Abstract
The amyloid formation of the folded segment of a variant of Exenatide (a marketed drug for type‐2 diabetes mellitus) was studied by electronic circular dichroism (ECD) and NMR spectroscopy. We found that the optimum temperature for E5 protein amyloidosis coincides with body temperature and requires well below physiological salt concentration. Decomposition of the ECD spectra and its barycentric representation on the folded‐unfolded‐amyloid potential energy surface allowed us to monitor the full range of molecular transformation of amyloidogenesis. We identified points of no return (e.g.; T=37 °C, pH 4.1, c E5=250 μm, c NaCl=50 mm, t>4–6 h) that will inevitably gravitate into the amyloid state. The strong B‐type far ultraviolet (FUV)‐ECD spectra and an unexpectedly strong near ultraviolet (NUV)‐ECD signal (Θ ≈275–285 nm) indicate that the amyloid phase of E5 is built from monomers of quasi‐elongated backbone structure (φ≈−145°, ψ≈+145°) with strong interstrand Tyr↔Trp interaction. Misfolded intermediates and the buildup of “toxic” early‐stage oligomers leading to self‐association were identified and monitored as a function of time. Results indicate that the amyloid transition is triggered by subtle misfolding of the α‐helix, exposing aromatic and hydrophobic side chains that may provide the first centers for an intermolecular reorganization. These initial clusters provide the spatial closeness and sufficient time for a transition to the β‐structured amyloid nucleus, thus the process follows a nucleated growth mechanism.
Highlights
Aggregation of proteins and peptide segments into amyloid fibrils have been studied intensively over the past decades since the process was shown to be associated with, or even trigger,[1,2,3] such illnesses as Alzheimer’s disease, type-2 diabetes mellitus, rheumatoid arthritis, or haemodialysis ass. amyloidosis.[4]
FUVECD measurements showed that amyloid formation is more effective in the presence than in the absence of salt, with the preferred protein concentration ranging from 80 to 160 mm, while the optimal pH is near 4.0
We propose that the enhanced positive near ultraviolet (NUV)-electronic circular dichroism (ECD) bands between 270 < l < 290 nm no longer arise from a pairwise, intramolecular shifted face-to-face p–p interaction, but rather from the interstrand interaction of Tyr and Trp side chains packed tighter in the supramolecular assembly of the amyloid phase
Summary
Aggregation of proteins and peptide segments into amyloid fibrils have been studied intensively over the past decades since the process was shown to be associated with, or even trigger,[1,2,3] such illnesses as Alzheimer’s disease, type-2 diabetes mellitus, rheumatoid arthritis, or haemodialysis ass. amyloidosis.[4]. E5 contains a b-turn, a polyproline-II helix and a hydrophobic center with a buried Trp, has a protein-like build-up and folds quite to a typical globular protein.[32]. We probed various regions of the f(T,pH,cprotein,cion,t) potential energy hypersurface of E5 by acquiring quantitative NUV- and FUV-ECD chiroptical information and NMR data complemented by MD simulations to pinpoint the reaction path that leads from the fully folded- to the amyloid-state. Based on these results, we were able to propose a mechanism that resembles that of well-folded proteins but relies on special features of the miniprotein. The methodology presented here gains significance because the amyloid state of E5 is ThT silent and presents an approach for dealing with such cases as well
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