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Core facility for protein biochemistry and spectroscopy

 

The core facility for protein spectroscopy in the Institute of Cytobiology provides high quality spectrometers, anaerobic facilities, stopped-flow and chromatography equipment. Applications and expertise are focussed on proteins with cofactors and on metalloproteins for which we have extensive experience (see publications below). We support all students and faculty of the Philipps University and their collaboration partners outside our university.

 

 
O. Stehling
 

Contact:

Dr. Oliver Stehling

Philipps-Universität Marburg
Institut für Zytobiologie
Robert-Koch-Str. 6, 35032 Marburg

Fon: ++6421-286 4044
Fax: ++6421-286 6414

E-mail: stehling@staff.uni-marburg.de

 

Equipment

 

Protein purification

An ÄKTA purifier 10 system (GE Healthcare), and an extensive collection of chromatography columns is available. The system is equipped with a very flexible fraction collector (Frac-950) and a Xenon flashlamp multiple-wavelength detector, all contained in a refrigerated cabinet.

 aekta


Anaerobic facilities

Two Coy anaerobic tents (2-5 % hydrogen in nitrogen) can be used for aqueous biochemistry of air-sensitive metallo-proteins. Extensive expertise is available to support such difficult experiments. The presence of spectrometers and spec-troscopists on site is an enormous advantage for the study of these systems.

 box


Differential Scanning Fluorimetry

A Nanotemper Prometheus NT.48 (nanoDSF) system allows label-free analysis of thermal and chemical protein stability in small-sized samples by measuring internal tryptophane fluorescence.

Prometheus NT.48

EPR spectroscopy

X-Band-EPR spectrometer (Bruker EMX-6/1) composed of EMX113 console, ER-041 XG X-band microwave bridge with ER-041-1161 microwave frequency counter and EMX-1101 magnet (11-640 mT). A standard-Universal TE102 rectangular (ER-4102) and a dual-mode cavity are available. Routinely, measurements at liquid nitrogen temperature are performed. The use of an Oxford instruments variable temperature Helium flow cryostat (4-200 K) is possible.

 EPR


CD spectroscopy

A Jasco J-815 CD spectrometer purged with argon allows to determine the content of secondary structural elements in the UV range. With the programmable PTC-423S Peltier-element thermostat melting curves can be recorded. The superb sensitivity of the Jasco machine makes time-dependent CD spectroscopyin the visible range for heme or [2Fe-2S] cluster-containing proteins possible.

 

 CD


Stopped flow kinetics

The SFM-20 stopped-flow system is routinely set up for spectroscopic detection of rapid kinetic processes. The system can be swapped to UV-Vis, fluorimetric and, if sensitivity allows, CD polarimetric detection. Since the removable syringes are air-tight and anaerobic tents are in the same building, we have the possibility to anaerobically study kinetic processes.

 SF


Microscale Thermophoresis (MST)

Using a Nanotemper Technologies Monolith NT.115 microscale thermophoresis instrument, complex formation between a fluorescently labelled protein and another protein or ligand can be detected with minimal effort requiring limited quantities of protein and/or ligand.

 thermo


Bio-Layer Interferometry (BLI)

The Pall ForteBio Octet K2 system allows the label-free analysis of biomolecular interactions between an immobilized compound at the tip of a biosensor and an analyte in solution. Binding assays enable the determination of complex formation, its kinetics, and the underlying affinities.

 Octet K2

 

Fluorescence spectroscopy

A Jasco FP-6300 fluorescence spectrometer is avalaible with computer interface for kinetics and scanning. For large numbers of samples the Tecan Infinite M200 plate reader, albeit of lower resolution, can be used.

 fluor


UV-Vis spectroscopy

In addition, several UV-Vis spectrometers are available at our institute and complement the equipment of the core facility: a Jasco V-550 interfaced with external computer, two Kontron Uvikon  spectrometers (922 and 933, internal computer), a Nanodrop spectrometer, and Tecan Infinite M200 plate readers. In the biggest of our two anaerobic gloveboxes a second Nanodrop spectrometer is present.

 UV

Publications of the Core facility


2018

  • Stehling O., Jeoung J.H., Freibert S.A., Paul V.D., Bänfer S., Niggemeyer B., Rösser R., Dobbek H., Lill R. (2018) Proc Natl Acad Sci U S A. 115, E9085. Function and crystal structure of the dimeric P-loop ATPase CFD1 coordinating an exposed [4Fe-4S] cluster for transfer to apoproteins.

  • Ben-Shimon L., Paul V.D., David-Kadoch G., Volpe M., Stümpfig M., Bill E., Mühlenhoff U., Lill R., Ben-Aroya S. (2018) J. Cell Sci. 131. Fe-S cluster coordination of the chromokinesin KIF4A alters its subcellular localization during mitosis.

  • Freibert S.A., Weiler B.D., Bill E., Pierik A.J., Mühlenhoff U., Lill R. (2018) Methods Enzymol. 59, 197. Biochemical Reconstitution and Spectroscopic Analysis of Iron-Sulfur Proteins.

  • Bänfer S., Schneider D., Dewes J., Strauss M.T., Freibert S.A., Heimerl T., Maier U.G., Elsässer H.P., Jungmann R., Jacob R. (2018) Proc Natl Acad Sci U S A., 115, E4396. Molecular mechanism to recruit galectin-3 into multivesicular bodies for polarized exosomal secretion.

 

2017

  • Boniecki M.T., Freibert S.A., Mühlenhoff U., Lill R., Cygler M. (2017) Nat. Commun. 8, 1287. Structure and functional dynamics of the mitochondrial Fe/S cluster synthesis complex.

  • Freibert S.A., Goldberg A.V., Hacker C., Molik S., Dean P., Williams T.A., Nakjang S., Long S., Sendra K., Bill E., Heinz E., Hirt R.P., Lucocq J.M., Embley T.M., Lill R. (2017) Nat. Commun. 8, 13932. Evolutionary conservation and in vitro reconstitution of microsporidian iron-sulfur cluster biosynthesis.

 

2016

  • Melber A., Na U., Vashisht A., Weiler B.D., Lill R., Wohlschlegel J.A., Winge D.R. (2016) Elife 5, e15991. Role of Nfu1 and Bol3 in iron-sulfur cluster transfer to mitochondrial clients.

  • Uzarska M.A., Nasta V., Weiler B.D., Spantgar F., Ciofi-Baffoni S., Saviello M.R., Gonnelli L., Mühlenhoff U., Banci L., Lill R. (2016) Elife 5, e16673. Mitochondrial Bol1 and Bol3 function as assembly factors for specific iron-sulfur proteins.

  • Netz D.J., Genau H.M., Weiler B.D., Bill E., Pierik A.J., Lill R. (2016) Biochem. J. 473, 2073-2085. The conserved protein Dre2 uses essential [2Fe-2S] and [4Fe-4S] clusters for its function in cytosolic iron-sulfur protein assembly.

 

2015

  • Moseler A., Aller I., Wagner S., Nietzel T., Przybyla-Toscano J., Mühlenhoff U., Lill R., Berndt C., Rouhier N., Schwarzländer M., Meyer A.J. (2015) PNAS 112, 13735-13740. The mitochondrial monothiol glutaredoxin S15 is essential for iron-sulfur protein maturation in Arabidopsis thaliana.

  • Paul V.D., Mühlenhoff U., Stümpfig M., Seebacher J., Kugler K.G., Renicke C., Taxis C., Gavin A.C., Pierik A.J., Lill R. (2015) Elife. 4, e08231. The deca-GX3 proteins Yae1-Lto1 function as adaptors recruiting the ABC protein Rli1 for iron-sulfur cluster insertion.

  • Glatt S., Zabel R., Vonkova I., Kumar A., Netz D.J., Pierik A.J., Rybin V., Lill R., Gavin A.C., Balbach J., Breunig K.D., Müller C.W. (2015) Structure 23, 149-160. Structure of the Kti11/Kti13 heterodimer and its double role in modifications of tRNA and eukaryotic elongation factor 2.

  • Rietzschel N., Pierik A.J., Bill E., Lill R., Mühlenhoff U. (2015) Mol. Cell. Biol. 35, 370-378. The basic leucine zipper stress response regulator Yap5 senses high-iron conditions by coordination of [2Fe-2S] clusters.

 

2014

  • Basu S., Netz D.J., Haindrich A.C., Herlerth N., Lagny T.J., Pierik A.J., Lill R., Lukeš J. (2014) Mol. Microbiol. 93, 897-910. Cytosolic iron-sulphur protein assembly is functionally conserved and essential in procyclic and bloodstream Trypanosoma brucei.

  • Webert H., Freibert S.A., Gallo A., Heidenreich T., Linne U., Amlacher S., Hurt E., Mühlenhoff U., Banci L., Lill R. (2014) Nat Commun. 5, 5013. Functional reconstitution of mitochondrial Fe/S cluster synthesis on Isu1 reveals the involvement of ferredoxin.

  • Srinivasan V., Pierik A.J., Lill R. (2014) Science 343, 1137-1140. Crystal structures of nucleotide-free and glutathione-bound mitochondrial ABC transporter Atm1.

 

2013

  • Uzarska M.A., Dutkiewicz R., Freibert S.A., Lill R., Mühlenhoff U. (2013) Mol. Biol. Cell. 24, 1830-1841. The mitochondrial Hsp70 chaperone Ssq1 facilitates Fe/S cluster transfer from Isu1 to Grx5 by complex formation.

  • Ringel P., Krausze J., van den Heuvel J., Curth U., Pierik A.J., Herzog S., Mendel R.R., Kruse T. (2013) J. Biol. Chem. 288, 14657-14671. Biochemical characterization of molybdenum cofactor-free nitrate reductase from Neurospora crassa.

 

2012

  • Soboh, B., Kuhns, M., Braussemann, M., Waclawek, M., Muhr, E., Pierik, A.J. & Sawers, R.G. (2012) Biochem Biophys. Res. Commun. 424, 158-163. Evidence for an oxygen-sensitive iron-sulfur cluster in an immature large subunit species of Escherichia coli [NiFe]-hydrogenase 2.

  • Netz, D. J. A., Pierik, A.J., Stümpfig, M., Bill, E., Sharma, A.K., Pallesen, L.J., Walden, W.E. & Lill, R. (2012) J. Biol. Chem. 287, 12365-12378. A bridging [4Fe-4S] cluster and nucleotide binding are essential for function of the Cfd1-Nbp35 complex as a scaffold in iron-sulfur protein maturation.

  • Netz, D. J. A., Stith, C. M., Stümpfig, M., Köpf, G., Vogel, D., Genau, H.M., Stodola, J.L., Lill, R., Burgers, P.M.J. & Pierik, A.J. (2012) Nat. Chem. Biol. 8, 125-132. Eukaryotic DNA polymerases require an iron-sulfur cluster for formation of active complexes.

  • Geisselbrecht, Y., Frühwirth, S., Schröder, C., Pierik, A.J., Klug, G., Essen, L.-O. (2012) EMBO Rep. 13, 223-229. CryB from Rhodobacter sphaeroides: a unique class of cryptochromes with new cofactors.

  • Hilberg, M., Pierik, A.J., Bill, E., Friedrich, T., Lippert, M.-L. & Heider, J. (2012) J. Biol. Inorg. Chem. 17, 49-56. Identification of FeS clusters in the glycyl-radical enzyme benzylsuccinate synthase via EPR and Mössbauer spectroscopy.

 

2011

  • Mühlenhoff, U., Richter, N., Pines, O., Pierik, A.J. & Lill, R. (2011) J. Biol. Chem. 286, 41205-41216. Specialized function of yeast Isa1 and Isa2 proteins in the maturation of mitochondrial [4Fe-4S] proteins.

  • Oberpichler, I., Pierik, A.J., Wesslowski, J., Pokorny, R., Rosen, R., Vugman, M., Zhang, F., Neubauer, O. Ron, E.Z., Batschauer, A. & Lamparter, T. (2011) PLoS ONE 6, e26775. A photolyase-like protein from Agrobacterium tumefaciens with an iron-sulfur cluster.

  • Parthasarathy, A., Pierik, A.J., Kahnt, J., Zelder, O. & Buckel, W. (2011) Biochemistry 50, 3540-3550. Substrate specificity of 2-hydroxyglutaryl-CoA dehydratase from Clostridium symbiosum: towards a bio-based production of adipic acid.

  • Miethke, M., Pierik, A.J., Peuckert, F., Seubert, A. & Marahiel, M.A. (2011) J. Biol. Chem. 286, 2245-2260. Identification and characterization of a novel-type ferric siderophore reductase from a Gram-positive extremophile.

 

2010 and before

  • Netz, D.J.A., Stümpfig, M., Doré, C., Mühlenhoff, U., Pierik, A.J. & Lill, R. (2010) Nat. Chem. Biol. 6, 758-765. Tah18 transfers electrons to Dre2 in cytosolic iron-sulfur protein biogenesis.

  • Sheftel, A.D., Stehling, O., Pierik, A.J., Elsässer, H.-P., Mühlenhoff, U., Webert, H., Hobler, A., Hannemann, F., Bernhardt, R. & Lill, R. (2010) Proc. Nat. Acad. Sci. USA 107, 11775-11780. Humans possess two mitochondrial ferredoxins, Fdx1 and Fdx2, with distinct roles in steroidogenesis, heme, and Fe/S cluster biosynthesis.

  • Kim, J., Pierik, A.J. & Buckel, W. (2010) ChemPhysChem 11, 1307-1312. A complex of 2-hydroxyisocaproyl-coenzyme A dehydratase and its activator from Clostridium difficile stabilized by aluminium tetrafluoride-adenosine diphosphate.

  • Albrecht, A.G., Netz, D.J.A., Miethke, M., Pierik, A.J., Burghaus, O., Peuckert, F., Lill, R. & Marahiel, M.A. (2010) J. Bacteriol. 192, 1643-1651. SufU is an essential iron-sulfur cluster scaffold protein in Bacillus subtilis.

  • Schwenkert, S., Netz, D.J.A., Frazzon, J., Pierik, A.J., Bill, E., Gross, J., Lill, R. & Meurer, J. (2010) Biochem. J. 425, 207-214. Chloroplast HCF101 is a scaffold protein for [4Fe-4S] cluster assembly.

  • Pierik, A.J., Netz, D.J.A. & Lill, R. (2009) Nat. Protoc. 4, 753-766. Analysis of iron-sulfur protein maturation in eukaryotes.

  •  Urzica, U., Pierik, A.J., Mühlenhoff, U. & Lill, R. (2009) Biochemistry 48, 4946-4958. Crucial role of conserved cysteine residues in the assembly of two iron-sulfur clusters on the CIA protein Nar1.

  • Bych, K., Netz, D.J.A., Vigani, G., Bill, E., Lill, R., Pierik, A.J. & Balk, J. (2008) J. Biol. Chem. 284, 35797-35804. The essential cytosolic iron-sulfur protein Nbp35 acts without Cfd1 partner in the green lineage.

  • Boyd, J.M., Pierik, A.J., Netz, D.J.A., Lill, R. & Downs, D.M. (2008) Biochemistry 47, 8195–8202. Bacterial ApbC can bind and effectively transfer iron−sulfur clusters.

  • Stehling, O., Netz, D.J.A., Niggemeyer, B., Rösser, R., Eisenstein, R.S., Puccio, H., Pierik, A.J. & Lill, R. (2008) Mol. Cell. Biol. 28, 5517-5528. Human Nbp35 is essential for both cytosolic iron-sulfur protein assembly and iron homeostasis.

  • Bych, K., Kerscher, S., Netz, D.J.A., Pierik, A.J., Zwicker, K., Huynen, M.A., Lill, R., Brandt, U. & Balk, J. (2008) EMBO J. 27, 1736–1746. The iron-sulphur protein Ind1 is required for effective complex I assembly.

  • Gelling, C., Dawes, I.W., Richhardt, N., Lill, R. & Mühlenhoff, U. (2008) Mol. Cell. Biol. 28, 1851-1861. Mitochondrial Iba57p is required for Fe/S cluster formation on aconitase and activation of radical SAM enzymes.

  • Kim, J., Darley, D.J., Buckel, W. & Pierik, A.J. (2008) Nature 452, 239-242. An allylic ketyl radical intermediate in clostridial amino-acid fermentation.

Zuletzt aktualisiert: 06.02.2019 · muehlenh

 
 
 
Fb. 20 - Medizin

Institut für Zytobiologie und Zytopathologie, Robert-Koch-Str. 6, D-35037 Marburg
Tel. +49 6421/28-66483, Fax +49 6421/28-66414, E-Mail: lesch@staff.uni-marburg.de

URL dieser Seite: https://www.uni-marburg.de/fb20/cyto/corefac

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