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Functional investigations of bacterial and eukaryotic membrane proteins

English title Functional investigations of bacterial and eukaryotic membrane proteins
Applicant von Ballmoos Christoph
Number 176154
Funding scheme Project funding (Div. I-III)
Research institution Departement für Chemie, Biochemie und Pharmazie Universität Bern
Institution of higher education University of Berne - BE
Main discipline Biochemistry
Start/End 01.12.2017 - 31.01.2022
Approved amount 700'000.00
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All Disciplines (2)

Discipline
Biochemistry
Biophysics

Keywords (7)

giant unilamellar vesicle; eukaryotic membrane transport; oxidative phosphorylation; lateral proton transfer; sugar transport; ATP synthesis; fluorescence microscopy

Lay Summary (German)

Lead
Biologische Membranen erlauben es der Zelle, Kompartimente mit verschiedenen chemischen Eigenschaften voneinander abzugrenzen. Transport und Kommunikation zwischen diesen Kompartimenten wird von Proteinen durchgeführt, welche fest in der biologischen Membran integriert sind. Der experimentelle Umgang mit diesen Membranproteinen ist allerdings erschwert und deren molekularer Mechanismus ist oft unbekannt. Durch ihre wichtige Rolle sind Membranproteine interessante "targets" von Medikamenten. Das vorgestellte Projekt trägt zur Erforschung einiger dieser Membranproteine bei.
Lay summary

Im ersten Teil des Projekts geht es darum, mithilfe fluoreszenzmikroskopischer Methoden neue Techniken für die funktionelle Untersuchung von eukaryotischen Membranproteinen zu entwickeln. Um die molekulare Funktion dieser Proteine im Detail zu verstehen, müssen diese vorgängig isoliert und in gereinigter Form wieder in eine membranähnliche Umgebung gebracht werden. Im Gegensatz zu bakteriellen Membranproteinen ist dieser Prozess bei eukaryotischen Proteinen durch die geringen Mengen an Ausgangsmaterial erschwert. Wir planen, diesen Prozess zu miniaturisieren und die funktionellen Messungen direkt unter einem Fluoreszenz-Mikroskop auszuführen. Im Fokus stehen dabei zwei Membranproteine, welche den Transport von kleinen Molekülen (Fructose, Natrium-Ionen) über die Membran katalysieren.

Im zweiten Projekt geht es darum, das molekulare Zusammenspiel derjenigen Proteinkomplexe zu untersuchen, welche für die Herstellung der zellulären Energie ATP zuständig sind. Obwohl dieser Prozess in seinen Grundsätzen seit rund vier Jahrzehnten bekannt ist und untersucht wird, fehlt uns weiterhin das Verständnis von vielen grundlegenden Zusammenhängen. Diese Proteinkomplexe befinden sich in der inneren Membran der Mitochondrien und eine Fehlfunktion des Energieumwandlungsprozesses wird mit vielen neurodegenerativen und altersbedingten Krankheiten in Verbindung gebracht. Unsere Grundlagenforschung soll zum detaillierten Verständnis beitragen, wie subtile Änderungen dieser Komplexe das delikate Energiegleichgewicht der Zelle beeinflussen.

Direct link to Lay Summary Last update: 28.11.2017

Responsible applicant and co-applicants

Employees

Publications

Publication
Current problems and future avenues in proteoliposome research
Amati Andrea Marco, Graf Simone, Deutschmann Sabina, Dolder Nicolas, von Ballmoos Christoph (2020), Current problems and future avenues in proteoliposome research, in Biochemical Society Transactions, 48(4), 1473.
CD31 (PECAM-1) Serves as the Endothelial Cell-Specific Receptor of Clostridium perfringens β-Toxin
Bruggisser Julia, Tarek Basma, Wyder Marianne, Müller Philipp, von Ballmoos Christoph, Witz Guillaume, Enzmann Gaby, Deutsch Urban, Engelhardt Britta, Posthaus Horst (2020), CD31 (PECAM-1) Serves as the Endothelial Cell-Specific Receptor of Clostridium perfringens β-Toxin, in Cell Host {&} Microbe, 28(1), 69.
Membrane‐Bound Superoxide Oxidase
Sjöstrand Dan, Högbom Martin, Ballmoos Christoph (2020), Membrane‐Bound Superoxide Oxidase, in Encyclopedia of Inorganic and Bioinorganic Chemistry, 1-10.
Bifunctional DNA Duplexes Permit Efficient Incorporation of pH Probes into Liposomes
Dolder Nicolas, Ballmoos Christoph (2020), Bifunctional DNA Duplexes Permit Efficient Incorporation of pH Probes into Liposomes, in ChemBioChem, 21(15), 2219-2224.
Kinetic coupling of the respiratory chain with ATP synthase, but not proton gradients, drives ATP production in cristae membranes
Toth Alexandra, Meyrat Axel, Stoldt Stefan, Santiago Ricardo, Wenzel Dirk, Jakobs Stefan, von Ballmoos Christoph, Ott Martin (2020), Kinetic coupling of the respiratory chain with ATP synthase, but not proton gradients, drives ATP production in cristae membranes, in Proceedings of the National Academy of Sciences, 117(5), 2412-2421.
The proton pumping bo oxidase from Vitreoscilla
Graf Simone, Brzezinski Peter, von Ballmoos Christoph (2019), The proton pumping bo oxidase from Vitreoscilla, in Scientific Reports, 9(1), 4766.
ATP synthesis at physiological nucleotide concentrations
Meyrat Axel, von Ballmoos Christoph (2019), ATP synthesis at physiological nucleotide concentrations, in Scientific Reports, 9(1), 3070.
Scavenging of superoxide by a membrane-bound superoxide oxidase
Lundgren Camilla A. K., Sjöstrand Dan, Biner Olivier, Bennett Matthew, Rudling Axel, Johansson Ann-Louise, Brzezinski Peter, Carlsson Jens, von Ballmoos Christoph, Högbom Martin (2018), Scavenging of superoxide by a membrane-bound superoxide oxidase, in Nature Chemical Biology, 14(8), 788-793.
Towards a Synthetic Mitochondrion
Biner Olivier, Schick Thomas, Ganguin Aymar Abel, von Ballmoos Christoph (2018), Towards a Synthetic Mitochondrion, in CHIMIA International Journal for Chemistry, 72(5), 291-296.
The lateral distance between a proton pump and ATP synthase determines the ATP-synthesis rate
Sjöholm Johannes, Bergstrand Jan, Nilsson Tobias, Šachl Radek, von Ballmoos Christoph, 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.
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.
Splitting of the O–O bond at the heme-copper catalytic site of respiratory oxidases
Poiana Federica, Ballmoos Christoph von, Gonska Nathalie, Blomberg Margareta R. A., Ädelroth Pia, Brzezinski Peter (2017), Splitting of the O–O bond at the heme-copper catalytic site of respiratory oxidases, in Sciences Advances, 3(6), 1.

Collaboration

Group / person Country
Types of collaboration
Horst Posthaus Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Exchange of personnel
Stavros Stavrakis, ETH Zürich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
- Exchange of personnel
Robert Gennis, Urbana Champaign United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Thorsten Friedrich, Universität Freiburg Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
Manuala Pereira, Universität Lissabon Portugal (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Exchange of personnel

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
GRC Bioenergetics Talk given at a conference Superoxide Oxidase - a new player in the respiratory chain 02.06.2019 Andover, United States of America Amati Andrea Marco; von Ballmoos Christoph;
EBEC 2018 Talk given at a conference Superoxide: quinone oxidoreductase – a new player in the respiratory chain? 25.08.2018 Budapest, Hungary Schick Thomas; Amati Andrea Marco; von Ballmoos Christoph;


Associated projects

Number Title Start Funding scheme
153351 Novel technologies for the functional investigation of membrane proteins 01.09.2014 Project funding (Div. I-III)
121481 Lateral proton diffusion along membrane surfaces: a fundamental principle in membrane bioenergetics 01.08.2008 Fellowships for advanced researchers

Abstract

Biological membranes play vital roles in all living organisms. They build a protective barrier with very limited permeability between different cellular components or to the extracellular space, and also actively participate in cellular processes. Transport of biological molecules (e.g. ions, nutrients, and neurotransmitter) across theses membranes are mediated and controlled by membrane proteins (MPs). In contrast to their soluble counter-parts, MPs are insoluble in aqueous solution until detergent is added, and for measuring transmembrane transport activities, they have to be embedded back into a membrane mimetic system. Our laboratory works with several MPs that are either involved in energy conservation or membrane transport of ions and solutes. Typically, we use a bottom-up process, reassembling a functional unit from purified components to understand the mechanistic details and cellular function.With the proposed experiments, we want to investigate both mechanical and technical aspects of a subset of these MPs. In Part A, we use methodologies that we developed over the last two years to reconstitute eukaryotic fructose (glut5) and glutamate transporter (vglut2) into giant unilamellar vesicles. Their functionality will be assessed by fluorescence microscopy assays, which we aim to develop here. This method requires very small amounts of proteins, which is ideal for eukaryotic proteins, where expression and purification is often cumbersome and pure protein is precious. With the results of this project, we hope to contribute experimental procedures that are attractive for many researchers in basic and applied research (e.g. drug screening). In part B, we propose kinetic experiments which are aimed to decipher the mechanistic details of proton transport and ATP synthesis during oxidative phosphorylation. In particular, we try to understand the role of the membrane during proton shuttling between cytochrome oxidase and ATP synthase. We recently have found that in isolated systems, membrane localized proton transfer between the two enzymes occurs, if the average distance between proton donor and acceptor is below a certain threshold, and that the average distance is affected by the lipid composition. Using rapid kinetic and spectroscopic techniques, we aim to understand the mechanistic details of this phenomenon, which could be universal for all energy conserving membranes. Furthermore, we aim to use electrochemical tools to quantitatively assess the efficiency of energy coupling between respiration and ATP synthesis in a minimal energy converting system.
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