Structural studies of the IAPP membrane-mediated aggregation pathway

Amyloid deposition in tissues is a common feature of several human diseases, including Parkinson's, Huntington's disease, and type II diabetes mellitus. In the case of type II diabetes mellitus, amyloid deposition is found in the pancreatic beta-cells of the patients, which contain fibrillar islet amyloid polypeptide (IAPP) as a main component. In all amyloid diseases a soluble protein is being misfolded and consequently forms beta-sheet fibrillar aggregates. This aggregation process occurs in several steps and is nucleation-dependent. The formation of several structural species precedes beta-sheet fibril deposition. The etiology why amyloid proteins misfold is not clear yet and neither is their mechanism of toxicity. However, recent evidence suggests that an oligomeric protein structure confers beta-cell toxicity via lipid membrane disruption.;The involvement of lipid membranes in the protein-misfolding pathway is increasingly being recognized. Both acceleration of amyloid protein aggregation and lipid membrane disruption have been witnessed upon interaction with lipid membranes. For IAPP, although the significance of lipid membranes in the aggregation pathway has also been established, a detailed structural understanding of such an interaction is missing.;As a first step toward obtaining structural information of the lipid membrane-mediated IAPP aggregation pathway, we investigated the detailed structure of monomeric IAPP upon membrane interaction by employing site-directed spin labeling. Our results indicate that, when membrane-bound, IAPP forms a single alpha-helix encompassing residues 9 to 22. This helix is flanked by the N- and C-terminal regions of the peptide, which are less ordered and do not take up a clearly detectable secondary structure.;In addition, we performed leakage studies to investigate the membrane disruption ability of different human IAPP analogs and rat IAPP in different conditions. According to these studies lipid membrane disruption by IAPP depends on the concentration of negatively charged phospholipids. When there is a high negatively charged phospholipid density the leakage by both human IAPP analogs and rat IAPP is very potent. In contrast, at low negatively charged phospholipid density, rat IAPP causes insignificant leakage while human IAPP is still potent but with a more complex disruption mechanism.;At last, expression and purification of a fusion IAPP protein (GB3-IAPP) is described in chapter 5. Large production of recombinant IAPP is important in studying the structural and molecular properties of the peptide. Thus, its expression is useful and will help in future studies requiring its use.