Determination of Transient Structure in Early Intermediates of Islet Amyloid Polypeptide Fiber Assembly

The protein islet amyloid polypeptide (IAPP) is a glucose metabolism associated hormone co-secreted with insulin by the beta-cells of the pancreas. In humans with type II diabetes, IAPP deposits as amyloid fibers. The assembly intermediates of this process are associated with beta-cell death. Formation of IAPP fibers in vitro is nucleation-dependent and kinetically characterized by a lag phase followed by a burst of fiber formation. Kinetic circular dichroism measurements in solution indicate IAPP transitions from the native random coil state to a highly ordered all beta-sheet amyloid. However, on negatively charged membrane bilayers, IAPP forms helical aggregates before transitioning to the fiber state. These helical states are associated with both the native function of the peptide as well as membrane toxicity. Structural measurements of assembly intermediates in solution and on membranes are essential to understanding both fiber assembly and pathology. In this work, NMR spectroscopy is used to characterize the structure of IAPP in solution. NMR is uniquely suited for atomic level measurements of transiently structured states. As isotopically labeled protein is required for most NMR studies, a bacterial expression system was developed. The system overcomes the aggregation propensity of IAPP and produces wild type protein, with all native posttranslational modifications. Full backbone resonance assignments for IAPP allows for characterization of IAPP structure under a variety of conditions. IAPP samples helical structure in its N-terminus directly associated with amyloid assembly. Kinetic analysis paired with structural study demonstrates that urea denatures the helix and inhibits amyloid assembly. Conversely, addition of fluorinated alcohol induces helical structure as well as accelerates assembly. On membrane bilayers, IAPP adopts a helical structure in the same region with transient helicity in solution. As membrane bilayers also accelerate amyloid formation, we propose that stabilization of helical structure is a common mechanism for IAPP amyloid assembly. This work characterizes the structure of IAPP under amyloidogenic conditions and determines residue-specific transitions important for amyloid assembly and toxicity.