Resolution of Oligomeric Species during the Aggregation of Aβ_1–40 Using ¹⁹F NMR

Abstract

In the commonly used nucleation-dependent model of protein aggregation, aggregation proceeds only after a lag phase in which the concentration of energetically unfavorable nuclei reaches a critical value. The formation of oligomeric species prior to aggregation can be difficult to detect by current spectroscopic techniques. By using real-time 19F NMR along with other techniques, we are able to show that multiple oligomeric species can be detected during the lag phase of Aβ1-40 fiber formation, consistent with a complex mechanism of aggregation. At least 6 types of oligomers can be detected by 19F NMR. These include the reversible formation of large β-sheet oligomer immediately after solubilization at high peptide concentration; a small oligomer that forms transiently during the early stages of the lag phase; and 4 spectroscopically distinct forms of oligomers with molecular weights between ~30–100 kDa that appear during the later stages of aggregation. The ability to resolve individual oligomers and track their formation in real-time should prove fruitful in understanding the aggregation of amyloidogenic proteins and in isolating potentially toxic non-amyloid oligomers.

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

“…3. Conformational free energy map for the dimerization of hIAPP in terms of order parameters Q [8][9][10][11][12][13][14][15][16] and Q [27][28][29][30][31][32][33][34][35] . Contour lines are drawn every 2.5 kJ/mol.…”

“…Solid-state NMR (ssNMR) and X-ray crystallography provide high-resolution structures of fibrils (4,5) and optical techniques can track structural changes in real time (6,7), but few techniques have both the structural and the temporal resolution to extract specific structural details about intermediates. Fragments have been trapped in intriguing oligomeric structures that may represent intermediate states (5,8) and transient secondary structures are known to exist from circular dichroism measurements and other experiments (9)(10)(11), but for full-length proteins it has been difficult to identify the specific residues that contribute to the secondary structure and thus understand their role in the aggregation mechanism. In this paper, we use 2D infrared (2D IR) spectroscopy and isotope labeling to monitor the structural evolution of the full-length human islet amyloid polypeptide (hIAPP or amylin), a 37-residue peptide implicated in type 2 diabetes.…”

Escherichia coli Topoisomerase IV E Subunit and an Inhibitor Binding Mode Revealed by NMR Spectroscopy

“…Based on these H-D exchange experiments, residues that are critical for and affected by 2 binding were revealed, suggesting that this method will also be useful for understanding protein-drug interactions. 19 F NMR experiments are widely used to study ligand-protein binding and dynamics (28,29). The simplicity and high sensitivity (natural abundance of 19 F ϭ 100%) makes the application of such techniques attractive (30).…”

Ibuprofen-Glutathione Conjugate as Anti-inflammatory and Anti-apoptotic Agent in Rat Brain Infused with β Amyloid (1-40)

“…Moreover, molecular dynamics simulations showed that the Aβ (1-40) monomer transiently binds to the Aβ (1-40) oligomer by non-native contacts with the side chains before being incorporated into the fiber through native contacts with the peptide backbone [14]. Combining 19F NMR with other spectroscopic techniques, more detailed information on the secondary structure of intermediates involved in amyloid formation could be obtained [15].…”