalized via clathrin-independent ultrafast endocytosis (UFE) within hundreds of milliseconds, while other studies have postulated a key role for clathrin-mediated endocytosis (CME) of SV proteins on a timescale of seconds to tens of seconds. Here we demonstrate using cultured hippocampal neurons as a model that at physiological temperature SV endocytosis occurs on several timescales from less than a second to several seconds, yet, is largely independent of clathrin. Clathrin-independent endocytosis (CIE) of SV membranes is mediated by actin-nucleating formins such as mDia1, which are required for the formation of presynaptic endosome-like vacuoles from which SVs reform. Our results resolve previous discrepancies in the field and suggest that SV membranes are predominantly retrieved via CIE mediated by formin-dependent actin assembly.

Direct Experimental Evidence for Halogen-Aryl pi Interactions in Solution from Molecular Torsion Balances
Sun, H., Horatscheck, A., Martos, V., Bartetzko, M., Uhrig, U., Lentz, D., Schmieder, P.; Nazare, M.
Angew Chem Int Ed Engl, 56:6454-6458
(2017)

Tags: Medicinal Chemistry (Nazare), Solution NMR (Schmieder), Computational Chemistry/ Drug Design (Kühne)

Abstract: We dissected halogen-aryl pi interactions experimentally using a bicyclic N-arylimide based molecular torsion balances system, which is based on the influence of the non-bonded interaction on the equilibria between folded and unfolded states. Through comparison of balances modulated by higher halogens with fluorine balances, we determined the magnitude of the halogen-aryl pi interactions in our unimolecular systems to be larger than -5.0 kJ mol-1 , which is comparable with the magnitude estimated in the biomolecular systems. Our study provides direct experimental evidence of halogen-aryl pi interactions in solution, which until now have only been revealed in the solid state and evaluated theoretically by quantum-mechanical calculations.

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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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info(at)fmp-berlin.de

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