NMR; amyloid; protein aggregation; Alzheimer's disease; membrane protein; 3D structures
Klammt C, Maslennikov I, Bayrhuber M, Eichmann C, Vajpai N, Chiu EJC, Blain KY, Esquivies L, Kwon JHJ, Balana B, Pieper U, Sali A, Slesinger PA, Kwiatkowski W, Riek R, Choe S (2012), Facile backbone structure determination of human membrane proteins by NMR spectroscopy, in NATURE METHODS
, 9(8), 834-834.
Mathur V, Seuring C, Riek R, Saupe SJ, Liebman SW (2012), Localization of HET-S to the Cell Periphery, Not to [Het-s] Aggregates, Is Associated with [Het-s]-HET-S Toxicity, in MOLECULAR AND CELLULAR BIOLOGY
, 32(1), 139-153.
Greenwald J, Riek R (2012), On the Possible Amyloid Origin of Protein Folds, in JOURNAL OF MOLECULAR BIOLOGY
, 421(4-5), 417-426.
Winner B, Jappelli R, Maji SK, Desplats PA, Boyer L, Aigner S, Hetzer C, Loher T, Vilar M, Campionic S, Tzitzilonis C, Soragni A, Jessberger S, Mira H, Consiglio A, Pham E, Masliah E, Gage FH, Riek R (2011), In vivo demonstration that alpha-synuclein oligomers are toxic, in PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
, 108(10), 4194-4199.
Reynolds NP, Soragni A, Rabe M, Verdes D, Liverani E, Handschin S, Riek R, Seeger S (2011), Mechanism of Membrane Interaction and Disruption by alpha-Synuclein, in JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
, 133(48), 19366-19375.
Bayrhuber M, Riek R (2011), Very simple combination of TROSY, CRINEPT and multiple quantum coherence for signal enhancement in an HN(CO)CA experiment for large proteins, in JOURNAL OF MAGNETIC RESONANCE
, 209(2), 310-314.
Greenwald J, Riek R (2010), Biology of Amyloid: Structure, Function, and Regulation, in STRUCTURE
, 18(10), 1244-1260.
Eichmann C, Preissler S, Riek R, Deuerling E (2010), Cotranslational structure acquisition of nascent polypeptides monitored by NMR spectroscopy, in PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
, 107(20), 9111-9116.
Goldschmidt L, Teng PK, Riek R, Eisenberg D (2010), Identifying the amylome, proteins capable of forming amyloid-like fibrils, in PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
, 107(8), 3487-3492.
Wang L, Schubert D, Sawaya MR, Eisenberg D, Riek R (2010), Multidimensional Structure Activity Relationship of a Protein in Its Aggregated States, in ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
, 49(23), 3904-3908.
Grace CRR, Perrin MH, Gulyas J, Rivier JE, Vale WW, Riek R (2010), NMR Structure of the First Extracellular Domain of Corticotropin-releasing Factor Receptor 1 (ECD1-CRF-R1) Complexed with a High Affinity Agonist, in JOURNAL OF BIOLOGICAL CHEMISTRY
, 285(49), 38580-38589.
Schuetz A, Wasmer C, Habenstein B, Verel R, Greenwald J, Riek R, Bockmann A, Meier BH (2010), Protocols for the Sequential Solid-State NMR Spectroscopic Assignment of a Uniformly Labeled 25 kDa Protein: HET-s(1-227), in CHEMBIOCHEM
, 11(11), 1543-1551.
Greenwald J, Buhtz C, Ritter C, Kwiatkowski W, Choe S, Maddelein ML, Ness F, Cescau S, Soragni A, Leitz D, Saupe SJ, Riek R (2010), The Mechanism of Prion Inhibition by HET-S, in MOLECULAR CELL
, 38(6), 889-899.
Chen Q, Prior M, Dargusch R, Roberts A, Riek R, Eichmann C, Chiruta C, Akaishi T, Abe K, Maher P, Schubert D, A Novel Neurotrophic Drug for Cognitive Enhancement and Alzheimer's Disease, in PLOS ONE
Project A: Structural Studies of AggregatesProtein aggregation is an omnipresent process in nature in which identical proteins self-associate into imperfectly ordered macroscopic entities with functional and/or toxic properties. They are generally classified as amorphous, lacking any long range order, or high-ordered fibrils. Protein fibrils are thereby composed of ß-sheet-rich aggregates termed amyloid-like 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 P. anserina. Although only little is known about the amorphous class of protein aggregates, our recent structural studies of bacterial inclusion bodies suggest that that they also comprise amino-acid sequence-specific cross-ß structure. These findings suggest that any kind of protein aggregates may be structured. It is poorly understood how the conformational transition and the formation of aggregates happen, which are the biophysical principles of aggregation and which conformational forms are toxic. It is the aim of this contuining proposal to get a detailed structural understanding of (1) the functional amyloid system of the het-s prion protein in P. anserina, (2) to determine the 3D structure of Aß(1-42) fibrils associated with Alzheimer’s disease, (3) to elucidate a structure-toxicity relationship of the amyloid system of a-synuclein associated with Parkinson’s disease, and (4) to get insights into the role of the environment in protein aggregation. The structural comparison between a functional amyloid system, disease-associated amyloid systems, and aggregates generated by various treatments may elucidate fundamental principels of protein aggregation including a structure-toxicity relationship in neurodegenerative diseases. Aim 1 is the structural studies of the het-s prion system: It is the aim to get structural insights into the components of the het-s prion system of P. anserina using solution-state and solid-state NMR (in collaboration with B. Meier, ETH), other biophysical techniques, and in vivo studies (in collaboration with S. Saupe, Bordeaux). Since the het-s prion system has been evolved to be a functional prion, its study is fundamental towards understanding protein infectivity and the process of amyloidosis. During our recent research we determined the 3D structure of the pHET-s(218-289) fibrils and showed that the amyloid structure is the infectious entity of the prion. Continuing our studies, (a) we will exploit the 3D structure of pHET-s(218-289) fibrils by mutagenesis to reveal the contribution of local interactions (i.e. hydrophobic interactions and hydrogen bonds) in the stability and infectivity of the fibrils. (b) We will also continue with our study on the different aggregation properties between pHET-S and pHET-s, and (c) study the structure of the toxic oligomeric complex between pHET-S and the amyloid form of pHET-s responsible for limited cell death.Aim 2 is the 3D structure determination of Aß(1-42) fibrils associated with Alzheimer’s disease: It is our long-term goal to elucidate the molecular mechanism of Aß amyloid formation associated with Alzheimer’s disease. Here, we propose to determine the 3D structure of amyloids of Aß(1-42) by solid state NMR in collaboration with B. Meier (ETH). Aim 3 is the elucidation of a structure-toxicity relationship of a-synuclein fibrils associated with Parkinson’s disease: It is the aim to establish a relationship between the various conformational entities of a-synuclein and toxicity. a-synuclein exists either in a helical monomeric membrane-attached, an unfolded monomeric soluble, a protofibril, or an amyloid fibril conformation. Using all structural information available conformation-trapped mutants (for example, mutants which can not form fibrils) will be generated to establish a conformation-toxicity relationship. The in vivo studies will be performed in the group of Dr. F. Gage (The Salk Institute) by applying Lenti Virus vectors for gene delivery into the substantia nigra of rats. The biophysical poperties of the a-synuclein mutants are studied at the ETH.Aim 4 is the structural studies of protein aggregates generated in vivo, chemically or physically: Protein aggregation is strongly environment-dependent. For example, aggregation may occur upon heat denaturation (physically), by TCA precipitation (chemically), or in E. coli by the formation of inclusion bodies. We will continue our study of the conformations of several protein aggregates generated by these procedures using quenched H/D exchange NMR, solid state NMR (in collaboration with B. Meier, ETH), and other biophysical techniques to elucidate whether the protein structures in these aggregates are completely heterogeneous or comprised of well folded environment-dependent segments. The structural knowledge obtained will be used towars establishing generic principles for protein aggregation.Project B: Structural Studies of Membrane Proteins30% of all the proteins in mammalian organisms are integral membrane proteins. They play a crucial role in many cellular processes. However, only ~30 folds of integral membrane proteins have been solved so far due to bottlenecks both in 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. We aim to further develop and refine these techniques and apply them to the prototypical potassium channel KcsA, to G-protein coupled receptors (GPCR) and to small E. coli membrane proteins. Aim 5 is the structural and dynamical studies of KcsA: It is our aim to continue our study on the dynamics and structures of KcsA at closed, intermediate, inactivated, and open states by NMR to further elucidate the molecular mechanism of ion selectivity and gating. Aim 6 is the structural and dynamical studies of small E. coli membrane proteins: The established strategy of NMR structure determination and dynamical studies of a-helical integral membrane proteins will be refined by the structure determination of two small E. coli membrane proteins for which NMR spectra of decent quality were obtained.Aim 7 is the expression and purification of functional GPCRs for structural studies: Mouse corticotroprin releasing factor receptor, a family B1 GPCR, and human somtatostatin receptors 1 - 5, family A GPCRs, are expressed either in E.coli or cell-free, solubilized in detergents or lipidis and tested for functionality by ligand binding assays.