Concurrent with the growth of the β-strand, 2 isotope-labeled features appear at 1,574 and 1,585 cm −1. The 1,617-cm −1 features are the signature of β-strand amyloid growth. As time progresses, the random coil doublet disappears, whereas a doublet grows in at ω pump = 1,617 cm −1. 2 are plotted as difference spectra, calculated by subtracting the 2D IR spectrum in Fig. To highlight the changes that occur in the 2D IR spectra during the aggregation processes, the remaining 2D IR spectra in Fig. At t = 5 min., the isotope-labeled absorption is very broad and weak, indicating that Ala-25 is conformationally disordered, which is consistent with the nature of the random coil state. 2), which provides information specific to the folding kinetics of Ala-25. The isotope-labeled features appear near 1,580 cm −1 (inside the red squares in Fig. The 2 broad peaks at t = 5 min are typical of random coil peptide structures with large structural distributions. In 2D IR spectra, vibrational modes create doublets in which the negative peak is located on the diagonal and can be interpreted much like a traditional infrared absorption peak, albeit with improved frequency resolution. At t = 5 min, the unlabeled features consists of 2 out-of-phase peaks at ω pump = 1,645 cm −1. The unlabeled peaks give information on the overall assembly kinetics of the amyloid, which were previously reported ( 5). The most prominent features in these spectra are between 1,617 and 1,670 cm −1 and are due to the unlabeled residues (inside the black squares in Fig. The 4 spectra are generated from many thousands of spectra that were collected. 2 are 4 2D IR spectra collected at successively later times during amyloid formation, starting from an initially unaggregated sample of hIAPP labeled at Ala-25. What is needed is a technique with sufficient time resolution to observe intermediates, provide residue-level structural information, is nonperturbing, and, ideally, can be used to test molecular dynamics simulations. Mechanistic information is vital to understand the mechanism of protein misfolding as well as to design inhibitors that subvert the pathway of amyloid formation. Other techniques, like electron spin resonance and fluorescence spectroscopy, require bulky labels that can perturb the structure and dynamics. Optical techniques that do have sufficient time resolution, such as circular dichroism spectroscopy, provide only a low-resolution view of structure. The difficulty arises because high-resolution techniques do not have the time resolution required to track the structural changes, nor can they be easily applied to aggregating systems. Although they have been the focus of numerous studies, details about these critical intermediates have been elusive, mostly because it is extraordinarily difficult to obtain structural and kinetic information for amyloid aggregation. In large quantities, organ function is disrupted by the formation of amyloid deposits, but for several amyloid diseases, there is evidence that the toxic entities are actually prefibril intermediates ( 2, 3). More than 20 different diseases are associated with proteins that form insoluble amyloid fibers ( 1). This experimental approach provides a detailed view of the aggregation pathway of hIAPP fibril formation as well as a general methodology for studying other amyloid forming proteins without the use of structure-perturbing labels. By monitoring the kinetics at 6 different labeled sites, we find that the peptides initially develop well-ordered structure in the region of the chain that is close to the ordered loop of the fibrils, followed by formation of the 2 parallel β-sheets with the N-terminal β-sheet likely forming before the C-terminal sheet. Using a recently invented method of collecting 2-dimensional infrared spectra and site-specific isotope labeling, we have measured the development of secondary structures for 6 residues during the aggregation process of the 37-residue polypeptide associated with type 2 diabetes, the human islet amyloid polypeptide (hIAPP). There is considerable interest in uncovering the pathway of amyloid formation because the toxic properties of amyloid likely stems from prefibril intermediates and not the fully formed fibrils.
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