Structure of Apolipoproteins B100 and VLDL-II

Plasma lipoproteins, which are macromolecular complexes of apolipoproteins, lipids and fat including cholesterol, play important roles in lipid metabolism and in the emergence of various diseases, in particular of coronary artery disease and atherosclerosis. Even though enormous progress has been made in the understanding of cellular regulation processes, the mechanisms of molecular recognition and uptake of lipoproteins by cells are not known in detail, due to the lack of information about the molecular structures of certain apolipoproteins that control lipoprotein function. However, this information would be of paramount interest for future strategies towards rational pharmaceutical developments for preventive and therapeutic treatment of atherosclerotic diseases. The aim of this project is to explore the three-dimensional domain structure of lipid-free apolipoprotein B100 (apo- B100), which is the only protein moiety of the primary human cholesterol carrier low density lipoprotein (LDL), and of defined fragments thereof, as well as of avian apo-VLDL-II, which is the most effective endogeneous inhibitor of the important enzyme lipoprotein lipase known. To achieve this goal with respect to apo-B100, we have decided to pursue a novel approach using detergent-like polymers and lipid-like peptides to solubilize and stabilize apo-B100 in its lipid-free form, either by isolation from native LDL particles or by detergent exchange. In contrast, naturally occurring cleavage products of apo-B100 will be obtained either by specific cleavage in-vitro or by isolation of in-vivo generated apo-B fragments from yolk. Similarly, the small apolipoprotein, apo-VLDL-II, will be isolated and purified from avian very low density lipoprotein (VLDL). In order to elucidate the atomic structure of proteins by X-ray crystallography the growth of well-ordered single crystals is indispensable, but at present it is still a technical challenge to solubilize and crystallize membrane- associated proteins. However, the necessary state-of-art equipment to face these problems as well as an automation of crystallisation procedures by the use of a robot is readily available. The latter permits efficient, systematic and reproducible screening of a broad array of crystallization conditions using minimum amounts of protein. In addition, biochemical and complementary biophysical methods such as UV/Vis, fluorescence-, CD-spectroscopy and small angle X-ray scattering will be applied for characterization of apolipoprotein complexes with polymers or peptides and for the evaluation of apolipoprotein integrity prior to crystallization. The project will be carried out in a national cooperation of research institutions from Graz and Vienna.

In this project we have identified novel structural and dynamic characteristics of pro- atherogenic lipoproteins, the primary carriers of cholesterol and fat in humans. Atherosclerosis is a chronic inflammatory disease that may start early in life, progresses insidiously over time accompanied by modification of pro-atherogenic lipoproteins, and eventually rupture of "vulnerable" plaques may lead to sudden death by heart attack or stroke. To date cardiovascular diseases are amongst the most common causes of mortality in Western societies. The results of our studies may be helpful towards development of future pharmaceutical interventions and novel therapeutic strategies. Within the project we explored the basic structural features of the small dimeric protein ApoVLDL-II, a powerful inhibitor of the key metabolic enzyme lipoprotein lipase which acts on triglyceride-rich lipoproteins. Furthermore, we showed that the lipid transitions within the core of lipoproteins occur fast enough to react to small changes in temperature, and that lipoprotein particles per se are in fact highly dynamic entities with pronounced intrinsic softness. Apart from lipid mobility and particle elasticity, we obtained strong evidence that the protein moiety, apolipoprotein B100 (apo-B100), displays different dynamics dependent on the lipoprotein it is bound to. To understand these molecular protein characteristics in more detail, we have intensified our efforts to elucidate the three-dimensional domain structure of apo-B100. Since classical detergents were not sufficient to stabilize the structure of lipid-free apo-B100, we have now pursued a new strategy by testing lipid-like self-assembling peptides as novel class of surfactants. These designer peptides reportedly exert rigidity-enhancing effects on proteins as a result of intermolecular hydrogen bonding. However, above a threshold concentration the amphipathic peptides form supermolecular structures by self- assembly. As a result, we are the first to report on a double-helical peptide structure which we revealed by Synchrotron X-ray scattering. However, only a few of the rationally designed peptides had sufficiently high membrane activity to have an impact on the lipid organization within lipoproteins. Nevertheless, we have now identified a promising peptide candidate to be used for crystallization trials with apo-B100. Thus, we have been successful in determining unique characteristics of lipoproteins likely to be important determinants for lipoprotein conversions and particle atherogenicity.