FMP Publications

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

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References
Light-Dark Adaptation of Channelrhodopsin Involves Photoconversion between the all-trans and 13-cis Retinal Isomers
Bruun(*), S., Stöppler, D., Keidel(*), A., Kuhlmann(*), U., Luck(*), M., Diehl, A., Geiger, M. A., Woodmansee(*), D., Trauner(*), D., Hegemann(*), P., Oschkinat, H., Hildebrandt(*), P.; Stehfes(*)t, K.
Biochemistry, 54:5389-5400
(2015)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Channelrhodopsins (ChR) are light-gated ion channels of green algae that are widely used to probe the function of neuronal cells with light. Most ChRs show a substantial reduction in photocurrents during illumination, a process named "light adaptation". The main objective of this spectroscopic study was to elucidate the molecular processes associated with light-dark adaptation. Here we show by liquid and solid-state nuclear magnetic resonance spectroscopy that the retinal chromophore of fully dark-adapted ChR is exclusively in an all-trans configuration. Resonance Raman (RR) spectroscopy, however, revealed that already low light intensities establish a photostationary equilibrium between all-trans,15-anti and 13-cis,15-syn configurations at a ratio of 3:1. The underlying photoreactions involve simultaneous isomerization of the C(13) horizontal lineC(14) and C(15) horizontal lineN bonds. Both isomers of this DAapp state may run through photoinduced reaction cycles initiated by photoisomerization of only the C(13) horizontal lineC(14) bond. RR spectroscopic experiments further demonstrated that photoinduced conversion of the apparent dark-adapted (DAapp) state to the photocycle intermediates P500 and P390 is distinctly more efficient for the all-trans isomer than for the 13-cis isomer, possibly because of different chromophore-water interactions. Our data demonstrating two complementary photocycles of the DAapp isomers are fully consistent with the existence of two conducting states that vary in quantitative relation during light-dark adaptation, as suggested previously by electrical measurements.

Sensitivity and resolution of proton detected spectra of a deuterated protein at 40 and 60 kHz magic-angle-spinning
Nieuwkoop, A. J., Franks, W. T., Rehbein, K., Diehl, A., Akbey, Ü., Engelke(*), F., Emsley(*), L., Pintacuda(*), G.; Oschkinat, H.
J Biomol NMR, 61:161-171
(2015)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: The use of small rotors capable of very fast magic-angle spinning (MAS) in conjunction with proton dilution by perdeuteration and partial reprotonation at exchangeable sites has enabled the acquisition of resolved, proton detected, solid-state NMR spectra on samples of biological macromolecules. The ability to detect the high-gamma protons, instead of carbons or nitrogens, increases sensitivity. In order to achieve sufficient resolution of the amide proton signals, rotors must be spun at the maximum rate possible given their size and the proton back-exchange percentage tuned. Here we investigate the optimal proton back-exchange ratio for triply labeled SH3 at 40 kHz MAS. We find that spectra acquired on 60 % back-exchanged samples in 1.9 mm rotors have similar resolution at 40 kHz MAS as spectra of 100 % back-exchanged samples in 1.3 mm rotors spinning at 60 kHz MAS, and for (H)NH 2D and (H)CNH 3D spectra, show 10-20 % higher sensitivity. For 100 % back-exchanged samples, the sensitivity in 1.9 mm rotors is superior by a factor of 1.9 in (H)NH and 1.8 in (H)CNH spectra but at lower resolution. For (H)C(C)NH experiments with a carbon-carbon mixing period, this sensitivity gain is lost due to shorter relaxation times and less efficient transfer steps. We present a detailed study on the sensitivity of these types of experiments for both types of rotors, which should enable experimentalists to make an informed decision about which type of rotor is best for specific applications.

A Multiplexed NMR-Reporter Approach to Measure Cellular Kinase and Phosphatase Activities in Real-Time
Thongwichian, R., Kosten, J., Benary(*), U., Rose, H. M., Stuiver, M., Theillet, F. X., Dose, A., Koch(*), B., Yokoyama(*), H., Schwarzer, D., Wolf(*), J.; Selenko, P.
J. Am. Chem. Soc., 137:6468-6471
(2015)

Tags: In-Cell NMR (Selenko), Protein Chemistry (Schwarzer)

Abstract: Cell signaling is governed by dynamic changes in kinase and phosphatase activities, which are difficult to assess with discontinuous readout methods. Here, we introduce an NMR-based reporter approach to directly identify active kinases and phosphatases in complex physiological environments such as cell lysates and to measure their individual activities in a semicontinuous fashion. Multiplexed NMR profiling of reporter phosphorylation states provides unique advantages for kinase inhibitor studies and reveals reversible modulations of cellular enzyme activities under different metabolic conditions.

The efficacy of trivalent cyclic hexapeptides to induce lipid clustering in PG/PE membranes correlates with their antimicrobial activity
Finger(*), S., Kerth(*), A., Dathe, M.; Blume(*), A.
Biochim Biophys Acta, 1848:2998-3006
(2015)

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

Abstract: Various models have been proposed for the sequence of events occurring after binding of specific antimicrobial peptides to lipid membranes. The lipid clustering model arose by the finding that antimicrobial peptides can induce a segregation of certain negatively charged lipids in lipid model membranes. Anionic lipid segregation by cationic peptides is initially an effect of charge interaction where the ratio of peptide and lipid charges is thought to be the decisive parameter in the peptide induced lipid demixing. However, the sequence of events following this initial lipid clustering is more complex and can lead to deactivation of membrane proteins involved in cell division or perturbation of lipid reorganization essential for cell division. In this study we used DSC and ITC techniques to investigate the effect of binding different cyclic hexapeptides with varying antimicrobial efficacy, to phosphatidylglycerol (PG)/phosphatidylethanolamine (PE) lipid membranes and their ability to induce lipid segregation in these mixtures. We found that these cyclic hexapeptides consisting of three charged and three aromatic amino acids showed indeed different abilities to induce lipid demixing depending on their amino acid composition and their sequence. The results clearly showed that the cationic amino acids are essential for electrostatic binding but that the three hydrophobic amino acids in the peptides and their position in the sequence also contribute to binding affinity and to the extent of induction of lipid clustering. The efficacy of these different hexapeptides to induce PG clusters in PG/PE membranes was found to be correlated with their antimicrobial activity.

Evidence for a novel mechanism of antimicrobial action of a cyclic R-,W-rich hexapeptide
Scheinpflug, K., Krylova, O., Nikolenko, H., Thurm, C.; Dathe, M.
Plos One, 10:e0125056
(2015)

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

Abstract: The development of antimicrobial peptides as new class of antibiotic agents requires structural characterisation and understanding of their diverse mechanisms of action. As the cyclic hexapeptide cWFW (cyclo(RRRWFW)) does not exert its rapid cell killing activity by membrane permeabilisation, in this study we investigated alternative mechanisms of action, such as peptide translocation into the cytoplasm and peptide interaction with components of the phospholipid matrix of the bacterial membrane. Using fluorescence microscopy and an HPLC-based strategy to analyse peptide uptake into the cells we could confirm the cytoplasmic membrane as the major peptide target. However, unexpectedly we observed accumulation of cWFW at distinct sites of the membrane. Further characterisation of peptide-membrane interaction involved live cell imaging to visualise the distribution of the lipid cardiolipin (CL) and isothermal titration calorimetry to determine the binding affinity to model membranes with different bacterial lipid compositions. Our results demonstrate a distribution of the cyclic peptide similar to that of cardiolipin within the membrane and highly preferred affinity of cWFW for CL-rich phosphatidylethanolamine (POPE) matrices. These observations point to a novel mechanism of antimicrobial killing for the cyclic hexapeptide cWFW which is neither based on membrane permeabilisation nor translocation into the cytoplasm but rather on preferred partitioning into particular lipid domains. As the phospholipids POPE/CL play a key role in the dynamic organisation of bacterial membranes we discuss the consequences of this peptide-lipid-interaction and outline the impact on antimicrobial peptide research.

Brain endothelial cell targeting via a peptide-functionalized liposomal carrier for xenon hyper-CEST MRI
Schnurr, M., Sydow, K., Rose, H. M., Dathe, M.; Schröder, L.
Advanced healthcare materials, 4:40-45
(2015)

Tags: Molecular Imaging (Schröder); Peptide-Lipid-Interaction/ Peptide Transport (Dathe)

Abstract: A nanoparticulate carrier system is used to efficiently deliver a contrast agent for highly sensitive xenon Hyper-CEST MRI. The carrier system not only improves the biocompatibility and solubility of the contrast agent, it also allows selective cell targeting as demonstrated by the discrimination of human brain capillary and aortic endothelial cells.

Challenges and Limits Using Antimicrobial Peptides in Boar Semen Preservation
Schulze(*), M., Grobbel(*), M., Müller(*), K., Junkes(*), C., Dathe, M., Rüdiger(*), K.; Jung(*), M.
Reprod Domest Anim, 50 Suppl 2:5-10
(2015)

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

Abstract: Antibiotics are of great importance for the preservation of ejaculates for livestock breading. The use of antibiotics, however, is not an appropriate compensation for a lack of hygiene standards in artificial insemination (AI) centres. Sophisticated hygiene management and the proper identification of hygienic critical control points (HCCPs) at AI centres provide the basis for counteracting the development of antibiotic resistance in contaminant bacteria and their settlement in AI centres. In recent years, efforts have been made to use antimicrobial peptides (AMPs) in the preservation of boar semen. Investigations have included the testing of synthetic magainin derivatives and cyclic hexapeptides. One prerequisite for the application of AMPs is that they have a minor impact on eukaryotic cells. Bacterial selectivity, proteolytic stability, thermodynamic resistance, and mechanisms including synergistic interaction with conventional antibiotics have made cyclic hexapeptides highly promising candidates for potential application as peptide antibiotics for semen preservation.

Anchoring dipalmitoyl phosphoethanolamine to nanoparticles boosts cellular uptake and fluorine-19 magnetic resonance signal
Waiczies(*), S., Lepore(*), S., Sydow, K., Drechsler(*), S., Ku(*), M. C., Martin(*), C., Lorenz, D., Schütz, I., Reimann(*), H. M., Purfurst(*), B., Dieringer(*), M. A., Waiczies(*), H., Dathe, M., Pohlmann(*), A.; Niendorf(*), T.
Sci Rep, 5:8427
(2015)

Tags: Peptide-Lipid-Interaction/ Peptide Transport (Dathe), Molecular Pharmacology and Cell Biology (Haucke), Cellular Imaging (Wiesner)

Abstract: Magnetic resonance (MR) methods to detect and quantify fluorine ((19)F) nuclei provide the opportunity to study the fate of cellular transplants in vivo. Cells are typically labeled with (19)F nanoparticles, introduced into living organisms and tracked by (19)F MR methods. Background-free imaging and quantification of cell numbers are amongst the strengths of (19)F MR-based cell tracking but challenges pertaining to signal sensitivity and cell detection exist. In this study we aimed to overcome these limitations by manipulating the aminophospholipid composition of (19)F nanoparticles in order to promote their uptake by dendritic cells (DCs). As critical components of biological membranes, phosphatidylethanolamines (PE) were studied. Both microscopy and MR spectroscopy methods revealed a striking (at least one order of magnitude) increase in cytoplasmic uptake of (19)F nanoparticles in DCs following enrichment with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE). The impact of enriching (19)F nanoparticles with PE on DC migration was also investigated. By manipulating the nanoparticle composition and as a result the cellular uptake we provide here one way of boosting (19)F signal per cell in order to overcome some of the limitations related to (19)F MR signal sensitivity. The boost in signal is ultimately necessary to detect and track cells in vivo.

Supramolecular Assays for Mapping Enzyme Activity by Displacement-Triggered Change in Hyperpolarized (129)Xe Magnetization Transfer NMR Spectroscopy
Schnurr, M., Sloniec-Myszk(*), J., Döpfert, J., Schröder, L.; Hennig(*), A.
Angew Chem Int Ed Engl, 54:13444-13447
(2015)

Tags: Molecular Imaging (Schröder)

Abstract: Reversibly bound Xe is a sensitive NMR and MRI reporter with its resonance frequency being influenced by the chemical environment of the host. Molecular imaging of enzyme activity presents a promising approach for disease identification, but current Xe biosensing concepts are limited since substrate conversion typically has little impact on the chemical shift of Xe inside tailored cavities. Herein, we exploit the ability of the product of the enzymatic reaction to bind itself to the macrocyclic hosts CB6 and CB7 and thereby displace Xe. We demonstrate the suitability of this method to map areas of enzyme activity through changes in magnetization transfer with hyperpolarized Xe under different saturation scenarios.

Orthogonal dual-modification of proteins for the engineering of multivalent protein scaffolds
Mühlberg(*), M., Hoesl(*), M. G., Kuehne(*), C., Dernedde(*), J., Budisa(*), N.; Hackenberger, C. P.
Beilstein J Org Chem, 11:784-791
(2015)

Tags: Chemical Biology II (Hackenberger)

Abstract: To add new tools to the repertoire of protein-based multivalent scaffold design, we have developed a novel dual-labeling strategy for proteins that combines residue-specific incorporation of unnatural amino acids with chemical oxidative aldehyde formation at the N-terminus of a protein. Our approach relies on the selective introduction of two different functional moieties in a protein by mutually orthogonal copper-catalyzed azide-alkyne cycloaddition (CuAAC) and oxime ligation. This method was applied to the conjugation of biotin and beta-linked galactose residues to yield an enzymatically active thermophilic lipase, which revealed specific binding to Erythrina cristagalli lectin by SPR binding studies.

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