Mechanisms of diabetic amyloid formation via 2D IR spectroscopy Abstract Type 2 diabetes afflicts nearly 26 million Americans and causes a larger economic loss than all cancers combined. It starts as insulin resistance, but ultimately the pancreatic β-cells that make insulin fail, resulting in overt diabetes. Failure is partially due to aggregation of the hormone known as the human islet amyloid polypeptide (hIAPP or amylin) into amyloid plaques that occupy up to 80% of the islet space. Surprisingly, the amyloid fibers themselves are not cytotoxic. Many researchers believe that the toxic species are oligomers of hIAPP, perhaps by interfering with receptor mediated processes or permeabilizing the membrane. As a result, there is much interest in understanding the mechanism by which hIAPP aggregates, because the aggregation pathway dictates the structures and populations of these cytotoxic intermediates. The first cryoEM structure of amylin was recently reported, but very little structural information exists about intermediates because applying most structural biology tools to kinetically evolving proteins is difficult. We discovered an oligomeric species by monitoring the aggregation kinetics of hIAPP using a technology that we invented, on-the-fly 2D IR spectroscopy. In doing so, we discovered that hIAPP forms oligomers with a parallel β-sheet in the FGAIL region, prior to restructuring into its fibrillar structure. The need to restructure results in a prolonged lifetime and stable population of the oligomers. We observed this “FGAIL oligomer” in 4 different mammalian species known to contract type 2 diabetes, strengthening our hypothesis that this intermediate is a key player in the disease. Most importantly, we realized that we could trap the oligomer with a few benignly placed mutations. Our trapped oligomers are nearly as toxic as wild-type hIAPP, but persist in vitro for days rather than hours. Because it is stable for so long, it enables many new structural, biochemical, and physiological assays not previously possible. And, it provides an intellectual basis to create a new knock-in mouse to investigate hIAPP oligomers in an animal model. With that goal in mind, we have begun working with humanized mice and developed the technology to image pancreas tissues with 2D IR microscopy. Specific Aim 1 will generate a series of trapped oligomers, each of which will be tested for its suitability as a model for hIAPP oligomers. Specific Aim 2 will investigate the aggregation pathway that leads to a recently reported cryoEM structure to determine if this polymorph is formed from a new or existing mechanistic pathway. In Aim 3, we link our in vitro observations to in vivo physiology via 2D IR imaging of two transgenic mouse models. We seek to understand hIAPP aggregation from a fundamental perspective, which is important for inhibitor design and hormone replacement therapies, and utilize that information to translate our in vitro work into in vivo anim...