Islet amyloid polypeptide: Identification of long-range contacts and localorder on the fibrillogenesis pathway

The pathology of type II diabetes includes deposition of amyloid in the ext ra cellular space surrounding the P-cells of the endocrine pancreas. The pr inciple component of these deposits is an insoluble fibrillar form of a nor mally soluble 37 residue peptide hormone, islet amyloid polypeptide. Multip le sequence analysis and peptide synthesis have identified a core set of re sidues (20 to 29) as intrinsically amyloidogenic. As the fibrillogenesis of the 20-29 peptide often requires conditions that deviate considerably from physiological, residues 20 to 29 may be necessary, but not sufficient, for amyloidosis. We aim to determine the structural role of residues outside t his core in the context of in vitro fibrillogenesis of the wild-type peptid e at physiological pH and ionic strength. Specifically, we make use of an i ntrinsic fluorescent probe, tyrosine 37 (Y37), to explore the role of the C terminus in fibrillogenesis. Our protocol permits steady state measurement of the lag phase and fiber conformational states of the protein under iden tical conditions. These are compared to a non-amyloi-dogenic variant of isl et amyloid polypeptide from rat and N-acetyl-tyrosinamide as models of the unfolded state under matched conditions. Spectral, quenching and anisotropi c properties of Y37 in the fiber state indicate that the C terminus is pack ed in a well-defined environment with near frozen rigidity. The presence of a fluorescence resonance energy transfer pathway shows Y37 is near F15 and F23. The lag-phase conformation, while considerably less ordered than the fiber, is more ordered than unfolded models. Differences in anisotropy betw een the lag and fiber state were used to monitor fibrillogenesis in real ti me. Parallel assessment of fiber formation using the histological dye, ThT, indicate that ordering at the C terminus of islet amyloid polypeptide is c oincident with, and thus indicative of, fiber formation. (C) 2001 Academic Press.