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Novel technologies for the functional investigation of membrane proteins

English title Novel technologies for the functional investigation of membrane proteins
Applicant von Ballmoos Christoph
Number 153351
Funding scheme Project funding (Div. I-III)
Research institution Departement für Chemie und Biochemie Universität Bern
Institution of higher education University of Berne - BE
Main discipline Biochemistry
Start/End 01.09.2014 - 30.11.2017
Approved amount 474'000.00
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All Disciplines (2)

Discipline
Biochemistry
Biophysics

Keywords (8)

liposome; reconstitution; ATP synthase; SNARE protein; membrane protein; respiratory chain; giant unilamellar vesicle; membrane fusion

Lay Summary (German)

Lead
Biologische Membranen bieten Zellen nicht nur Schutz vor der Außenwelt, sondern erlauben ihnen auch, ihr Inneres in Kompartimente mit verschiedenen chemischen Eigenschaften zu unterteilen. Die Unterschiedlichkeit dieser Kompartimente ist eine wichtige Voraussetzung für die Vielzahl an Prozessen, die in einer lebenden Zelle stattfinden. Der Transport und die Kommunikation zwischen diesen Kompartimenten wird von Proteinen durchgeführt, welche fest in der biologischen Membran integriert sind, welche die Kompartimente trennt. Durch ihre wichtige Rolle sind Membranproteine das Ziel von mehr als 50% der neueren Medikamente. Das vorgestellte Projekt trägt zur Erforschung von diesen Membranproteinen bei.
Lay summary

Das Ziel dieses Projekt ist es, neue biochemische und biophysikalische Methoden zu entwickeln, um die molekulare Erforschung von Membranproteinen voranzutreiben. Um die Komplexität einer biologischen Zelle zu reduzieren, isolieren wir einige, wenige Membranproteine und stellen diese rein dar. Um ihren Mechanismus zu erforschen, werden die Proteine wieder zurück in eine chemisch definierte Membran gebracht und mit diversen Methoden untersucht. Während des Projekts werden wir versuchen, komplexe Systeme mit mehreren Proteinen zu erarbeiten, um das Zusammenspiel dieser Proteine zu untersuchen. Die Möglichkeit, mehrere Proteine gleichzeitig zu untersuchen, ist eine wichtige Voraussetzung, um biologische Kaskaden zu verstehen. Unsere Methoden beinhalten eine Kombination von Erkenntnissen aus der Materialchemie, unserer Expertise mit Membranproteinen und Visualisierungstechniken wie Spektroskopie und Fluoreszenzmikroskopie.
Die Ergebnisse des vorgelegten Projekts werden helfen, neue, komplexere Fragestellungen mithilfe von biochemischen Experimenten zu beantworten. Dabei werden wir unsere gewonnen methodischen Fortschritte zur Erforschung der Proteine der Atmungskette in Bakterien anwenden, welche potentielle Angriffspunkte für Inhibitoren hat, die als Antibiotikaersatz in Frage kommen.

Direct link to Lay Summary Last update: 23.09.2014

Responsible applicant and co-applicants

Employees

Publications

Publication
Activation of Proton Translocation by Respiratory Complex I.
Belevich Nikolai, von Ballmoos Christoph, Verkhovskaya Marina (2017), Activation of Proton Translocation by Respiratory Complex I., in Biochemistry, 56(42), 5691-5697.
The lateral distance between a proton pump and ATP synthase determines the ATP-synthesis rate.
Sjöholm Johannes, Bergstrand Jan, Nilsson Tobias, Šachl Radek, Ballmoos Christoph von, Widengren Jerker, Brzezinski Peter (2017), The lateral distance between a proton pump and ATP synthase determines the ATP-synthesis rate., in Scientific reports, 7(1), 2926-2926.
Delivery of membrane proteins into small and giant unilamellar vesicles by charge-mediated fusion.
Biner Olivier, Schick Thomas, Müller Yannic, von Ballmoos Christoph (2016), Delivery of membrane proteins into small and giant unilamellar vesicles by charge-mediated fusion., in FEBS letters, 590(14), 2051-2062.
Isolation of yeast complex IV in native lipid nanodiscs.
Smirnova Irina A, Sjöstrand Dan, Li Fei, Björck Markus, Schäfer Jacob, Östbye Henrik, Högbom Martin, von Ballmoos Christoph, Lander Gabriel C, Ädelroth Pia, Brzezinski Peter (2016), Isolation of yeast complex IV in native lipid nanodiscs., in Biochimica et biophysica acta, 1858(12), 2984-2992.
Lipid-mediated Protein-protein Interactions Modulate Respiration-driven ATP Synthesis.
Nilsson Tobias, Lundin Camilla Rydström, Nordlund Gustav, Ädelroth Pia, von Ballmoos Christoph, Brzezinski Peter (2016), Lipid-mediated Protein-protein Interactions Modulate Respiration-driven ATP Synthesis., in Scientific reports, 6, 24113-24113.
Mimicking respiratory phosphorylation using purified enzymes.
von Ballmoos Christoph, Biner Olivier, Nilsson Tobias, Brzezinski Peter (2016), Mimicking respiratory phosphorylation using purified enzymes., in Biochimica et biophysica acta, 1857(4), 321-31.
SNARE-fusion mediated insertion of membrane proteins into native and artificial membranes.
Nordlund Gustav, Brzezinski Peter, von Ballmoos Christoph (2014), SNARE-fusion mediated insertion of membrane proteins into native and artificial membranes., in Nature communications, 5, 4303-4303.

Collaboration

Group / person Country
Types of collaboration
Peter Brzezinski, Universtät Stockholm Sweden (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
David Drew, Stockholm Universität Sweden (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Exchange of personnel
Reinhard Jahn, MPI Göttingen Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
European Bioenergetics Conference Poster Towards synthetic respiration – methods for the co-reconstitution of respiratory chain enzymes 02.01.2018 Riva del Garda, Italy Schick Thomas;
International Conference of Molecular Systems Engineering Poster Towards unidirectional reconstitution of membrane proteins into liposomes. 27.08.2017 Basel, Switzerland Amati Andrea Marco;
19th IUPAB congress and 11th EBSA congress Poster Delivery of membrane proteins into small and giant unilamellar vesicles by charge-mediated fusion 16.07.2017 edinburgh, Great Britain and Northern Ireland Schick Thomas;
Gordon Conference Bioenergetics Poster Delivery of respiratory chain enzymes into membrane mimicking systems by charge-mediated fusion 04.07.2017 Andover, United States of America von Ballmoos Christoph; Graf Simone;
Annual Meeting of the German Biophysical Society Poster Towards Unidirectional Reconstitution Of Membrane Proteins Into Liposomes 25.09.2016 Erlange, Germany Amati Andrea Marco;
CEF‐Symposium: Structures, Mechanism and Dynamics of Membrane Complexes Talk given at a conference Membrane fusion as a tool for co-reconstitution of membrane proteins into liposomes 24.09.2015 Bad Homburg, Germany von Ballmoos Christoph;


Communication with the public

Communication Title Media Place Year

Associated projects

Number Title Start Funding scheme
176154 Functional investigations of bacterial and eukaryotic membrane proteins 01.12.2017 Project funding (Div. I-III)
157704 Direct electron detector and phase plate for cryo-transmission electron microscopy of biological samples 01.12.2014 R'EQUIP

Abstract

Goals:To establish new technologies that allow the functional investigation of membrane proteins on a molecular level. A special emphasis is given on the interplay of more than one membrane protein. Methods are developed to investigate the membrane proteins in their native environment, a lipid bilayer. The developed methods are tested with membrane proteins from bacterial respiratory chains.Specific goals:•The controlled incorporation of more than one membrane protein by a “building block” strategy. Every membrane protein is first optimally reconstituted in a small liposome before they are fused into a single membrane, mediated by SNARE proteins. •Reconstituted in giant unilamellar vesicles, membranes proteins at work are directly observed under a microscope, using fluorescent techniques.•Reconstruction of an “artificial energy cell” from purified components.Scientific impact an relevance:In all life forms, biological membranes separate the contents of different compartments. Thereby, transport of molecules and communication between these compartments and thus across membranes is crucial. This task is performed by membrane proteins, the doors and windows of biological membranes. Due to their important role, MPs are the targets of more than 50% of modern drugs, making their molecular mechanism interesting for both basic and applied research. The complexity of an intact membrane, however, complicates the interpretation of many experiments, including drug binding studies to membrane bound receptors.In vitro experiments with purified components are thus necessary to elucidate the molecular details of cellular processes. The technologies outlined in this proposal are not specific for the respiratory enzymes investigated here. Instead, they are generally applicable to all kind of membrane proteins and their interaction partners. Experimental methods:Mainly spectroscopic techniques will be used to follow enzyme activities in situ. For ion translocation processes, specific fluorescent dyes will be used and the changes analyzed according to their time scale (stopped flow or normal mixing). For visualization of giant vesicles, phase contrast and fluorescent (confocal) microscopy will be used.
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