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Structural Studies of Aggregates and Membrane Proteins

English title Structural Studies of Aggregates and Membrane Proteins
Applicant Riek Roland
Number 144444
Funding scheme Project funding
Research institution Laboratorium für Physikalische Chemie D-CHAB ETH Zürich
Institution of higher education ETH Zurich - ETHZ
Main discipline Physical Chemistry
Start/End 01.01.2013 - 31.12.2015
Approved amount 595'306.00
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All Disciplines (2)

Discipline
Physical Chemistry
Biophysics

Keywords (1)

3D structure, NMR, membrane protein, aggregation

Lay Summary (English)

Lead
Structural Studies of Protein Aggregates
Lay summary

Lay Summary

Protein aggregation is an omnipresent process in nature in which identical proteins self-associate into imperfectly ordered macroscopic entities with toxic and/or functional activities. Protein aggregates are in generally composed of a b-sheet-rich entity termed cross-b-sheet structure and are associated with several pathological conditions in humans including Alzheimer’s disease and Parkinson’s disease, but may be also be of functional nature as the het-s prion system in Podospora anserina and hormone storage in secretory granules in mammals. It is the aim of this proposal to get a detailed structure-activity and structure-toxicity relationship of several amyloid systems. These include the structural elucidation of the toxic entity of the HET-s/HET-s prion system, the determination of a structure-toxicity relationship of the amyloid system of a-synuclein associated with Parkinson’s disease, and to determine the 3D structures of several hormone amyloids with the goal to get insights into the role of the amyloid entity in the formation and release of secretory granules. The structural and mechanistic comparison between functional amyloid system and disease-associated amyloid systems may elucidate fundamental principles of protein aggregation including a structure-toxicity relationship in neurodegenerative diseases.

An independent aim of this proposal is the 3D structure determination of membrane proteins by NMR. Integral membrane proteins constitute more than 20% of all the proteins in mammalian organisms. However, membrane protein structure determination is still a challenge attributed to the two main bottlenecks: protein preparation and structure determination. Recent developments in recombinant membrane protein production and solution-state NMR technology open novel perspectives towards the study of the 3D structures and dynamics of membrane proteins. Here it is aimed to further develop and refine these techniques and apply them to the corticotropin releasing factor receptor (CRF-R), which belongs to the family B G-protein coupled receptors (GPCR). 

 

Direct link to Lay Summary Last update: 19.12.2012

Responsible applicant and co-applicants

Employees

Publications

Publication
alpha-Synuclein Insertion into Supported Lipid Bilayers As Seen by in Situ X-ray Reflectivity
Haehl Hendrik, Moeller Isabelle, Kiesel Irena, Campioni Silvia, Riek Roland, Verdes Dorinel, Seeger Stefan (2015), alpha-Synuclein Insertion into Supported Lipid Bilayers As Seen by in Situ X-ray Reflectivity, in ACS CHEMICAL NEUROSCIENCE, 6(3), 374-379.
Dymanic Assebmly and Disassembly of Functional beta Endorphin Amyloid Fibrils
Nespovitaya Nadezhda (2015), Dymanic Assebmly and Disassembly of Functional beta Endorphin Amyloid Fibrils, in J. Am. Chem. Soc., 846-856.
Structure based aggregation studies reveal the presence of helix-rich intermediate during alpha-Synuclein aggregation
Ghosh Dhiman, Singh Pradeep K., Sahay Shruti, Jha Narendra Nath, Jacob Reeba S., Sen Shamik, Kumar Ashutosh, Riek Roland, Maji Samir K. (2015), Structure based aggregation studies reveal the presence of helix-rich intermediate during alpha-Synuclein aggregation, in SCIENTIFIC REPORTS, 5, 1-15.
Contribution of Specific Residues of the beta-Solenoid Fold to HET-s Prion Function, Amyloid Structure and Stability
Daskalov Asen, Gantner Matthias, Waelti Marielle Aulikki, Schmidlin Thierry, Chi Celestine N., Wasmer Christian, Schuetz Anne, Ceschin Johanna, Clave Corinne, Cescau Sandra, Meier Beat, Riek Roland, Saupe Sven J. (2014), Contribution of Specific Residues of the beta-Solenoid Fold to HET-s Prion Function, Amyloid Structure and Stability, in PLOS PATHOGENS, 10(6), 1-16.
Expression and Functional Characterization of Membrane-Integrated Mammalian Corticotropin Releasing Factor Receptors 1 and 2 in Escherichia coli
Jappelli Roberto, Perrin Marilyn H., Lewis Kathy A., Vaughan Joan M., Tzitzilonis Christos, Rivier Jean E., Vale Wylie W., Riek Roland (2014), Expression and Functional Characterization of Membrane-Integrated Mammalian Corticotropin Releasing Factor Receptors 1 and 2 in Escherichia coli, in PLOS ONE, 9(1), 1-16.
The Presence of an Air-Water Interface Affects Formation and Elongation of alpha-Synuclein Fibrils
Campioni Silvia, Carret Guillaume, Jordens Sophia, Nicoud Lucrece, Mezzenga Raffaele, Riek Roland (2014), The Presence of an Air-Water Interface Affects Formation and Elongation of alpha-Synuclein Fibrils, in JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 136(7), 2866-2875.
The production of recombinant N-15, C-13-labelled somatostatin 14 for NMR spectroscopy
Nespovitaya Nadezhda, Barylyuk Konstantin, Eichmann Cedric, Zenobi Renato, Riek Roland (2014), The production of recombinant N-15, C-13-labelled somatostatin 14 for NMR spectroscopy, in PROTEIN EXPRESSION AND PURIFICATION, 99, 78-86.

Collaboration

Group / person Country
Types of collaboration
The Seeger group/ University Zurich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
The Mezzenga group Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
The Liebman group United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
The Vale group/ The Salk Institute United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
The Rivier group/ The Salk Institute United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
The Meier group/ETH Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
The Saupe group/University Bordeaux France (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
The Maji group India (Asia)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Associated projects

Number Title Start Funding scheme
163284 Structural Studies of Aggregates and Membrane Proteins 01.01.2016 Project funding
154461 The Time- and Spatially Resolved Aggregation of a-Synuclein and its Relationship to Cell-Cell Transmissibility 01.10.2014 Sinergia
126488 Structural Studies of Aggregates and Membrane Proteins 01.01.2010 Project funding
148329 In cell NMR study of a-Syn and its familial disease-associated mutants. 01.05.2013 International short research visits

Abstract

Project A: Structural Studies of Protein AggregatesProtein aggregation is an omnipresent process in nature in which identical proteins self-associate into imperfectly ordered macroscopic entities with toxic and/or functional activities. Protein aggregates are in generally composed of a ß-sheet-rich entity termed cross-ß-sheet structure and are associated with several pathological conditions in humans including Parkinson’s disease, but may be also be of functional nature as hormone storage in secretory granules in mammals. It is the aim of this continuing proposal to get a detailed structural understanding of the toxic entity of the HET-s/HET-s prion system, to elucidate a structure-toxicity relationship of the amyloid system of a-synuclein associated with Parkinson’s disease, and to determine a few amyloid structures of hormones resembling their state in secretory granules. The structural and mechanistic comparison between functional and disease-associated amyloid systems may elucidate fundamental principles of protein aggregation including a structure-toxicity relationship in neurodegenerative diseases. Aim 1 is the study of the toxic mechanism of the het-s prion system: The Podospora anserina HET-s prion system is of functional nature being involved in a primitive immune system. It appeared to be an excellent model system to prove the protein-only hypothesis of prions, to determine the 3D structure of the amyloid of HET-s, and to elucidate the mechanism of prion inhibition by its natural counterpart HET-S. Here, we aim to study the mechanism of toxicity of the HET-S - HET-s prion system at atomic resolution. The preliminary data indicate that it is a two components system in which the HET-s prion is a template on which HET-S refolds from a soluble to an integral membrane protein that upon oligomerisation causes membrane leakage indicative of pore-forming toxins. Aim 2 is the biophysical study of a-synuclein (a-syn) aggregation on membranes: While a-syn aggregates in lewy bodies is a pathological hallmark of Parkinson’s disease, neither the mechanism of aggregation nor the mechanism of toxicity is known. Furthermore, recently it got established that a-syn aggregation in vitro is based on the physiological irrelevant water-air interface. Hence, the biophysical and structural study of a-syn aggregation and its relationship to toxicity must be restarted. To do so biophysically (including NMR), we will make use of the designed artificial variants of a-syn which are much more toxic in a rat model of Parkinson’s disease than wild-type (in collaboration with Dr. F. Gage, The Salk Institute) and of the observation that a-syn aggregates on membranes, accompanied by lipid extraction (in collaboration with Dr. S. Seeger, University Zurich). The latter studies also suggested that it is the process of protein aggregation on the membrane that is toxic rather than a specific structural entity, which is regarded as a paradigm shift. Aim 3 is the 3D structure determination of hormone amyloids: We recently proposed that protein hormones are stored in mammalian secretory granules as amyloids-like entities. Secretory granules are membrane-enclosed electron-dense entities in neuroendocrine cells in order to store secretory protein and peptides for extended time periods in a highly concentrated fashion ready to be secreted upon a trigger. It is the aim to investigate the 3D structures of amyloids of several peptide hormones by biophysical methods such as electron microscopy (EM), quenched H/D exchange NMR and solid-state NMR in collaboration with B. Meier (ETH Zurich). The targets are the small peptide hormones somatostatin, ß-endorphin, ACTH, and a member of the corticotropin releasing factor (CRF) family. Project B: Structural Studies of Membrane ProteinsIntegral membrane proteins constitute more than 20% of all the proteins in mammalian organisms. However, membrane protein structure determination is still a challenge attributed to the two main bottlenecks: protein preparation and structure determination. Recent developments in recombinant membrane protein production and NMR technology open novel perspectives towards the study of the 3D structures and dynamics of membrane proteins. We aim to further develop and refine these techniques and apply them to the CRF receptor (CRF-R), which belongs to the family B G-protein coupled receptors (GPCR). Aim 4: Structure determination of multiple conformations of the CRF family peptide: Towards establishing a structural landscape of CRF (analog), it is the aim to determine its 3D structures in solution (using exact NOE rates), when bound to membrane, the amyloid state (aim 3), and in in complex with one of its receptors using either transferred NOE on a weakly interacting system or a high affine agonist labeled with 2H, 13C, and 15N. The CRF receptors used will be either purified from mammalian cells or from E. coli. Aim 5: Structural studies of the hormone-receptor complex in full-length CRFR2/1: Extending our structural work on the complex between CRF analogs and fragments of the receptor, we aim to study the structural and dynamical changes of full-length CRFR2/1 free and in complex with ligands in order to get insights into hormone binding and receptor activation. 13C,15N,2H-labeled functional CRF receptors will be expressed in E. coli, purified and solubilized in either detergent micelles or nanodiscs. This is a long-term project.
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