FMP Publications

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

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Real-Time Monitoring of Membrane-Protein Reconstitution by Isothermal Titration Calorimetry
Jahnke, N., Krylova, O. O., Hoomann(*), T., Vargas(*), C., Fiedler(*), S., Pohl(*), P.; Keller(*), S.
Anal Chem, 86:920-927

Tags: Biophysics of Membrane Proteins (Keller)

Abstract: Phase diagrams offer a wealth of thermodynamic information on aqueous mixtures of bilayer-forming lipids and micelle-forming detergents, providing a straightforward means of monitoring and adjusting the supramolecular state of such systems. However, equilibrium phase diagrams are of very limited use for the reconstitution of membrane proteins because of the occurrence of irreversible, unproductive processes such as aggregation and precipitation that compete with productive reconstitution. Here, we exemplify this by dissecting the effects of the K+ channel KcsA on the process of bilayer self-assembly in a mixture of Escherichia coli polar lipid extract and the nonionic detergent octyl-beta-D-glucopyranoside. Even at starting concentrations in the low micromolar range, KcsA has a tremendous impact on the supramolecular organization of the system, shifting the critical lipid/detergent ratios at the onset and completion of vesicle formation by more than 2-fold. Thus, equilibrium phase diagrams obtained for protein-free lipid/detergent mixtures would be misleading when used to guide the reconstitution process. To address this issue, we demonstrate that, even under such nonequilibrium conditions, high-sensitivity isothermal titration calorimetry can be exploited to monitor the progress of membrane-protein reconstitution in real time, in a noninvasive manner, and at high resolution to yield functional proteoliposomes with a narrow size distribution for further downstream applications.

Site-Specific Copper-Catalyzed Oxidation of alpha-Synuclein: Tightening the Link between Metal Binding and Protein Oxidative Damage in Parkinson's Disease
Miotto(*), M. C., Rodriguez(*), E. E., Valiente-Gabioud(*), A. A., Torres-Monserrat(*), V., Binolfi, A., Quintanar(*), L., Zweckstetter(*), M., Griesinger(*), C.; Fernandez(*), C. O.
Inorg Chem, 53:4350-4358

Tags: In-Cell NMR (Selenko)

Abstract: Amyloid aggregation of a-synuclein (AS) has been linked to the pathological effects associated with Parkinson's disease (PD). Cu-II binds specifically at the N-terminus of AS and triggers its aggregation. Site-specific Cu-I-catalyzed oxidation of AS has been proposed as a plausible mechanism for metal-enhanced AS amyloid formation. In this study, Cu-I binding to AS was probed by NMR spectroscopy, in combination with synthetic peptide models, site-directed mutagenesis, and C-terminal-truncated protein variants. Our results demonstrate that both Met residues in the motif (MDVFM5)-M-1 constitute key structural determinants for the high-affinity binding of Cu-I to the N-terminal region of AS. The replacement of one Met residue by Ile causes a dramatic decrease in the binding affinity for Cu-I, whereas the removal of both Met residues results in a complete lack of binding. Moreover, these Met residues can be oxidized rapidly after air exposure of the AS-Cu-I complex, whereas Met-116 and Met-127 in the C-terminal region remain unaffected. Met-1 displays higher susceptibility to oxidative damage compared to Met-5 because it is directly involved in both Cu-II and Cu-I coordination, resulting in closer exposure to the reactive oxygen species that may be generated by the redox cycling of copper. Our findings support a mechanism where the interaction of AS with copper ions leads to site-specific metal-catalyzed oxidation in the protein under physiologically relevant conditions. In light of recent biological findings, these results support a role for AS-copper interactions in neurodegeneration in PD.

Bioinorganic chemistry of synucleinopathies: deciphering the binding features of Met motifs and His-50 in AS-Cu(I) interactions
Miotto(*), M. C., Binolfi, A., Zweckstetter(*), M., Griesinger(*), C.; Fernandez(*), C. O.
J Inorg Biochem, 141:208-211

Tags: In-Cell NMR (Selenko)

Abstract: The aggregation of alpha-synuclein (AS) is a critical step in the etiology of Parkinson's disease (PD) and other neurodegenerative synucleinopathies. This process is selectively enhanced by copper in vitro and the interaction is proposed to play a potential role in vivo. Presently, the identity of the Cu(I) binding sites in AS and their relative affinities are under debate. In this work we have addressed unresolved details related to the structural binding specificity and affinity of Cu(I) to full-length AS. We demonstrated conclusively that: (i) the binding preferences of Cu(I) for the Met-binding sites at the N- (Kd=20 muM) and C-terminus (Kd=270 muM) of AS are widely different: (ii) the imidazole ring of His-50 acts as an effective anchoring residue (Kd=50 muM) for Cu(I) binding to AS; and (iii) no major structural rearrangements occur in the protein upon Cu(I) binding. Overall, our work shows that Cu(I) binding to the N- and C-terminal regions of AS are two independent events, with substantial differences in their affinities, and suggest that protein oxidative damage derived from a misbalance in cellular copper homeostasis would target preferentially the N-terminal region of AS. This knowledge is key to understanding the structural-aggregation basis of the copper catalyzed oxidation of AS.

A missense mutation accelerating the gating of the lysosomal Cl-/H+-exchanger ClC-7/Ostm1 causes osteopetrosis with gingival hamartomas in cattle
Sartelet(*), A., Stauber, T., Coppieters(*), W., Ludwig, C. F., Fasquelle(*), C., Druet(*), T., Zhang(*), Z. Y., Ahariz(*), N., Cambisano(*), N., Jentsch, T. J.; Charlier(*), C.
Dis Model Mech, 7:119-128

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: Chloride-proton exchange by the lysosomal anion transporter ClC7/Ostm1 is of pivotal importance for the physiology of lysosomes and bone resorption. Mice lacking either ClC-7 or Ostm1 develop a lysosomal storage disease and mutations in either protein have been found to underlie osteopetrosis in mice and humans. Some human disease-causing CLCN7 mutations accelerate the usually slow voltage-dependent gating of ClC-7/Ostm1. However, it has remained unclear whether the fastened kinetics is indeed causative for the disease. Here we identified and characterized a new deleterious ClC-7 mutation in Belgian Blue cattle with a severe symptomatology including perinatal lethality and in most cases gingival hamartomas. By autozygosity mapping and genome-wide sequencing we found a handful of candidate variants, including a cluster of three private SNPs causing the substitution of a conserved tyrosine in the CBS2 domain of ClC-7 by glutamine. The case for ClC-7 was strengthened by subsequent examination of affected calves that revealed severe osteopetrosis. The Y750Q mutation largely preserved the lysosomal localization and assembly of ClC-7/Ostm1, but drastically accelerated its activation by membrane depolarization. These data provide first evidence that accelerated ClC-7/Ostm1 gating per se is deleterious, highlighting a physiological importance of the slow voltage-activation of ClC-7/Ostm1 in lysosomal function and bone resorption.

ClC-7 expression levels critically regulate bone turnover, but not gastric acid secretion
Supanchart(*), C., Wartosch, L., Schlack(*), C., Kühnisch(*), J., Felsenberg, D., Fuhrmann(*), J. C., de Vernejoul(*), M. C., Jentsch, T. J.; Kornak(*), U.
Bone, 58:92-102

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: Mutations in the 2Cl(-)/1H(+)-exchanger ClC-7 impair osteoclast function and cause different types of osteodastrich osteopetrosis. However, it is unknown to what extent ClC-7 function has to be reduced to become rate-limiting for bone resorption. In osteoclasts from osteopetrosis patients expression of the mutated ClC-7 protein did not correlate with disease severity and resorption impairment. Therefore, a series of transgenic mice expressing ClC-7 in osteoclasts at different levels was generated. Crossing of these mice with Clat7(-/-) mutants rescued the osteopetrotic phenotype to variable degrees. One resulting double transgenic line mimicked human autosomal dominant osteopetrosis. The trabecular bone of these mice showed a reduction of osteoblast numbers, osteoid, and osteoblast marker gene expression indicative of reduced osteoblast function. In osteoclasts from these mutants ClC-7 expression levels were 20 to 30% of wildtype levels. These reduced levels not only impaired resorptive activity, but also increased numbers, size and nucleus numbers of osteoclasts differentiated in vitro. Although ClC-7 was expressed in the stomach and PTH levels were high in Clcn7(-/-) mutants loss of ClC-7 did not entail a relevant elevation of gastric pH. In conclusion, we show that in our model a reduction of ClC-7 function by approximately 70% is sufficient to increase bone mass, but does not necessarily enhance bone formation. ClC-7 does not appear to be crucially involved in gastric acid secretion, which explains the absence of an osteopetrorickets phenotype in CLCN7-related osteopetrosis.

Physicochemical properties of cells and their effects on intrinsically disordered proteins (IDPs)
Theillet, F. X., Binolfi, A., Frembgen-Kesner(*), T., Hingorani(*), K., Sarkar(*), M., Kyne(*), C., Li(*), C., Crowley(*), P. B., Gierasch(*), L., Pielak(*), G. J., Elcock(*), A. H., Gershenson(*), A.; Selenko, P.
Chem Rev, 114:6661-6714

Tags: In-Cell NMR (Selenko)

Nanometer-resolution fluorescence electron microscopy (nano-EM) in cultured cells
Watanabe(*), S., Lehmann, M., Hujber(*), E., Fetter(*), R. D., Richards(*), J., Sohl-Kielczynski(*), B., Felies(*), A., Rosenmund(*), C., Schmoranzer, J.; Jorgensen(*), E. M.
Methods Mol Biol, 1117:503-526

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Nano-resolution fluorescence electron microscopy (nano-fEM) pinpoints the location of individual proteins in electron micrographs. Plastic sections are first imaged using a super-resolution fluorescence microscope and then imaged on an electron microscope. The two images are superimposed to correlate the position of labeled proteins relative to subcellular structures. Here, we describe the method in detail and present five technical advancements: the use of uranyl acetate during the freeze-substitution to enhance the contrast of tissues and reduce the loss of fluorescence, the use of ground-state depletion instead of photoactivation for temporal control of fluorescence, the use of organic fluorophores instead of fluorescent proteins to obtain brighter fluorescence signals, the use of tissue culture cells to broaden the utility of the method, and the use of a transmission electron microscope to achieve sharper images of ultrastructure.

A dataset comprising 141 magnetic resonance imaging scans of 98 extant sea urchin species
Ziegler(*), A., Faber(*), C., Mueller(*), S., Nagelmann(*), N.; Schröder, L.
GigaScience, 3:21

Tags: Molecular Imaging (Schröder)

Abstract: BACKGROUND: Apart from its application in human diagnostics, magnetic resonance imaging (MRI) can also be used to study the internal anatomy of zoological specimens. As a non-invasive imaging technique, MRI has several advantages, such as rapid data acquisition, output of true three-dimensional imagery, and provision of digital data right from the onset of a study. Of particular importance for comparative zoological studies is the capacity of MRI to conduct high-throughput analyses of multiple specimens. In this study, MRI was applied to systematically document the internal anatomy of 98 representative species of sea urchins (Echinodermata: Echinoidea). FINDINGS: The dataset includes raw and derived image data from 141 MRI scans. Most of the whole sea urchin specimens analyzed were obtained from museum collections. The attained scan resolutions permit differentiation of various internal organs, including the digestive tract, reproductive system, coelomic compartments, and lantern musculature. All data deposited in the GigaDB repository can be accessed using open source software. Potential uses of the dataset include interactive exploration of sea urchin anatomy, morphometric and volumetric analyses of internal organs observed in their natural context, as well as correlation of hard and soft tissue structures. CONCLUSIONS: The dataset covers a broad taxonomical and morphological spectrum of the Echinoidea, focusing on 'regular' sea urchin taxa. The deposited files significantly expand the amount of morphological data on echinoids that are electronically available. The approach chosen here can be extended to various other vertebrate and invertebrate taxa. We argue that publicly available digital anatomical and morphological data gathered during experiments involving non-invasive imaging techniques constitute one of the prerequisites for future large-scale genotype-phenotype correlations.

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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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