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Structural Studies of Aggregates and Membrane Proteins

English title Structural Studies of Aggregates and Membrane Proteins
Applicant Riek Roland
Number 182800
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 01.01.2019 - 31.12.2022
Approved amount 1'280'000.00
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All Disciplines (2)

Discipline
Physical Chemistry
Biophysics

Keywords (9)

Parkinson's Disease; Protein Dynamics; Protein Aggregation; Protein Folding; Alzheimer's Disease; Structural Biology; High Density Lipoprotein Particles (HDL); Nuclear Magnetic Resonance ; Membrane Proteins

Lay Summary (German)

Lead
Das Wissen der dreidimensionalen Gestalt von Proteinen ist essentiell für das Verstehen Ihrer Aktivität im kranken als auch im gesunden Körper. Die Bestimmung der drei dimensionalen Struktur von Proteinaggregaten assoziiert mit neurodegenerativen Erkrankungen als auch von Protein-Lipid Komplexen involviert im Cholesteroltransport im Blut sind das Haupt-Ziel dieser Forschungsarbeit.
Lay summary

Proteinaggregation ist ein Prozess, bei dem identische Eiweisse verklumpen. Dabei verklumpen sie in einer bestimmten Art und Weise und formen sogenannte Beta-Blatt Strukturen, die Amyloide genannt werden. Bei der Alzheimerkrankheit findet man diese Amyloide bestehend aus dem Eiweiss Aβ(1-42) als Ablagerungen im Hirn und man glaubt, dass diese Amyloide für die Uebertragung der Krankheit von einer Zelle zur anderen wichtig sind. Es ist das Ziel dieser Forschungsarbeit, die drei dimensionale Gestalt mit atomarer Auflösung von familiären Mutationen dieses physiologisch relevanten Amyloids zu bestimmen. Dabei werden verschiedene Techniken angewendet insbesondere auch die Festkörper Kernspin Resonanz Spektroskopie (solid state NMR). Auch bei der Parkinsonschen Erkrankung aggregiert ein Protein, welches a-Synuclein heisst. Die ersten Schritte der Aggregation von a-synuclein werden auch studiert.

HDL (high density lipoportein) Partikel bestehen sowohl aus Eiweissen als auch Lipiden. Sie sind wichtig für den Cholesteroltransport im Blut, oder Entsorgung von schädlichen Eiweissen, wie z. Bsp. Aβ(1-42). Es ist das Ziel dieser Forschungsarbeit, die drei dimensionale Struktur vo HDL und HDL-ähnlichen mit Hilfe von NMR und Elektronen paramagnetische Resonanz Spektroskopie (EPR) zu bestimmen. Die Bestimmung dieseren Strukturen werden Einsichten geben, wieso HDL das sogenanntes „gute Cholesterol“ in unserem Körper ist und wieso diese in Alzheimer involviert sein könnten.

Im Weiteren wird die Dynamik von Eiweissen mit atomarer Auflösung studiert und Methoden für die Strukturbiologie in der lebenden Zelle mittels flüssig NMR entwickelt.

Direct link to Lay Summary Last update: 16.10.2018

Responsible applicant and co-applicants

Employees

Associated projects

Number Title Start Funding scheme
163284 Structural Studies of Aggregates and Membrane Proteins 01.01.2016 Project funding

Abstract

1. Summary of Research ProjectThe presented proposal covers the main scientific projects A-D of the Riek group. Project A: Structural Studies of Protein AggregatesProtein aggregation is a process in which identical proteins self-associate into imperfectly ordered macroscopic entities. These cross-ß-sheet containing aggregates, termed amyloids, are the pathological hallmarks of several diseases including Alzheimer’s, and Parkinson’s disease, but are also associated with functional states. It is the aim of this continuing proposal to study protein aggregation from a structural point of view with a particular focus on two disease-associated amyloid systems. Aim 1 is the 3D structure determination of fibrils of familial mutants of Aß(1-42) associated with Alzheimer’s disease: The recent structure determination of a disease-relevant Aß(1-42) fibril by solid state NMR (in collaboration with B. Meier, ETH) revealed that all of the familial mutations within the 3D fibril structure of Aß(1-42) are located at the structurally frustrated segment A20-D23 and most of these mutations are expected to release at least in part this frustration indicating a more stable structure. To find support of this hypothesis the 3D structure determination of two familial mutants with best spectroscopic properties will be determined.Aim 2 is the structural and dynamical studies of a-synuclein bound to an aggregation-inducing membrane surface: The aggregation of human a-synuclein (a-Syn) into amyloid fibrils is related to the onset of multiple diseases termed synucleinopathies including Parkinson’s disease. Albeit a-Syn aggregates in vitro easily, aggregation of a-Syn in absence of a water-air interface at physiological buffer conditions (~150 mM NaCl) has not been obtained yet. It is the goal to study a-Syn aggregation on the surface of a lipid bilayer by solution NMR at atomic resolution under physiological conditions using nanodiscs composed of a diverse set of lipids as a model membrane. Project B: Structural studies on membrane-protein interactionsThe interplay between membrane lipids and proteins are crucial not only for integral membrane proteins, but also for membrane binding proteins, and high density lipoportein (HDL) particles. Aim 3 is the 3D structure determination of full-length discoidal HDL particle: HDL particles are the major cholesterol and lipid transport containers. Recently, we determined the three-dimensional structure of a reconstituted discoidal HDL (rdHDL) particles using a shortened construct of human apolipoprotein A-I. Here, it is the aim to determine the 3D structure of the full-length discoidal HDL particle (in collaboration with S. Bibow, Biozentrum Basel).Aim 4 is the investigation between bilayer composition and dynamics of membrane proteins: The lipid-composition of a biological membrane is fundamental for membrane protein functions. A dynamical interplay between lipid bilayers and embedded membrane proteins may thereby by of critical relevance. To get insights into potential lipid-modulated membrane protein backbone dynamics in saturated, unsaturated and cholesterol-containing lipid bilayers 15N relaxation measurements will be investigated on a protoypic ?-barrel membrane protein embedded in the lipid bilayer of nanodiscs. In parallel, lipid-dependent ion binding of the K+ channel KcsA will be studied by 15NH4 NMR experiments.Aim 5 is the structural investigation of ApoE-HDL-like particle and its interaction with Aß(1-42): The gene of ApoE is a major risk factor for Alzheimer’s disease and ApoE-HDL-like particles are proposed to be involved in the clearance of Aß from the brain. Following procedures established for Apo A-HDL-like particles structural investigation on ApoE-HDL-like particles will be initiated. In addition, the interaction between Aß(1-42) and ApoE HDL-like particles will be studied by solution state NMR. Project C: Multi-state structure determination of proteinsAim 6 is the folding of the WW domain at atomic resolution using eNOEs: Because of the accuracy of eNOE-derived distances the determination of multi-state structures is now feasible. This method is applied to the WW domain of Pin1 with a melting temperature of ca 53 °C to determine several multi-state structures at temperatures between 4-40 °C with the emphasis to reveal the temperature-dependent unfolding of the WW at atomic resolution.Project D: In cell NMRIn cell NMR has the promise to reveal the structure and dynamics of a protein within a cellular environment at residue-resolution.Aim 7 is In cell NMR of folded proteins: It is the goal to exploit in cell NMR to study the structures and dynamics of two model proteins (i.e. ubiquitin and GB1) and their transient interactions with other biomolecules at near atomic resolution upon modulation of the genetics of the cell, change in the cellular environment, and change of cell type.Aim 8 is In cell NMR of tau: It is proposed to investigate by in cell NMR the 441-residue neuronal Tau protein, which forms intracellular deposits, an important pathological hallmark of Alzheimer disease AD. Tau is a prototypic intrinsically disordered protein that binds to cytoskeletal proteins and regulates dynamic instability. In the course of neurodegenerative diseases such as Alzheimer’s disease, the interaction of Tau with its physiological binding partners is impaired in the neuronal cells. It is the goal to investigate such effects by in cell NMR by modulation of the genetics or the environment of the cell.
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