Abstract
Apolipoprotein (apo) A-I is a major component of high density lipoprotein (HDL) which removes and transports cholesterol from peripheral cells to the liver to be excreted in a process known as reverse cholesterol transport (RCT). Plasma apo A-I levels are correlated with reduced cardiovascular disease (CVD) risk ( , ), presumably due to the role of apo A-I in RCT, as well as apo A-I’s demonstrated anti-inflammatory and anti-oxidant properties ( ). However, mutations in apo A-I result in fibril formation, which can lead to severe diseases including atherosclerosis, neuropathy, nephropathy, and peptic ulcer disease ( , ). These mutations cluster in two regions, within the first 100 amino acid residues of the protein and in a small cluster spanning residues 173-178. The two clusters appear to be associated with tissue specific deposition of fibrils ( ). Two full-length fibril-forming proteins with a mutation in either the N-terminal region (G26R) or the C-terminal region (L178H) have been studied. The G26R mutation leads to the formation of amyloid fibrils with increased β-strand structure ( ) that causes hereditary systemic amyloidosis, resulting in neuropathy, nephropathy and visceral amyloidosis. The L178H mutation results in helical fibril formation leading to fibril deposits in the larynx and in cardiac tissue (6). A first step in understanding differences between the two mutation clusters and how they are manifested in vivo is to explore the properties of variants from each cluster. To that end, we are exploring the structure and stability of L178H. A common feature of fibril-forming proteins is that they are generally less stable, and therefore more prone to misfolding, than their wild-type (WT) counterparts ( - ). The goal of this thesis project is to analyze the stability of L178H in comparison to that of wild-type apo A-I using equilibrium solvent denaturation. In this work, the protein stability and cooperativity of unfolding were measured through the intrinsic fluorescence of tryptophan residues. From intrinsic fluorescence measurements, the Gibbs free energy of protein unfolding (ΔG), effectiveness of unfolding (m), and the midpoint of denaturation (D1/2) were obtained. The results indicate L178H is less stable and unfolds less cooperatively than WT protein. In addition, the stability of L178H under all conditions tested is dominated more by hydrophobic interactions than electrostatic interactions compared to WT apo A-I. These factors are likely responsible for the propensity of this mutant to form fibrillar structures in vivo.