Two-dimensional infrared spectroscopy reveals the complex behaviour of an amyloid fibril inhibitor

Abstract

While amyloid formation has been implicated in the pathology of over twenty human diseases, the rational design of amyloid inhibitors is hampered by a lack of structural information about amyloid-inhibitor complexes. We use isotope labeling and two-dimensional infrared spectroscopy to obtain a residue-specific structure for the complex of human amylin, the peptide responsible for islet amyloid formation in type 2 diabetes, with a known inhibitor, rat amylin. Based on its sequence, rat amylin should block formation of the C-terminal β-sheet, but at 8 hours after mixing rat amylin blocks the N-terminal β-sheet instead. At 24 hours after mixing, rat amylin blocks neither β-sheet and forms its own β-sheet most likely on the outside of the human fibrils. This is striking because rat amylin is natively disordered and not previously known to form amyloid β-sheets. The results show that even seemingly intuitive inhibitors may function by unforeseen and complex structural processes.

Mechanism of IAPP amyloid fibril formation involves an intermediate with a transient β-sheet

“…It is possible that the early β-sheets are limited to residues 23-27, which would be consistent with early fragment studies showing that hIAPP [23][24][25][26][27] is the smallest fragment of hIAPP capable of forming amyloid fibrils (15). However, the hIAPP 22-27 fragment aggregates 40 times faster than hIAPP [23][24][25][26][27] and hIAPP [20][21][22][23][24][25][26][27][28][29] aggregates faster still, suggesting that the oligomeric β-sheets might extend beyond our labeled region to include the serines (15). Nonetheless, we know that the β-sheets must be small because, like typical random coil peptides, the unlabeled region of the spectra has only a minimal peak at 1,620 cm −1 (and does not extend to V17 or G33).…”

“…Within an in-register parallel β-sheet conformation, the labeled residues form a coupled linear chain along the length of the sheet, shifting the isotope mode frequency to between 1,570 cm −1 and 1,588 cm −1 , depending on the precise coupling strength and structural order of the labeled residue. Isotope dilution experiments, in which only 25% of the peptides are labeled, provide an independent test of β-sheet structure by eliminating the effects of coupling and its characteristic spectral shift without changing the structure (26,27). This labeling strategy has previously been used to study amyloid structure, aggregation kinetics, and inhibitor binding (26,28,29).…”

“…Isotope dilution experiments, in which only 25% of the peptides are labeled, provide an independent test of β-sheet structure by eliminating the effects of coupling and its characteristic spectral shift without changing the structure (26,27). This labeling strategy has previously been used to study amyloid structure, aggregation kinetics, and inhibitor binding (26,28,29).…”

“…Early work revealed a correlation between the ability to form amyloid in vitro and the sequence in the 20-29 region (13,14). The fragment hIAPP [20][21][22][23][24][25][26][27][28][29] (SNNFGAILSS) readily forms amyloid fibrils, as do smaller fragments such as hIAPP 23-27 (13-17) and hIAPP [21][22][23][24][25][26][27] ; the latter has been characterized with X-ray crystallography (5). Molecular dynamics simulations corroborate the assembly of such fragments into β-sheet structures (18)(19)(20).…”

Microscopic insights into the protein-stabilizing effect of trimethylamine N-oxide (TMAO)

“…For an IR probe that is able to interact with water via H bonding, measurement of its FFCF can provide, sometimes in a site-specific manner, detailed information about the hydration dynamics of the protein molecule of interest. For example, this approach has been used to identify the existence of mobile water molecules inside Aβ40 amyloid fibrils (31,32) and to interrogate the water-assisted drug-binding mechanism of HIV-1 reverse transcriptase (33), among many other applications (34)(35)(36)(37)(38)(39). In the current study, we capitalize on the established sensitivity of the nitrile stretching vibration (C≡N) to local hydration and electrostatic environment (40) and use the unnatural amino acid p-cyano-phenyalanine (Phe CN ) as a local IR reporter.…”