Structural and functional studeies of angiopoietin/Tie2 signalling and a novelangiopoietin-like protein (360G-Wellcome-074343_Z_04_B)

Detailed knowledge of protein-protein interactions is crucial for understanding protein structure and function, as well as the origin and progression of certain fatal disorders, such as amyloid diseases. Because an understanding of these interactions is often hindered by the nature of the interacting proteins or the process of interest per se, protein engineering and protein de novo design have traditionally been used to study complicated processes in a simplified manner. In these studies, the a-helical coiled-coil structural motif has frequently been used for protein engineering purposes, and its simplicity and regularity has also attracted much attention to elucidate and validate fundamental principles of protein folding by de novo protein design. We already successfully demonstrated the usefulness of the coiled-coil structural motif to address key open issues of amyloid fibril formation and angiopoietin function. Based on the coiled-coil structural motif, we recently succeeded in designing de novo a model peptide that folds into a coiled-coil conformation under ambient conditions but transforms into amyloid fibrils at elevated temperatures. The long-term goal of this proposal is to obtain an understanding of angiopoietin function and amyloid diseases at the molecular level. The specific objectives of this proposal are (a) to systematically dissect the driving forces of amyloid formation (e.g. native state stability, physiochemical properties and residual structure of a peptide/protein in its unfolded state, kinetics of fibril formation, fibril packing and stability) and evaluate their relative contribution to the process, (b) to elucidate the role of amyloid fibril packing in human disorders, (c) to define the mechanism of interaction of angiopoietins and their splice variants with the Tie2 receptor, (d) and to identify by mutageneis and X-ray crystallography the regions that are important for angiopoietin function. Biophysical, biochemical and cell biological method will be used to address these important issues.