Projects & Collaborations 12 foundShow per page10 10 20 50 Efficient Characterization of Biomolecular Interactions by Automated Isothermal Titration Calorimetry Research Project | 5 Project MembersInhalt und Ziel des Forschungsprojekts Im Gegensatz zu anderen Methoden ist die isothermale Titrationskalorimetrie (ITC) in einzigartiger Weise geeignet, die Wechselwirkung von unmodifizierten Bindungspartnern anhand der aufgenommenen oder abgegebenen Bindungswärme zu messen. Messungen mittels ITC erlauben dabei eine thermodynamische Charakterisierung der Wechselwirkungen zwischen Bindungspartnern. Diese erlaubt, zusammen mit Strukturinformationen, die Mechanismen der Bindung im Detail zu verstehen und zu erklären, warum verschiedene Bindungspartner unterschiedlich stark interagieren. In diesem Projekt wird ein automatisiertes System für ITC Messungen aufgebaut, das hilft, wesentliche Nachteile von manuellen Messungen hinsichtlich des Durchsatzes und der Datenqualität zu überkommen. Das neue ITC System wird in der Biophysik Forschungsserviceeinheit (BF) des Biozentrums der Universität Basel installiert und betrieben, und steht dort auch externen Nutzern zur Verfügung. Erste Anwendungen des neuen Geräts sind in den Bereichen Antibiotikaentwicklung und Antibiotikaresistenzen, sowie der molekularen und zellulären Krebsforschung vorgesehen. Wissenschaftlicher und gesellschaftlicher Kontext Wir erwarten, dass dieses Projekt zu einem besseren Verständnis fundamentaler biologischer Prozesse beitragen wird und dass die entsprechenden Erkenntnisse einen wertvollen Beitrag zu der Entwicklung von Molekülen leisten, die als Werkzeuge in der Biologie oder als Vorstufen zukünftiger Medikamente dienen. Structure, dynamics and function of biological macromolecules by NMR Research Project | 2 Project MembersNo Description available Structure, dynamics and function of CCR5-arrestin interactions Research Project | 3 Project MembersThe human CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that plays a major role in general inflammatory processes by recruiting and activating leukocytes. CCR5 is also the principal HIV coreceptor, is involved in the pathology of both cancer and neuroinflammation, and has been im plicated in the inflammatory complications of COVID-19. Binding of ligands to GPCRs results in the activation of G protein-, arrestin-mediated and other signaling pathways. The Grzesiek lab has recently solved the cryo-EM structure of an agonist chemokine CCL5-CCR5-G protein complex, which delineates the G protein activation pathway triggered by chemokine agonists within CCR5. While meanwhile a number GPCR-G protein complexes have been solved, much less is known on the structural basis of GPCR arrestin signaling. In particular, no structure of a chemokine receptor-arrestin complex exists. The Shukla is one of the world leading labs in the structural and functional analysis of arrestins. We propose here to combine the expertises and capabilities of these two groups to (A) solve the structure of a CCR5-ß arrestin 1 complex, (B) investigate the dynamics of interactions between CCR5-derived peptides and arrestin by NMR and other biophysical techniques, as well as (C) characterize the CCR5-ß arrestin 1 interactions by cell-based assays and develop new synthetic antibody fragments to stabilize the CCR5-ß arrestin 1 complex. The structural and functional insights from these combined experiments should decipher key elements of chemokine-induced CCR5-arrestin signaling and reveal differences to the G protein signaling pathway. This may pave the way for the development of directed therapeutics targeting either pathway. Due to the homology of CCR5 to a number of other chemokine receptors, the results obtained may serve as a paradigm for other chemokine receptor/ligand systems. Structures, dynamics and interactions of disease-relevant proteins and new developments in NMR technology Research Project | 6 Project MembersBiological 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. However, it is limited in the molecular size and sensitivity. In the last years we have combined NMR, cryo-EM, single-molecule FRET and other biophysical techniques to derive mechanistic insights at the atomic level into the function and interactions of a number of disease-relevant protein systems. In the next years we want to further develop and conclude several of these projects, which address (1) G protein-coupled receptors (GPCRs), (2) Abelson kinase, (3) c-di-GMP binding proteins, and (4) NMR technology. Subproject (1) addresses the further dynamical characterization by NMR and other techniques of the function of the beta1-adrenergic receptor (b1AR), which is one of the main targets of beta-blockers, as well as the characterization of functional complexes by cryo-EM and NMR of the human chemokine receptor CCR5, which is also the coreceptor for HIV. Subproject (2) addresses the further dynamical characterization by single-molecule FRET and NMR of Abelson kinase (Abl), which is a large multi-domain protein and important leukemia drug target. We have now established labeling with fluorophore pairs at arbitrary sites by using unnatural amino acid technology. We want to use this development to study the dynamical behavior of single Abl molecules within (cancer) cells by FRET and combine the obtained information with high-resolution NMR. Subproject (3) addresses the interactions of the bacterial virulence factor cyclic di-guanosine-monophosphate (c-di-GMP). We have determined the NMR structure and dynamics of several complexes of c-di-GMP binding proteins and want to continue to develop a genetically encodable c-di-GMP fluorescence sensor based on our structural data. Subproject (4) foresees further developments in NMR technology addressing the rapid assignment of large proteins and the improvement of isotope labeling techniques in higher eukaryotes. Biomacromolecular structures, dynamics and interactions by NMR and new developments in NMR technology Research Project | 1 Project MembersBiological 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 1 H- 15 N 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 % 15 N and 13 C as well as >60 % 2 H labeling with very good yields. We want to improve this method by increasing the level of 2 H incorporation and cost efficiency for 13 C 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 2 H incorporation to cellular behavior by a proteomics analysis of the response to growth on 2 H 2 O for E. coli , yeast and higher eukaryotes. Structural and dynamical basis of allosteric regulation and inhibition of abelson tyrosine kinase a drug target in the treatment of chronic mylogeneous leukaemia Research Project | 2 Project MembersAbelson tyrosine kinase (Abl) in its healthy state is a tightly regulated, human protein involved in many cellular processes. An abnormal rearrangement of chromosomes leads to the formation of the aberrant fusion protein Bcr-Abl. Bcr-Abl is highly active and causes uncontrolled production of immature blood cells, which ultimately results in the blood cancers chronic myelogenous leukemia (CML) or acute lymphoblastic leukemia (ALL). Drugs like imatinib (gleevec), nilotinib (tasigna), and dasatinib (sprycel) have been developed to bind to a specific location on Bcr-Abl (ATP-binding pocket) thereby blocking its abnormal activity. They are highly successful in the clinic. However, after prolonged treatment a fraction of the cancers become resistant to these drugs by spontaneous mutations in Bcr-Abl, making the treatment ineffective. Recently, a new class of inhibitors has been discovered that bind to a different location on Bcr-Abl. These so-called allosteric inhibitors have promise to overcome drug resistance in combination with conventional ATP-site inhibitors. The mode of action of this combination is unclear. Bcr-Abl consists of many subdomains and their relative movement and interplay is thought to be responsible for its regulation. We have recently been able to detect such movements at atomic resolution by Nuclear Magnetic Resonance (NMR) methods. We now want to build on these initial studies and determine Abl's molecular movements and interactions in response to various inhibitors and disease- or functionally relevant mutations by a combination of NMR, single molecule fluorescence resonance energy transfer (FRET) and other biophysical techniques. The results should provide an atomic-level understanding of the mechanism of Abl regulation and a rationale for the improvement of existing and the development of new therapeutic strategies for Bcr-Abl inhibition. Purchase of a 900 MHz High-Resolution NMR Instrument Research Project | 5 Project MembersBackground - Biological function results from time-dependent interactions between biomolecules. It is ultimately encoded in the primary chemical structures of molecules, which determine the positions and movements of their atoms in space. 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 chemical structure, three-dimensional structure, dynamics and ultimately function. The application of NMR spectroscopy to large interesting biomolecular systems is limited by sensitivity and resolution, both of which increase strongly with magnetic field strength. The planned new building for the Biozentrum of the University Basel provides the unique opportunity to upgrade the present 800 MHz NMR instrument to a state-of-the-art 900 MHz instrument. This will substantially improve the scientific capacities of the Biozentrum's high-field NMR center. The new installation will benefit from the strong technical and biological expertise of the principal applicants and the high demand for NMR characterization of exciting biological projects inside and outside of the University of Basel. Research proposal - Expertise, maintenance, running, building and a large part of the total purchase costs will be provided by the Biozentrum and the University of Basel. The core users will use the instrument to A) study structures and interactions of disease-relevant biomolecules and to further develop high-resolution NMR methods (Grzesiek, principal applicant); B) characterize structure, function and folding mechanisms of large biomacromolecules and their complexes with a focus on integral membrane proteins (Hiller); C) investigate proteins, interaction and functional mechanisms in cyclic-di-GMP signaling (Schirmer, Jenal); D) study large protein complexes (Maier, Hiller). Further research projects comprise 1) interactions of the uropathogenic E. coli protein FimH with glycoproteins from the urothelial cell surface (Ernst); 2) interactions of bioactive natural products with target proteins (Hamburger); 3) the catalytic activity of artificial metalloenzymes (Ward, Häussinger); 4) dynamics and interaction of G-protein coupled receptors (Schertler); 5) interactions and dynamics of histone-binding proteins and their role in heterochromatin silencing (Bühler); 6) alterations in cellular metabolism of eukaryotic cells upon infection with pathogens (Bumann). Expected value - Structures and motions of biomolecules need to be determined at atomic resolution to understand their mechanisms from first principles. This information can be provided by NMR analysis. This is a fundamental prerequisite to unravel the connection between biomolecular structure, dynamics and function, as well as for the rational intervention into biological processes such as drug design. Justification of the needed equipment - The new instrument will provide significant increases in sensitivity and resolution for the NMR analysis of structures and motions of biomolecules. The reduced required concentrations and the larger achievable molecular sizes will further extend its applicability to highly challenging biomolecular systems. The instrument will satisfy the high demand for such NMR characterizations within the University of Basel and from outside collaborations. The foreseen research projects comprise several medically relevant G-protein coupled receptors (GPCRs), cancer drug targets, pathogenic bacterial systems, large molecular machines such as membrane protein assembly and polypeptide translocation complexes, as well as general questions of protein folding. The new instrument will ensure that this research can be carried out at an internationally competitive level. Biomacromolecular structures, dynamics and interactions by NMR and new developments in NMR technology Research Project | 1 Project MembersBiological function results from time-dependent interactions between biomolecules. It is ultimately encoded within the primary chemical structure of molecules resulting in defined three-dimensional atom positions and movements. 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 primary structure, tertiary structure, dynamics and function. It is the goal of this proposal to apply and further develop these strengths of NMR technology with the aim to reveal general principles of protein structure function relations. The proposal is divided into two subprojects: Subproject A is directed towards the determination of structure, dynamics, and interactions in four medically important systems, for which we have made significant progress in recent years: (1) Abl kinase, which is an important leukemia drug target. We have obtained assignment of a large, multidomain Abl construct that is the minimal autoregulatory fragment. Our data show that binding of different classes of inhibitors induces distinct domain rearrangements, which shed light on the mechanism of kinase regulation. We now want to reveal the atomic causes of these allosteric rearrangements, investigate several medically and functionally relevant mutants, and extend the studies to single molecule FRET. (2) the HIV-1 coreceptor and G-protein coupled receptor (GPCR) CCR5. We have obtained large-scale functional expression of CCR5 in insect cells and E. coli, which permits effective isotope labeling. We seek to apply new solid-state and solution NMR techniques for structure determination and to develop general strategies for GPCR analysis by a comparison to other available GPCRs. (3) Interactions of the bacterial virulence factor cyclic di-guanosine-monophosphate (c-di-GMP). We have determined the structure of the PilZ homolog PA4608 in complex with c-di-GMP and the kinetics of c-di-GMP oligomer formation in solution. We now want to solve the structures of a complex c-di-GMP with the chemotactic protein CC3300 and of higher c-di-GMP oligomers. (4) Lipopolysaccharide (LPS), the causative agent of endotoxic shock. We have developed a method to study LPS by solution NMR and obtained expression of the lipopolysaccharide binding protein, which is the first receptor of the endotoxic recognition cascade. We intend to study its interaction with LPS. Subproject B is directed towards the NMR characterization of unfolded states of proteins and their relation to the folded structure with an emphasis on pressure denaturation and structural modeling of unfolded ensembles. The overarching goal is to rationalize protein folding by correlating high resolution experimental data on unfolded states with primary, secondary and tertiary structure information. (1) We have obtained unique data on the pressure/cold-denatured state of ubiquitin, which shows similar subpopulations of partial native structures as an alcohol-denatured state. By addition of non-denaturing concentrations of alcohol, full pressure-induced unfolding can be achieved at room temperature in a completely reversible way. This allows following the unfolding transition at very high resolution. We want to extend this analysis to a larger set of about 10 proteins representing different folds and sequences with the aim to correlate unfolding behavior with sequence or structure and to understand the nature of pressure unfolding. (2) We have developed an effective method to calculate structural ensembles of unfolded proteins from large sets of NMR data. We want to continue these efforts with the aim to include FRET and chemical shift data in order to obtain highly defined quantitative models for larger sets of unfolded proteins. NMR studies of GPCRs: Structure, dynamics and interactions with ligands and signaling proteins Research Project | 1 Project MembersG protein-coupled receptors (GPCRs) are integral membrane proteins that transmit extracellular neural, endocrine, olfactory and visual signals across the plasma membrane. When activated by light or a ligand GPCRs undergo a conformational change leading to activation of a G protein, arrestin and other signaling pathways. Over 30% of compounds used in medicine today modulate the activity of GPCRs. Some GPCRs can bind a range of ligands, some of which, instead of fully activating the receptor, rather bias the signal transduction cascade toward distinct intracellular pathways. The currently available X-ray structures of several GPCRs and of a 2-adrenoreceptor/Gs signaling complex provide a frozen snapshot of a signal transduction event, but do not fully explain how the ligand selectivity is achieved, and how the ligand binding results in preferential binding of a specific signaling protein such as Gi, Gs or arrestin. Understanding the precise structural and dynamic nature of these phenomena is critical to knowing the mechanism of GPCR activation and will help further development of pharmaceuticals with desired pharmacological properties. We propose here to provide the missing dynamical information on GPCR function by Nuclear magnetic resonance spectroscopy (NMR), which shall determine the conformational changes and dynamics of the receptors and signaling complexes in solution. This should then a yield a comprehensive view of GPCR activation of and signal transduction. We will initially focus on rhodopsin and 1-adrenoreceptor ( 1AR), as well as their signaling complexes with G proteins and arrestin, and later apply developed expertise to the vasopressin receptor V2R and its signaling complexes. In particular, we propose: a) to label GPCRs for NMR analysis; b) to obtain a dynamic view of the conformational changes in GPCRs induced by a range of ligands with different activities including agonists, antagonists and biased ligands; c) to investigate the effect of G proteins and arrestin binding on the receptors; d) to identify the molecular basis for signaling selectivity via a comparative NMR and biophysical analysis of receptor binding to the various ligands, G proteins and arrestin. Due to the high sequence conservation among G protein-coupled receptor proteins our results are expected to have wide implications for signal transduction by GPCRs in general. Biomacromolecular structures, dynamics and interactions by NMR and new developments in NMR technology. Research Project | 1 Project MembersSummary Biological function is almost uniquely exerted via interactions of biomacromolecules. Such interactions are still poorly understood and often involve large conformational changes, in extreme cases even from completely unfolded to folded structures. High resolution NMR has emerged as one of the most versatile tools for a precise description of biomolecules and their interactions, not only in terms of static structures, but also by kinetic, energetic, and thermodynamic parameters at the atomic level. It is the goal of this proposal to use and to develop the unique strengths of NMR to arrive at a more quantitative understanding of biomolecules and their interactions. The proposal is divided into two subprojects: Subproject A is directed towards the determination of structure, dynamics, and interactions in a number of protein systems where we have detailed biological information from in-house or external collaborations, but no or insufficient structural and dynamical data are available, and where solution NMR methods are expected to yield unique new information. Specifically, this subproject addresses protein-protein and protein-ligand interactions in cadherin-mediated cellular adhesion, antiobiotic resistance in Streptomyces, proteins of the Yersinia injectisome, the Bartonella type IV secretion system, and proteins involved in transcriptional elongation as well as nematocyst wall formation. In addition, we want to characterize the interactions of lipopolysaccharides (endotoxins) by novel NMR methods. Subproject B is directed towards the development of novel NMR methods with a specific focus on the precise description of folding in model systems and of hydrogen bonds. In particular, it seems now possible to obtain a complete geometric description of the folding of peptides and small proteins in terms of angular distributions and order parameters from residual dipolar couplings. This could finally lead to a complete thermodynamic description of folding transitions in peptides and small proteins. We also want to continue our research on the spectroscopy of biomolecular hydrogen bonds with an emphasis on the influence of biomolecular dynamics on hydrogen bond parameters and a comparison between the thermodynamic behavior of protein and nucleic acid hydrogen bonds. The goal is to determine to what extent H-bonds contribute to the overall thermodynamic properties of biomolecules. Finally, we also want to start an exploratory project in solid state NMR of biomolecules with the goal to describe the influence of crystalline and non-crystalline environment on the line width of biomolecules in solid state NMR spectra. 12 12
Efficient Characterization of Biomolecular Interactions by Automated Isothermal Titration Calorimetry Research Project | 5 Project MembersInhalt und Ziel des Forschungsprojekts Im Gegensatz zu anderen Methoden ist die isothermale Titrationskalorimetrie (ITC) in einzigartiger Weise geeignet, die Wechselwirkung von unmodifizierten Bindungspartnern anhand der aufgenommenen oder abgegebenen Bindungswärme zu messen. Messungen mittels ITC erlauben dabei eine thermodynamische Charakterisierung der Wechselwirkungen zwischen Bindungspartnern. Diese erlaubt, zusammen mit Strukturinformationen, die Mechanismen der Bindung im Detail zu verstehen und zu erklären, warum verschiedene Bindungspartner unterschiedlich stark interagieren. In diesem Projekt wird ein automatisiertes System für ITC Messungen aufgebaut, das hilft, wesentliche Nachteile von manuellen Messungen hinsichtlich des Durchsatzes und der Datenqualität zu überkommen. Das neue ITC System wird in der Biophysik Forschungsserviceeinheit (BF) des Biozentrums der Universität Basel installiert und betrieben, und steht dort auch externen Nutzern zur Verfügung. Erste Anwendungen des neuen Geräts sind in den Bereichen Antibiotikaentwicklung und Antibiotikaresistenzen, sowie der molekularen und zellulären Krebsforschung vorgesehen. Wissenschaftlicher und gesellschaftlicher Kontext Wir erwarten, dass dieses Projekt zu einem besseren Verständnis fundamentaler biologischer Prozesse beitragen wird und dass die entsprechenden Erkenntnisse einen wertvollen Beitrag zu der Entwicklung von Molekülen leisten, die als Werkzeuge in der Biologie oder als Vorstufen zukünftiger Medikamente dienen.
Structure, dynamics and function of biological macromolecules by NMR Research Project | 2 Project MembersNo Description available
Structure, dynamics and function of CCR5-arrestin interactions Research Project | 3 Project MembersThe human CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that plays a major role in general inflammatory processes by recruiting and activating leukocytes. CCR5 is also the principal HIV coreceptor, is involved in the pathology of both cancer and neuroinflammation, and has been im plicated in the inflammatory complications of COVID-19. Binding of ligands to GPCRs results in the activation of G protein-, arrestin-mediated and other signaling pathways. The Grzesiek lab has recently solved the cryo-EM structure of an agonist chemokine CCL5-CCR5-G protein complex, which delineates the G protein activation pathway triggered by chemokine agonists within CCR5. While meanwhile a number GPCR-G protein complexes have been solved, much less is known on the structural basis of GPCR arrestin signaling. In particular, no structure of a chemokine receptor-arrestin complex exists. The Shukla is one of the world leading labs in the structural and functional analysis of arrestins. We propose here to combine the expertises and capabilities of these two groups to (A) solve the structure of a CCR5-ß arrestin 1 complex, (B) investigate the dynamics of interactions between CCR5-derived peptides and arrestin by NMR and other biophysical techniques, as well as (C) characterize the CCR5-ß arrestin 1 interactions by cell-based assays and develop new synthetic antibody fragments to stabilize the CCR5-ß arrestin 1 complex. The structural and functional insights from these combined experiments should decipher key elements of chemokine-induced CCR5-arrestin signaling and reveal differences to the G protein signaling pathway. This may pave the way for the development of directed therapeutics targeting either pathway. Due to the homology of CCR5 to a number of other chemokine receptors, the results obtained may serve as a paradigm for other chemokine receptor/ligand systems.
Structures, dynamics and interactions of disease-relevant proteins and new developments in NMR technology Research Project | 6 Project MembersBiological 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. However, it is limited in the molecular size and sensitivity. In the last years we have combined NMR, cryo-EM, single-molecule FRET and other biophysical techniques to derive mechanistic insights at the atomic level into the function and interactions of a number of disease-relevant protein systems. In the next years we want to further develop and conclude several of these projects, which address (1) G protein-coupled receptors (GPCRs), (2) Abelson kinase, (3) c-di-GMP binding proteins, and (4) NMR technology. Subproject (1) addresses the further dynamical characterization by NMR and other techniques of the function of the beta1-adrenergic receptor (b1AR), which is one of the main targets of beta-blockers, as well as the characterization of functional complexes by cryo-EM and NMR of the human chemokine receptor CCR5, which is also the coreceptor for HIV. Subproject (2) addresses the further dynamical characterization by single-molecule FRET and NMR of Abelson kinase (Abl), which is a large multi-domain protein and important leukemia drug target. We have now established labeling with fluorophore pairs at arbitrary sites by using unnatural amino acid technology. We want to use this development to study the dynamical behavior of single Abl molecules within (cancer) cells by FRET and combine the obtained information with high-resolution NMR. Subproject (3) addresses the interactions of the bacterial virulence factor cyclic di-guanosine-monophosphate (c-di-GMP). We have determined the NMR structure and dynamics of several complexes of c-di-GMP binding proteins and want to continue to develop a genetically encodable c-di-GMP fluorescence sensor based on our structural data. Subproject (4) foresees further developments in NMR technology addressing the rapid assignment of large proteins and the improvement of isotope labeling techniques in higher eukaryotes.
Biomacromolecular structures, dynamics and interactions by NMR and new developments in NMR technology Research Project | 1 Project MembersBiological 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 1 H- 15 N 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 % 15 N and 13 C as well as >60 % 2 H labeling with very good yields. We want to improve this method by increasing the level of 2 H incorporation and cost efficiency for 13 C 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 2 H incorporation to cellular behavior by a proteomics analysis of the response to growth on 2 H 2 O for E. coli , yeast and higher eukaryotes.
Structural and dynamical basis of allosteric regulation and inhibition of abelson tyrosine kinase a drug target in the treatment of chronic mylogeneous leukaemia Research Project | 2 Project MembersAbelson tyrosine kinase (Abl) in its healthy state is a tightly regulated, human protein involved in many cellular processes. An abnormal rearrangement of chromosomes leads to the formation of the aberrant fusion protein Bcr-Abl. Bcr-Abl is highly active and causes uncontrolled production of immature blood cells, which ultimately results in the blood cancers chronic myelogenous leukemia (CML) or acute lymphoblastic leukemia (ALL). Drugs like imatinib (gleevec), nilotinib (tasigna), and dasatinib (sprycel) have been developed to bind to a specific location on Bcr-Abl (ATP-binding pocket) thereby blocking its abnormal activity. They are highly successful in the clinic. However, after prolonged treatment a fraction of the cancers become resistant to these drugs by spontaneous mutations in Bcr-Abl, making the treatment ineffective. Recently, a new class of inhibitors has been discovered that bind to a different location on Bcr-Abl. These so-called allosteric inhibitors have promise to overcome drug resistance in combination with conventional ATP-site inhibitors. The mode of action of this combination is unclear. Bcr-Abl consists of many subdomains and their relative movement and interplay is thought to be responsible for its regulation. We have recently been able to detect such movements at atomic resolution by Nuclear Magnetic Resonance (NMR) methods. We now want to build on these initial studies and determine Abl's molecular movements and interactions in response to various inhibitors and disease- or functionally relevant mutations by a combination of NMR, single molecule fluorescence resonance energy transfer (FRET) and other biophysical techniques. The results should provide an atomic-level understanding of the mechanism of Abl regulation and a rationale for the improvement of existing and the development of new therapeutic strategies for Bcr-Abl inhibition.
Purchase of a 900 MHz High-Resolution NMR Instrument Research Project | 5 Project MembersBackground - Biological function results from time-dependent interactions between biomolecules. It is ultimately encoded in the primary chemical structures of molecules, which determine the positions and movements of their atoms in space. 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 chemical structure, three-dimensional structure, dynamics and ultimately function. The application of NMR spectroscopy to large interesting biomolecular systems is limited by sensitivity and resolution, both of which increase strongly with magnetic field strength. The planned new building for the Biozentrum of the University Basel provides the unique opportunity to upgrade the present 800 MHz NMR instrument to a state-of-the-art 900 MHz instrument. This will substantially improve the scientific capacities of the Biozentrum's high-field NMR center. The new installation will benefit from the strong technical and biological expertise of the principal applicants and the high demand for NMR characterization of exciting biological projects inside and outside of the University of Basel. Research proposal - Expertise, maintenance, running, building and a large part of the total purchase costs will be provided by the Biozentrum and the University of Basel. The core users will use the instrument to A) study structures and interactions of disease-relevant biomolecules and to further develop high-resolution NMR methods (Grzesiek, principal applicant); B) characterize structure, function and folding mechanisms of large biomacromolecules and their complexes with a focus on integral membrane proteins (Hiller); C) investigate proteins, interaction and functional mechanisms in cyclic-di-GMP signaling (Schirmer, Jenal); D) study large protein complexes (Maier, Hiller). Further research projects comprise 1) interactions of the uropathogenic E. coli protein FimH with glycoproteins from the urothelial cell surface (Ernst); 2) interactions of bioactive natural products with target proteins (Hamburger); 3) the catalytic activity of artificial metalloenzymes (Ward, Häussinger); 4) dynamics and interaction of G-protein coupled receptors (Schertler); 5) interactions and dynamics of histone-binding proteins and their role in heterochromatin silencing (Bühler); 6) alterations in cellular metabolism of eukaryotic cells upon infection with pathogens (Bumann). Expected value - Structures and motions of biomolecules need to be determined at atomic resolution to understand their mechanisms from first principles. This information can be provided by NMR analysis. This is a fundamental prerequisite to unravel the connection between biomolecular structure, dynamics and function, as well as for the rational intervention into biological processes such as drug design. Justification of the needed equipment - The new instrument will provide significant increases in sensitivity and resolution for the NMR analysis of structures and motions of biomolecules. The reduced required concentrations and the larger achievable molecular sizes will further extend its applicability to highly challenging biomolecular systems. The instrument will satisfy the high demand for such NMR characterizations within the University of Basel and from outside collaborations. The foreseen research projects comprise several medically relevant G-protein coupled receptors (GPCRs), cancer drug targets, pathogenic bacterial systems, large molecular machines such as membrane protein assembly and polypeptide translocation complexes, as well as general questions of protein folding. The new instrument will ensure that this research can be carried out at an internationally competitive level.
Biomacromolecular structures, dynamics and interactions by NMR and new developments in NMR technology Research Project | 1 Project MembersBiological function results from time-dependent interactions between biomolecules. It is ultimately encoded within the primary chemical structure of molecules resulting in defined three-dimensional atom positions and movements. 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 primary structure, tertiary structure, dynamics and function. It is the goal of this proposal to apply and further develop these strengths of NMR technology with the aim to reveal general principles of protein structure function relations. The proposal is divided into two subprojects: Subproject A is directed towards the determination of structure, dynamics, and interactions in four medically important systems, for which we have made significant progress in recent years: (1) Abl kinase, which is an important leukemia drug target. We have obtained assignment of a large, multidomain Abl construct that is the minimal autoregulatory fragment. Our data show that binding of different classes of inhibitors induces distinct domain rearrangements, which shed light on the mechanism of kinase regulation. We now want to reveal the atomic causes of these allosteric rearrangements, investigate several medically and functionally relevant mutants, and extend the studies to single molecule FRET. (2) the HIV-1 coreceptor and G-protein coupled receptor (GPCR) CCR5. We have obtained large-scale functional expression of CCR5 in insect cells and E. coli, which permits effective isotope labeling. We seek to apply new solid-state and solution NMR techniques for structure determination and to develop general strategies for GPCR analysis by a comparison to other available GPCRs. (3) Interactions of the bacterial virulence factor cyclic di-guanosine-monophosphate (c-di-GMP). We have determined the structure of the PilZ homolog PA4608 in complex with c-di-GMP and the kinetics of c-di-GMP oligomer formation in solution. We now want to solve the structures of a complex c-di-GMP with the chemotactic protein CC3300 and of higher c-di-GMP oligomers. (4) Lipopolysaccharide (LPS), the causative agent of endotoxic shock. We have developed a method to study LPS by solution NMR and obtained expression of the lipopolysaccharide binding protein, which is the first receptor of the endotoxic recognition cascade. We intend to study its interaction with LPS. Subproject B is directed towards the NMR characterization of unfolded states of proteins and their relation to the folded structure with an emphasis on pressure denaturation and structural modeling of unfolded ensembles. The overarching goal is to rationalize protein folding by correlating high resolution experimental data on unfolded states with primary, secondary and tertiary structure information. (1) We have obtained unique data on the pressure/cold-denatured state of ubiquitin, which shows similar subpopulations of partial native structures as an alcohol-denatured state. By addition of non-denaturing concentrations of alcohol, full pressure-induced unfolding can be achieved at room temperature in a completely reversible way. This allows following the unfolding transition at very high resolution. We want to extend this analysis to a larger set of about 10 proteins representing different folds and sequences with the aim to correlate unfolding behavior with sequence or structure and to understand the nature of pressure unfolding. (2) We have developed an effective method to calculate structural ensembles of unfolded proteins from large sets of NMR data. We want to continue these efforts with the aim to include FRET and chemical shift data in order to obtain highly defined quantitative models for larger sets of unfolded proteins.
NMR studies of GPCRs: Structure, dynamics and interactions with ligands and signaling proteins Research Project | 1 Project MembersG protein-coupled receptors (GPCRs) are integral membrane proteins that transmit extracellular neural, endocrine, olfactory and visual signals across the plasma membrane. When activated by light or a ligand GPCRs undergo a conformational change leading to activation of a G protein, arrestin and other signaling pathways. Over 30% of compounds used in medicine today modulate the activity of GPCRs. Some GPCRs can bind a range of ligands, some of which, instead of fully activating the receptor, rather bias the signal transduction cascade toward distinct intracellular pathways. The currently available X-ray structures of several GPCRs and of a 2-adrenoreceptor/Gs signaling complex provide a frozen snapshot of a signal transduction event, but do not fully explain how the ligand selectivity is achieved, and how the ligand binding results in preferential binding of a specific signaling protein such as Gi, Gs or arrestin. Understanding the precise structural and dynamic nature of these phenomena is critical to knowing the mechanism of GPCR activation and will help further development of pharmaceuticals with desired pharmacological properties. We propose here to provide the missing dynamical information on GPCR function by Nuclear magnetic resonance spectroscopy (NMR), which shall determine the conformational changes and dynamics of the receptors and signaling complexes in solution. This should then a yield a comprehensive view of GPCR activation of and signal transduction. We will initially focus on rhodopsin and 1-adrenoreceptor ( 1AR), as well as their signaling complexes with G proteins and arrestin, and later apply developed expertise to the vasopressin receptor V2R and its signaling complexes. In particular, we propose: a) to label GPCRs for NMR analysis; b) to obtain a dynamic view of the conformational changes in GPCRs induced by a range of ligands with different activities including agonists, antagonists and biased ligands; c) to investigate the effect of G proteins and arrestin binding on the receptors; d) to identify the molecular basis for signaling selectivity via a comparative NMR and biophysical analysis of receptor binding to the various ligands, G proteins and arrestin. Due to the high sequence conservation among G protein-coupled receptor proteins our results are expected to have wide implications for signal transduction by GPCRs in general.
Biomacromolecular structures, dynamics and interactions by NMR and new developments in NMR technology. Research Project | 1 Project MembersSummary Biological function is almost uniquely exerted via interactions of biomacromolecules. Such interactions are still poorly understood and often involve large conformational changes, in extreme cases even from completely unfolded to folded structures. High resolution NMR has emerged as one of the most versatile tools for a precise description of biomolecules and their interactions, not only in terms of static structures, but also by kinetic, energetic, and thermodynamic parameters at the atomic level. It is the goal of this proposal to use and to develop the unique strengths of NMR to arrive at a more quantitative understanding of biomolecules and their interactions. The proposal is divided into two subprojects: Subproject A is directed towards the determination of structure, dynamics, and interactions in a number of protein systems where we have detailed biological information from in-house or external collaborations, but no or insufficient structural and dynamical data are available, and where solution NMR methods are expected to yield unique new information. Specifically, this subproject addresses protein-protein and protein-ligand interactions in cadherin-mediated cellular adhesion, antiobiotic resistance in Streptomyces, proteins of the Yersinia injectisome, the Bartonella type IV secretion system, and proteins involved in transcriptional elongation as well as nematocyst wall formation. In addition, we want to characterize the interactions of lipopolysaccharides (endotoxins) by novel NMR methods. Subproject B is directed towards the development of novel NMR methods with a specific focus on the precise description of folding in model systems and of hydrogen bonds. In particular, it seems now possible to obtain a complete geometric description of the folding of peptides and small proteins in terms of angular distributions and order parameters from residual dipolar couplings. This could finally lead to a complete thermodynamic description of folding transitions in peptides and small proteins. We also want to continue our research on the spectroscopy of biomolecular hydrogen bonds with an emphasis on the influence of biomolecular dynamics on hydrogen bond parameters and a comparison between the thermodynamic behavior of protein and nucleic acid hydrogen bonds. The goal is to determine to what extent H-bonds contribute to the overall thermodynamic properties of biomolecules. Finally, we also want to start an exploratory project in solid state NMR of biomolecules with the goal to describe the influence of crystalline and non-crystalline environment on the line width of biomolecules in solid state NMR spectra.
