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Biomacromolecular structures, dynamics and interactions by NMR and new developments in NMR technology

English title Biomacromolecular structures, dynamics and interactions by NMR and new developments in NMR technology
Applicant Grzesiek Stephan
Number 173089
Funding scheme Project funding (Div. I-III)
Research institution Abteilung Strukturbiologie und Biophysik Biozentrum Universität Basel
Institution of higher education University of Basel - BS
Main discipline Biophysics
Start/End 01.04.2017 - 31.03.2021
Approved amount 1'112'000.00
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Keywords (13)

cyclic di-GMP; HIV; GPCR; protein interaction; structure; Abelson kinase; protein; CCR5; adrenergic receptor; protein folding; dynamics; nuclear magnetic resonance; cancer

Lay Summary (German)

Lead
Biologische Funktion folgt aus den zeitabhängigen Wechselwirkungen zwischen biologischen Molekülen. Diese können durch Kernspinresonanzspektroskopie (NMR) mit atomarer Auflösung bestimmt werden. In diesem Projekt soll diese Methode auf medizinisch relevante Proteine wie G-Protein gekoppelte Rezeptoren, das Leukämie-Protein Abelson-Kinase, und bakterielle Virulenzproteine angewendet und weiterentwickelt werden. Dies leistet einen Beitrag zum Verständnis ihrer Funktion und zu ihrer möglichen Beeinflussung durch Medikamente.
Lay summary

Teilprojekt A hat zum Ziel Struktur, Dynamik und Wechselwirkungen von mehreren medizinisch relevanten Systemen zu untersuchen, für die wir in den letzten Jahren Vorarbeiten geleistet haben: (1) zwei G-Protein gekoppelte Rezeptoren, der beta1-adrenerge Rezeptor und der HIV-Korezeptor CCR5. Für beide sind die Signalübertragungsmechanismen weitgehend unbekannt und die bisher erhaltenen Spektren geben zur Hoffnung Anlass, dass wesentliche Teile der Signalübertragung durch NMR sichtbar gemacht werden können. (2)  Zyklisches di-GMP (c-di-GMP) ist ein bakterieller Virulenzfaktor, der für Biofilmentstehung verantwortlich ist. Wir haben bereits die Struktur und Dynamik von mehreren c-di-GMP Rezeptorproteinen aufgeklärt. In dem Projekt soll nun ein kritischer Signalweg, der den bakteriellen Flagellenmotor durch c-di-GMP steuert, untersucht werden. (3) Das Protein Abelson Kinase ist ein Zielprotein für Medikamente gegen chronische myeloische Leukämie (CML). Wir haben durch NMR bereits den Einfluss von Medikamenten, anderen kleinen Liganden und Mutationen auf Bewegung von mehreren Abelson Teildomänen sichtbar machen können. Dies soll nun mit höherer Auflösung auf der Kinaseteildomäne untersucht werden. Zudem sollen Fluoreszenzmethoden entwickelt werden, um diese Bewegung am Einzelmolekül sichtbar zu machen.

Teilprojekt B hat zum Ziel verbesserte Methoden zur Markierung von Proteinen mit den stabilen Isotopen 13C, 15N, 2H in eukaryotischen Zellen zu entwickeln. Dies ist eine wichtige Voraussetzung für die Untersuchung komplizierterer, menschlicher Proteine durch Kernspinresonanz.

Direct link to Lay Summary Last update: 11.04.2017

Responsible applicant and co-applicants

Employees

Publications

Publication
Versatile modules enable automated multi-column purifications on the ÄKTA pure chromatography system
Franke Bastian, Frigård Tuomo, Grzesiek Stephan, Isogai Shin (2020), Versatile modules enable automated multi-column purifications on the ÄKTA pure chromatography system, in Journal of Chromatography A, 460846-460846.
High Pressure Shifts the β1-Adrenergic Receptor to the Active Conformation in the Absence of G Protein
Abiko Layara Akemi, Grahl Anne, Grzesiek Stephan (2019), High Pressure Shifts the β1-Adrenergic Receptor to the Active Conformation in the Absence of G Protein, in Journal of the American Chemical Society, 141(42), 16663-16670.
Rapid determination of general cell status, cell viability, and optimal harvest time in eukaryotic cell cultures by impedance flow cytometry
Opitz Christian, Schade Grit, Kaufmann Silvan, Berardino Marco Di, Ottiger Marcel, Grzesiek Stephan (2019), Rapid determination of general cell status, cell viability, and optimal harvest time in eukaryotic cell cultures by impedance flow cytometry, in Applied Microbiology and Biotechnology, 103(20), 8619-8629.
Deuterium induces a distinctive Escherichia coli proteome that correlates with the reduction in growth rate
Opitz Christian, Ahrné Erik, Goldie Kenneth N., Schmidt Alexander, Grzesiek Stephan (2018), Deuterium induces a distinctive Escherichia coli proteome that correlates with the reduction in growth rate, in Journal of Biological Chemistry, 294(7), 2279-2292.
Production of isotope-labeled proteins in insect cells for NMR
Franke Bastian, Opitz Christian, Isogai Shin, Grahl Anne, Delgado Leonildo, Gossert Alvar D., Grzesiek Stephan (2018), Production of isotope-labeled proteins in insect cells for NMR, in Journal of Biomolecular {NMR}, 71(3), 173-184.
ATP Site Ligands Determine the Assembly State of the Abelson Kinase Regulatory Core via the Activation Loop Conformation
Sonti Rajesh, Hertel-Hering Ines, Lamontanara Allan Joaquim, Hantschel Oliver, Grzesiek Stephan (2018), ATP Site Ligands Determine the Assembly State of the Abelson Kinase Regulatory Core via the Activation Loop Conformation, in Journal of the American Chemical Society, 140(5), 1863-1869.
Cyclic di-GMP differentially tunes a bacterial flagellar motor through a novel class of CheY-like regulators.
Schirmer Tilman, Nesper Jutta, Hug Isabelle, Hee Chee Seng, Habazettl Judith Maria, Manfredi Pablo, Grzesiek Stephan, Emonet Thierry, Jenal Urs, Kato Setsu (2017), Cyclic di-GMP differentially tunes a bacterial flagellar motor through a novel class of CheY-like regulators., in eLife, 6, e28842- e28842.

Collaboration

Group / person Country
Types of collaboration
J. Lewandowski, U. Warwick Great Britain and Northern Ireland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
T. Schirmer/U. Basel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
H. Stahlberg/U. Basel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
- Exchange of personnel
T. Maier/U. Basel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
O. Hantschel/EPFL Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
O. Hartley/U. Geneva Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
A. Gossert/ETHZ Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
G. Schertler/Paul-Scherrer-Institut Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
C. Seidel/U. Duesseldorf Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
- Exchange of personnel
R. Neher/U. Basel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
W. Jahnke/Novartis NIBR Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Industry/business/other use-inspired collaboration
U. Jenal/U. Basel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
D. Haeussinger/U. Basel Switzerland (Europe)
- 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
EMBO Practical Course: Structure determination of biological macromolecules by solution NMR Talk given at a conference Lectures on relaxation and residual dipolar couplings. 26.08.2019 München, Germany Grzesiek Stephan;
EUROISMAR 2019 Talk given at a conference A high-resolution description of functional dynamics and allosteric coupling of the β1-adrenergic receptor from backbone NMR 25.08.2019 Berlin, Germany Grahl Anne;
33rd Annual Symposium of The Protein Society Talk given at a conference Dynamics of GPCR Signal Transmission and Allosteric Regulation Detected by NMR 30.06.2019 Seattle, WA, United States of America Grzesiek Stephan;
Pasteur International Conference “NMR: a tool for Biology“ Talk given at a conference Insights into allosteric regulation by solution NMR 28.01.2019 Paris, France Grzesiek Stephan;
CECAM workshop “Multiscale simulations of allosteric regulatory mechanisms in cancer-associated proteins and signaling protein networks” Talk given at a conference Insights into allosteric regulation of Abelson kinase and GPCRs by solution NMR. 15.10.2018 Lugano, Switzerland Grzesiek Stephan;
EUROMAR 2018 Talk given at a conference Isotope labeling in higher eukaryotes makes more proteins amenable to NMR: applications to signal transmission in GPCRs 01.07.2018 Nantes, France Grzesiek Stephan;
XXII Swiss NMR Symposium Talk given at a conference Deciphering allosteric signaling by NMR 16.01.2018 Zürich, Switzerland Grzesiek Stephan;
EMBO Practical Course: Structure determination of biological macromolecules by solution NMR Talk given at a conference Lectures on relaxation and residual dipolar couplings. 05.08.2017 Basel, Switzerland Grzesiek Stephan;
20th ISMAR Conference Talk given at a conference Insights into allosteric regulation of GPCRs by solution NMR. 23.07.2017 Québec City, Canada Grzesiek Stephan;


Self-organised

Title Date Place

Communication with the public

Communication Title Media Place Year
Print (books, brochures, leaflets) Kann man die Biologie vorausberechnen?, in Natura Obscura, Natf. Ges. i. Basel German-speaking Switzerland 2017

Awards

Title Year
Suraj Manrao Student Poster Award at the XXVIII International Conference on Magnetic Resonance in Biological Systems, ICMRBS, 2018 on 19-24 August in Dublin, Ireland 2019
Elected Vice President of the International Society of Magnetic Resonance (ISMAR) for the years 2018-2021 2018

Associated projects

Number Title Start Funding scheme
141898 NMR studies of GPCRs: Structure, dynamics and interactions with ligands and signaling proteins 01.12.2012 Sinergia
149927 Biomacromolecular structures, dynamics and interactions by NMR and new developments in NMR technology 01.10.2013 Project funding (Div. I-III)
168031 Elucidating allosteric signal transmission in the beta1-adrenergic receptor 01.04.2017 Ambizione

Abstract

Biological function results from time-dependent interactions between biomolecules. NMR spectroscopy is the only experimental method, which yields both structural and dynamical information on biomolecules at atomic resolution with minimal invasiveness and at close to natural conditions. As such it can provide unique information to understand the connection between structure, dynamics and function. It is the goal of this proposal to apply and further develop these strengths of NMR technology in two subprojects.Subproject A is directed towards the determination of structure, dynamics, and interactions in several medically important systems, for which we have made significant progress in recent years: (1) two G-protein coupled receptors (GPCRs), i.e. the beta1-adrenergic receptor (b1AR) and the chemokine receptor CCR5, which is also the coreceptor for HIV. For b1AR, we could show that it is possible to follow signal transduction by 1H-15N backbone resonances. We now want to carry out a full NMR dynamics characterization comprising the binding of drug, G protein and arrestin ligands in different membrane environments. Beside solution NMR, this will also entail the study in full lipid bilayers by solid state NMR. Thus we hope to establish b1AR as a reference system for studies of GPCRs by NMR. We have recently obtained solution spectra for CCR5 of comparable quality to b1AR. We could also detect many resonances of the engineered chemokine ligand 5P12-RANTES in complex with CCR5 by solid state NMR. 5P12-RANTES is currently in clinical trials as an HIV entry inhibitor. We want to carry out a solution and solid state NMR analysis of this receptor with 5P12-RANTES and engineered RANTES ligands that elicit different arrestin and G-protein signaling activities with the aim to provide the structural and dynamical explanation for this differing signaling behavior. (2) Interactions of the bacterial virulence factor cyclic di-guanosine-monophosphate (c-di-GMP). We have solved the structures of several c-di-GMP protein complexes and c-di-GMP oligomers as well as determined the kinetics of c-di-GMP oligomer formation in solution. In particular, we could recently solve the structure of the c-di-GMP recognition region of the chemotactic protein CleD in complex with c-di-GMP. C-di-GMP binding to CleD induces formation of a ternary complex with the flagellar motor switch protein FliM thereby controlling bacterial flagellar motor function. We now want to solve the structure of the full c-di-GMP•CleD complex and study the formation of the ternary c-di-GMP•CleD•FliM complex. (3) Abl kinase, which is an important leukemia drug target. We have shown that binding of different classes of inhibitors to a large, multidomain Abl construct induces distinct domain rearrangements, which shed light on the mechanism of kinase regulation. We have now assessed the influence of a number of medically relevant point mutations and studied a larger class of inhibitors. In particular, the opening of the multidomain structure is correlated to the conformation of the activation loop in the catalytic domain. We now want to obtain an in-depth dynamic description of the catalytic domain, which is possible due to an advance in labeling techniques, as well as continue to develop suitable labeling of Abl for single molecule FRET studies.Subproject B is directed towards NMR technique development. Often proteins from higher eukaryotes cannot be expressed in functional form in E. coli, but only in higher eukaryotic expression systems. The difficulties of isotope labeling in such systems presents a bottleneck for the analysis by modern heteronuclear NMR. We have recently developed a robust method for isotope labeling in insect cells by feeding isotope-labeled yeast extract. The method currently yields 90 % 15N and 13C as well as >60 % 2H labeling with very good yields. We want to improve this method by increasing the level of 2H incorporation and cost efficiency for 13C labeling as well as extend it to other eukaryotic systems. We also want to obtain an in-depth understanding of the often adverse effects of 2H incorporation to cellular behavior by a proteomics analysis of the response to growth on 2H2O for E. coli, yeast and higher eukaryotes.
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