Efforts towards elucidating hIAPP aggregation pathway by 2D IR spectroscopy

Two-dimensional infrared (2D IR) spectroscopy has been used to monitor structural change and kinetics during hIAPP aggregation. With unlabeled peptides, we have followed the folding process of random coil and other secondary structures turning into fibrils rich in parallel beta-sheet in absence and in presence of membranes. An intermediate is detected by 2D IR during hIAPP aggregation with membranes with evidences of classic intermediate mediated kinetics, linewidth narrowing and anharmonicity shift. The intermediate is found to have some ordered structure evidenced by its smaller linewidth and anharmonicity. The intermediate is also shown to bind to membranes by comparison of its rise-and-fall kinetics with membrane-binding kinetics of rIAPP, a variant known to bind to membranes but not form amyloid fibrils. But structural origin of the intermediate is not revealed with the unlabeled hIAPP studies because the structural resolution is too low.;In order to elucidate more details in the picture of hIAPP aggregation such as what the intermediate is, which residue is in the intermediate and binds to membranes, we have synthesized hIAPPs doubly labeled with 1- 13C=18O in the most promising positions with high purity. The double-labeling strategy allows us to differentiate between alpha-helix, parallel-beta-sheet and alpha-sheet intermediates and to detect beta-hairpin oligomer. IR spectra of these structures in model systems have been simulated with a TDC mode to prove the feasibility of the double-labeling schemes.