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).