Abstract
The top two leading causes of death worldwide in 2004 (stroke and ischemic heart attack) are both cardiovascular diseases (CVD). High-density lipoprotein and its major protein apolipoprotein A-I (apoA-I, 243 residues) are each inversely associated with risk of CVD and therefore believed to have cardio-protective properties. However, fibrillar apoA-I has been found in arterial plaque, indicating that fibrillogenesis of apoA-I may actually contribute to the development of CVD. Fourteen fibril forming variants of apoA-I are known whose mutations cluster in two regions of the protein's N-terminal helical domain, the "inside" cluster (residues 26-107) and the "outside" cluster (residues 154-178). A mutant from the "inside" cluster, G26R, was previously characterized by members of our research group (Lagerstedt, J.O., Cavigiolio, G., Roberts, L.M., Hong, H.S., Jin, L.W., Fitzgerald, P.G., Oda, M.N., Voss, J.C. 2007. Biochemistry 46, 9693-9699). The G26R protein was found to exhibit an increase in beta-strand structure, N-terminal protease sensitivity and hydrophobic surface area compared to wild-type apoA-I. We proposed that the alteration at position 26 disrupts the N-terminal helical bundle, destabilizing it and promoting the formation of misfolded fibrils. To determine whether mutations in the "outside" cluster produce similar effects on the protein, and therefore similar misfolded fibrils, the structure of the full-length L178H variant was studied using ANS binding, limited proteolysis, and intrinsic fluorescence quenching. In this thesis work, it is shown that the L178H mutation results in increased N-terminal protease sensitivity and hydrophobic surface area, but with no change in the nonpolar environment of the protein's tryptophan residues (Petrlova, J., Duong, T., Cochran, M., Axelsson, A., Morgelin, M., Roberts, L.M., Lagerstedt, J.O. 2012 J. Lipid Res. 53, 390-398). Thus, mutations from the two clusters result in similar structural effects on the protein. ANS binding was also used to analyze conformational changes over time in protein incubated at either 4oC or 37oC. L178H exhibited earlier and more substantial changes in hydrophobic surface area at both temperatures compared to wild-type apoA-I, which likely reflects the increased propensity of L178H to form fibrils. The effects of the L178H mutation are reviewed in the context of recent structural models of apoA-I.