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Gleichzeitiges hochauflösendes Abbilden von Membranproteinen und dreidimensionales Kartieren der Wechselwirkungskräfte und Energielandschaften mit Liganden

English title Simultaneous high-resolution imaging of membrane proteins and three-dimensional mapping of their interaction forces and energy landscape with ligands
Applicant Müller Daniel Jobst
Number 160199
Funding scheme Project funding (Div. I-III)
Research institution Computational Systems Biology Department of Biosystems, D-BSSE ETH Zürich
Institution of higher education ETH Zurich - ETHZ
Main discipline Other disciplines of Physics
Start/End 01.03.2016 - 29.02.2020
Approved amount 516'232.00
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All Disciplines (4)

Discipline
Other disciplines of Physics
Biochemistry
Biophysics
Molecular Biology

Keywords (7)

Membrane Proteins; Molecular Biophysics; High-resolution Microscopy; Quantitative Measurements; Bionanotechnology; Biological Membranes; Receptor-Ligand Interactions

Lay Summary (German)

Lead
Membranproteine statten biologische Zellen mit einzigartigen und lebenswichtigen Funktionen aus. Deshalb wird Membranproteinen erhebliches Interesse in der biologischen, medizinischen und pharmakologischen Forschung entgegengebracht. Eine wichtige Frage gilt dabei zu verstehen wie Liganden (oder molekulare Wirkstoffe) an Membranproteine binden, um deren Funktion zu beeinflussen. Obwohl etablierte biophysikalische Methoden die Struktur von ligandengebundenen Membranproteinen lösen können, gibt es gegenwärtig keine Möglichkeit einzelne Membranproteine abzubilden und währenddessen deren Wechselwirkungen mit Liganden zu quantifizieren und strukturell zu lokalisieren. Auch gibt es keine Methode Membranproteine und deren freie Energielandschaft mit Liganden abzubilden. Solche Einblicke würden fundamentale Erkenntnissen beitragen können zum Verständnis wie Membranproteine mit ihrer Umgebung wechselwirken.
Lay summary

In diesem Projekt beabsichtigen wir eine Methode zu entwickeln welche es ermöglicht, einzelne Membranproteine in ihrer nativen Umgebung abzubilden und gleichzeitig deren Wechselwirkungen mit Liganden räumlich abzutasten. Diese räumliche Abtastung von Struktur und Wechselwirkung wird den Beobachter in die Lage versetzen quantitativ zu verstehen wie ein Ligand zu einem Membranrezeptor findet und diesen funktionell moduliert. Die Methode welche unter anderem auf humanen G-Protein gekoppelten Rezeptoren etabliert wird, wird selbst die freie Energielandschaft der Membranrezeptoren kartieren können und somit einzigartige thermodynamische und kinetische Einsichten in spezifische Wechselwirkungen der Membranproteine liefern. Diese Einsichten dazu beitragen die Wirkungsweise der Membranproteine quantitativ zu erfassen und verständlich zu machen.

Direct link to Lay Summary Last update: 27.12.2015

Lay Summary (English)

Lead
Membrane proteins provide unique and essential functions for basic cellular processes. This is the main reason why membrane proteins reach a high interest in biological, medical and pharmacological research. One key question of these fields is how ligands (or drugs or molecular compounds) bind to native membrane proteins thereby modulating their functional state. Although well-established methods can solve the structure of liganded membrane proteins, microscopic (or more suitable ‘nanoscopic’) methods that allow imaging single membrane proteins at nanometer resolution (˜1 nm) and at the same time maps the molecular interactions and energy landscape of a binding ligand do not exist. Such insight would contribute significantly to the chemical and physical understanding of how ligands, drugs and other molecular compounds interact with native membrane proteins.
Lay summary

Here we intend to develop and establish a multifunctional high-resolution microscopy method to image single native membrane proteins at sub-nanometer resolution and to simultaneously quantify and map their interaction forces and free energy landscape of ligand binding. Because the method will be applicable to quantify and to structurally map the interaction of any given molecule with membrane proteins or other biological surfaces it is of general interest for a plethora of biological systems. Once developed, this multifunctional and nanotechnological tool will be applicable to gain unique structural, kinetic and energetic insights of ligands interacting with membrane receptors, or of any given molecule interacting with any biological system.

Direct link to Lay Summary Last update: 27.12.2015

Responsible applicant and co-applicants

Employees

Publications

Publication
High-Resolution Imaging of Maltoporin LamB while Quantifying the Free-Energy Landscape and Asymmetry of Sugar Binding
Mulvihill Estefania, Pfreundschuh Moritz, Thoma Johannes, Ritzmann Noah, Müller Daniel J. (2019), High-Resolution Imaging of Maltoporin LamB while Quantifying the Free-Energy Landscape and Asymmetry of Sugar Binding, in Nano Letters, 19(9), 6442-6453.
Seeing and sensing single G protein-coupled receptors by atomic force microscopy
Sapra K Tanuj, Spoerri Patrizia M, Engel Andreas, Alsteens David, Müller Daniel J (2019), Seeing and sensing single G protein-coupled receptors by atomic force microscopy, in Current Opinion in Cell Biology, 57, 25-32.
Insertion and folding pathways of single membrane proteins guided by translocases and insertases
Serdiuk Tetiana, Steudle Anja, Mari Stefania A., Manioglu Selen, Kaback H. Ronald, Kuhn Andreas, Müller Daniel J. (2019), Insertion and folding pathways of single membrane proteins guided by translocases and insertases, in Science Advances, 5(1), eaau6824-eaau6824.
Protein-enriched outer membrane vesicles as a native platform for outer membrane protein studies
Thoma Johannes, Manioglu Selen, Kalbermatter David, Bosshart Patrick D., Fotiadis Dimitrios, Müller Daniel J. (2018), Protein-enriched outer membrane vesicles as a native platform for outer membrane protein studies, in Communications Biology, 1(1), 23-23.
Single-Molecule Force Spectroscopy of Transmembrane β-Barrel Proteins
Thoma Johannes, Sapra K. Tanuj, Müller Daniel J. (2018), Single-Molecule Force Spectroscopy of Transmembrane β-Barrel Proteins, in Annual Review of Analytical Chemistry, 11(1), 375-395.
Mechanism of membrane pore formation by human gasdermin‐D
Mulvihill Estefania, Sborgi Lorenzo, Mari Stefania A, Pfreundschuh Moritz, Hiller Sebastian, Müller Daniel J (2018), Mechanism of membrane pore formation by human gasdermin‐D, in The EMBO Journal, 37(14), e98321.
Structural Properties of the Human Protease-Activated Receptor 1 Changing by a Strong Antagonist
Spoerri Patrizia M., Kato Hideaki E., Pfreundschuh Moritz, Mari Stefania A., Serdiuk Tetiana, Thoma Johannes, Sapra K. Tanuj, Zhang Cheng, Kobilka Brian K., Müller Daniel J. (2018), Structural Properties of the Human Protease-Activated Receptor 1 Changing by a Strong Antagonist, in Structure, 26(6), 829-838.e4.
Tau protein liquid–liquid phase separation can initiate tau aggregation
Wegmann Susanne, Eftekharzadeh Bahareh, Tepper Katharina, Zoltowska Katarzyna M, Bennett Rachel E, Dujardin Simon, Laskowski Pawel R, MacKenzie Danny, Kamath Tarun, Commins Caitlin, Vanderburg Charles, Roe Allyson D, Fan Zhanyun, Molliex Amandine M, Hernandez‐Vega Amayra, Muller Daniel, Hyman Anthony A, Mandelkow Eckhard, Taylor J Paul, Hyman Bradley T (2018), Tau protein liquid–liquid phase separation can initiate tau aggregation, in The EMBO Journal, 37(7), e98049.
High-Resolution Imaging and Multiparametric Characterization of Native Membranes by Combining Confocal Microscopy and an Atomic Force Microscopy-Based Toolbox
Laskowski Pawel R., Pfreundschuh Moritz, Stauffer Mirko, Ucurum Zöhre, Fotiadis Dimitrios, Müller Daniel J. (2017), High-Resolution Imaging and Multiparametric Characterization of Native Membranes by Combining Confocal Microscopy and an Atomic Force Microscopy-Based Toolbox, in ACS Nano, 11(8), 8292-8301.
Maltoporin LamB Unfolds β Hairpins along Mechanical Stress-Dependent Unfolding Pathways
Thoma Johannes, Ritzmann Noah, Wolf Dominik, Mulvihill Estefania, Hiller Sebastian, Müller Daniel J. (2017), Maltoporin LamB Unfolds β Hairpins along Mechanical Stress-Dependent Unfolding Pathways, in Structure, 25(7), 1139-1144.e2.
Detecting Ligand-Binding Events and Free Energy Landscape while Imaging Membrane Receptors at Subnanometer Resolution
Pfreundschuh Moritz, Harder Daniel, Ucurum Zöhre, Fotiadis Dimitrios, Müller Daniel J. (2017), Detecting Ligand-Binding Events and Free Energy Landscape while Imaging Membrane Receptors at Subnanometer Resolution, in Nano Letters, 17(5), 3261-3269.
Imaging modes of atomic force microscopy for application in molecular and cell biology
Dufrêne Yves F., Ando Toshio, Garcia Ricardo, Alsteens David, Martinez-Martin David, Engel Andreas, Gerber Christoph, Müller Daniel J. (2017), Imaging modes of atomic force microscopy for application in molecular and cell biology, in Nature Nanotechnology, 12(4), 295-307.
Multiparametric Atomic Force Microscopy Imaging of Biomolecular and Cellular Systems
Alsteens David, Müller Daniel J., Dufrêne Yves F. (2017), Multiparametric Atomic Force Microscopy Imaging of Biomolecular and Cellular Systems, in Accounts of Chemical Research, 50(4), 924-931.
Unraveling the Pore-Forming Steps of Pneumolysin from Streptococcus pneumoniae
van Pee Katharina, Mulvihill Estefania, Müller Daniel J., Yildiz Özkan (2016), Unraveling the Pore-Forming Steps of Pneumolysin from Streptococcus pneumoniae, in Nano Letters, 16(12), 7915-7924.
YidC assists the stepwise and stochastic folding of membrane proteins
Serdiuk Tetiana, Balasubramaniam Dhandayuthapani, Sugihara Junichi, Mari Stefania A, Kaback H Ronald, Müller Daniel J (2016), YidC assists the stepwise and stochastic folding of membrane proteins, in Nature Chemical Biology, 12(11), 911-917.
SAS-6 engineering reveals interdependence between cartwheel and microtubules in determining centriole architecture
Hilbert Manuel, Noga Akira, Frey Daniel, Hamel Virginie, Guichard Paul, Kraatz Sebastian H. W., Pfreundschuh Moritz, Hosner Sarah, Flückiger Isabelle, Jaussi Rolf, Wieser Mara M., Thieltges Katherine M., Deupi Xavier, Müller Daniel J., Kammerer Richard A., Gönczy Pierre, Hirono Masafumi, Steinmetz Michel O. (2016), SAS-6 engineering reveals interdependence between cartwheel and microtubules in determining centriole architecture, in Nature Cell Biology, 18(4), 393-403.

Collaboration

Group / person Country
Types of collaboration
Prof. Dr. Krystof Palczewsky, Cleveland University, School of Medicine, Dept. Pharmacology United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Dr. Xiaodan Li, PSI Villingen Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Dr. Ron Kaback, UC Los Angeles United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Dr. Gina Sosinsky, University of California San Diego, School of Medicine United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
Prof. Dr. Brian Kobilka, Stanford University, Dept. Cellular and Molecular Physiology United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Annual Meeting of the Biophysical Society 2020 Talk given at a conference Revealing a Multistep Binding Mechanism of Insertases for Membrane Protein Insertion and Folding 15.02.2020 San Diego, United States of America Laskowski Pawel Ryszard;
Colloquium on Nano-LifeScience Talk given at a conference Quantifying and directing cell biological processes 06.02.2020 Berlin, Germany Müller Daniel Jobst;
International Scanning Probe Microscopy (ISPM 2019) Talk given at a conference Quantifying and directing cell biological processes 26.05.2019 Louvain, Belgium Müller Daniel Jobst;
EMBO Workshop, Current advances in protein translocation across membranes Talk given at a conference Multistep binding mechanism of YidC insertase 23.03.2019 Barcelona, Spain Laskowski Pawel Ryszard;
Annual Meeting of the Swizz Nanoscience Institute Talk given at a conference Quantifying and directing cell biological processes 13.09.2018 Lenserheide, Switzerland Müller Daniel Jobst;
Oswalt-Kolloquium 35 Years Membrane Biophysics Talk given at a conference Monitoring single membrane protein folding and insertion 15.08.2018 Frankfurt a Main, Germany Müller Daniel Jobst;
Dutch Biophysics Meeting Individual talk Quantifying and directing cell biological processes 29.10.2017 Veldhoven, Netherlands Müller Daniel Jobst;
Conference ‘New Horizon in Membrane Transport and Communication’ Talk given at a conference Quantifying and directing cell biological processes 04.10.2017 Frankfurt a Main, Germany Müller Daniel Jobst;
Annual Meeting of the German Biophysical Society (DGfB) Talk given at a conference AFM imaging and spectroscopy of single membrane proteins 26.09.2016 Erlangen, Germany Müller Daniel Jobst;
Membrane Protein Gordon Research Conference (GRC) Talk given at a conference Single membrane protein folding and insertion processes 10.09.2016 Easton, United States of America Müller Daniel Jobst;
Biomembrane Days Talk given at a conference AFM imaging and spectroscopy of single membrane proteins 05.09.2016 Berlin, Germany Müller Daniel Jobst;
Swiss Nanoconvention Individual talk Imaging, quantifying and directing biological processes 30.06.2016 Basel, Switzerland Müller Daniel Jobst;
Gordon Research Conference (GRC) on Biointerface Science Talk given at a conference Quantifying and directing cell biological processes 13.06.2016 Les Diablerets, Switzerland Müller Daniel Jobst;
NovAliX Conference ‘Biophysics in Drug Discovery 2016 Talk given at a conference Imaging and manipulating single membrane proteins by AFM 06.06.2016 Strasbourg, France Müller Daniel Jobst;


Self-organised

Title Date Place

Knowledge transfer events

Active participation

Title Type of contribution Date Place Persons involved
Filmfestival Locarno Talk 09.08.2019 Locarno, Switzerland Müller Daniel Jobst;
Filmfestival Locarno Workshop 07.08.2019 Locarno, Switzerland Manioglu Selen;


Communication with the public

Communication Title Media Place Year
Media relations: radio, television RSI Locarno Film Festival Interview Daniel Müller und Barbara Treutlein RSI Italian-speaking Switzerland 2019

Awards

Title Year
Wissenschaftlcihes Mitglied der Max Planck Gesellschaft, Deutschland 2020
Marsilius Medallie der Universiutät Heidelberg 2019
Elected EMBO Member 2016

Associated projects

Number Title Start Funding scheme
134521 Multifunktionelles hochauflösendes Abbilden und Kartieren der Kraftfelder biologischer Membranen und Membranproteine 01.03.2012 Project funding (Div. I-III)
189807 Multiphoton Confocal Microscope for High-Speed and High-Resolution Imaging 01.09.2020 R'EQUIP

Abstract

Membrane proteins are prominent actors in living cells providing unique and essential functions for basic cellular processes. This is the main reason why membrane proteins reach a high interest in biological, medical and pharmacological research. One key question of these fields is how ligands (or drugs or molecular compounds) bind to native membrane proteins thereby modulating their functional state. Although well-established methods can solve the structure of liganded membrane proteins, microscopic (or more suitable ‘nanoscopic’) methods that allow imaging single membrane proteins at nanometer resolution (˜1 nm) and at the same time structurally quantifying the interaction forces of a given ligand hardly exist. Moreover, high-resolution nanoscopic and spectroscopic approaches that enable to structurally observe membrane proteins at nanometer resolution and to simultaneously map the energy landscape of a binding ligand do not exist. Importantly, such insight would contribute significantly to the chemical and physical understanding of how ligands, drugs and other molecular compounds interact with native membrane proteins.Here we intend to develop and establish multifunctional high-resolution atomic force microscopy (AFM) that allows to image single native membrane proteins at sub-nanometer resolution and at the same time to quantify the interaction forces of a given ligand with a membrane protein. Therefore, we will employ force-distance (FD) curve-based AFM (FD-based AFM), which we have recently established to image the surface of biological membranes and membrane proteins at a resolution of =1 nm and to simultaneously map the interactions of single membrane proteins in three dimensions. We intend to further develop this method to detect how a ligand finds its way to specifically bind and to unbind from the receptor’s binding pocket. To do so a ligand is tethered to the AFM stylus and the interaction forces of the ligand with the membrane protein are localized and quantified while imaging the membrane protein at high-resolution. These interaction forces are then mapped in three-dimensions (3D) to the membrane protein at sub-nanometer resolution and pico-newton sensitivity. Because the method will also be applicable to quantify and to structurally map the interaction of any given molecule with membrane proteins or other biological surfaces it is of general interest for a plethora of biological systems. In initial experiments we could show the feasibility of this method at a relatively low resolution of ˜5 nm. However, because the interaction forces of ligands and membrane proteins (or of any other biological system) depend on the rate at which they are probed [9], the quantification of forces is relative. An approach towards providing an absolute measure would be to determine the energy landscape of a ligand interacting with a membrane protein. Thus, we take the challenge to develop FD-based AFM into a method, which allows imaging native proteins at high-resolution and to structurally map the energy landscape of a ligand interacting with a native protein. Once developed, this multifunctional and nanotechnological tool will be applicable to gain unique structural, kinetic and energetic insights of ligands interacting with membrane receptors, or of any given molecule interacting with any biological system.
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