Molecular structure of apolipoproteins

The aim of this project is the 3D-structure determination of apolipoproteins and of a receptor protein at atomic level. This knowledge should contribute to a better understanding of molecular and biomedical-relevant mechanisms in lipoprotein metabolism and help to explain pathological functions which are due to structural changes. In particular, the studies on apolipoprotein H will be continued and the crystallisation of apolipoprotein B100 and of the scavenger receptor B1 will be started. All three project parts will be performed in parallel and the final priorities will strongly depend on the success rate to be reached in the different parts of the project. The 3D- structure of human apolipoprotein H has already been solved within the scope of the preceding project (EMBO J., 1999), likewise we have investigated the interaction of the protein with lipids (Biochemistry, 2001) and the structure in solution (JMB, 2002). In the proposed project we intend to solve the structure of apolipoprotein H from other mammalian species in order to understand their different ways of action. In addition, crystallisation of complexes with lipids and/or autoantibodies will be performed in order to determine structural changes with potential pathological effects. Another task of the project is to solubilize apolipoprotein B100 in a lipid-free form with detergents from native low density lipoprotein particles, to characterize the micellar complex and to grow single crystals for X-ray crystallography. Finally, the scavenger receptor class B, type 1 (SRB1) shall be crystallized after optimisation of expression and purification procedures, in order to gain insight into specific ligand-receptor interaction mechanisms. As main methodology for structure determination X-ray crystallography will be applied, whereas the X-ray diffraction measurements will be performed either at a Synchrotron radiation source or inhouse. The necessary technical state-of-art equipment and essential computer resources are readily available. The automation of crystallisation procedures by the use of a robot will be of utmost advantage. This permits efficient and reproducible screening of a broad array of crystallization conditions using minimum amounts of protein. Complementary biophysical methods will be applied to evaluate protein integrity, to investigate the structure of the protein in solution and to answer questions on interaction mechanisms with lipids, detergents and antibodies. UV/Vis, fluorescence-, CD-spectroscopy, as well as small angle X-ray scattering and microcalorimetry will be used. The project will be carried out in a national cooperation with research institutions from Vienna, Innsbruck and Graz.

The aim of this project is the 3D-structure determination of apolipoproteins and of a receptor protein at atomic level. This knowledge should contribute to a better understanding of molecular and biomedical-relevant mechanisms in lipoprotein metabolism and help to explain pathological functions which are due to structural changes. In particular, the studies on apolipoprotein H will be continued and the crystallisation of apolipoprotein B100 and of the scavenger receptor B1 will be started. All three project parts will be performed in parallel and the final priorities will strongly depend on the success rate to be reached in the different parts of the project. The 3D- structure of human apolipoprotein H has already been solved within the scope of the preceding project (EMBO J., 1999), likewise we have investigated the interaction of the protein with lipids (Biochemistry, 2001) and the structure in solution (JMB, 2002). In the proposed project we intend to solve the structure of apolipoprotein H from other mammalian species in order to understand their different ways of action. In addition, crystallisation of complexes with lipids and/or autoantibodies will be performed in order to determine structural changes with potential pathological effects. Another task of the project is to solubilize apolipoprotein B100 in a lipid-free form with detergents from native low density lipoprotein particles, to characterize the micellar complex and to grow single crystals for X-ray crystallography. Finally, the scavenger receptor class B, type 1 (SRB1) shall be crystallized after optimisation of expression and purification procedures, in order to gain insight into specific ligand-receptor interaction mechanisms. As main methodology for structure determination X-ray crystallography will be applied, whereas the X-ray diffraction measurements will be performed either at a Synchrotron radiation source or inhouse. The necessary technical state-of-art equipment and essential computer resources are readily available. The automation of crystallisation procedures by the use of a robot will be of utmost advantage. This permits efficient and reproducible screening of a broad array of crystallization conditions using minimum amounts of protein. Complementary biophysical methods will be applied to evaluate protein integrity, to investigate the structure of the protein in solution and to answer questions on interaction mechanisms with lipids, detergents and antibodies. UV/Vis, fluorescence-, CD-spectroscopy, as well as small angle X-ray scattering and microcalorimetry will be used. The project will be carried out in a national cooperation with research institutions from Vienna, Innsbruck and Graz.