Deamidation accelerates amyloid formation and alters amylin fiber structure

Abstract

Deamidation of asparagine and glutamine is the most common nonenzymatic, post-translational modification. Deamidation can influence the structure, stability, folding, and aggregation of proteins and has been proposed to play a role in amyloid formation. However there are no structural studies of the consequences of deamidation on amyloid fibers, in large part because of the difficulty of studying these materials using conventional methods. Here we examine the effects of deamidation on the kinetics of amyloid formation by amylin, the causative agent of type 2 diabetes. We find that deamidation accelerates amyloid formation and the deamidated material is able to seed amyloid formation by unmodified amylin. Using site-specific isotope labeling and two-dimensional infrared (2D IR) spectroscopy, we show that fibers formed by samples that contain deamidated polypeptide contain reduced amounts of β-sheet. Deamidation leads to disruption of the N-terminal β-sheet between Ala-8 and Ala-13, but β-sheet is still retained near Leu-16. The C-terminal sheet is disrupted near Leu-27. Analysis of potential sites of deamidation together with structural models of amylin fibers reveals that deamidation in the N-terminal β-sheet region may be the cause for the disruption of the fiber structure at both the N- and C-terminal β-sheet. Thus, deamidation is a post-translational modification that creates fibers that have an altered structure but can still act as a template for amylin aggregation. Deamidation is very difficult to detect with standard methods used to follow amyloid formation, but isotope-labeled IR spectroscopy provides a means for monitoring sample degradation and investigating the structural consequences of deamidation.

Figure 1

Mechanism for deamidation of L-Asn.

Figure 2

a. Sequence of hIAPP. Potential sites for deamidation are shown in red and residues that were isotope labeled for this study are shown with an overbar. There is a disulfide bond between Cys-2 and Cys-7, and the C-terminus is amidated. Underlined amino acids have parallel β-sheet structure, according to Tycko. b. Structural model for an isomorphic structure of amylin fiber from solid-state NMR. Asparagines and glutamine are shown in red. The four isotope labeled residues incorporated into deamidated amylin are shown in blue. Val-17 is shown in green. The perspective of the figure is looking down the fiber axis.

Figure 3

Model slice through the diagonal of a 2D IR spectrum of an isotopically labeled parallel β-sheet. The unlabeled amide I mode absorbs at 1620 cm⁻¹, while the strongly coupled, in-register isotope labeled amino acid absorbs at ~1575 cm⁻¹.

Figure 4

a. TEM image of deamidated Ala-13 fibers. b. TEM image of unmodified Ala-13 fibers. c. Statistical analysis of the apparent width of deamidated Ala-13 fibers. d. Statistical analysis of the apparent width of unmodified Ala-13 fibers. Scale bars on TEM represent 200 nm.

Figure 5

Normalized CD spectra of deamidated Ala-13 (blue) and unmodified Ala-13 (red).

Figure 6

a. Aggregation kinetics of human amylin monitored by following the amide I peak intensity in unmodified amylin. b. Aggregation kinetics of human amylin monitored by following the amide I peak in deamidated amylin.

Figure 7

a. Diagonal slice through the 2D IR spectrum of unmodified Ala-13. b. 2D IR spectrum of unmodified Ala-13. c. Diagonal slice through the 2D IR spectrum of deamidated Ala-13. d. 2D IR spectrum of deamidated Ala-13.

Figure 8

Slices through 2D IR spectra. a. Unmodified Ala-8. b. Deamidated Ala-8. c. Unmodified Ala-13. d. Deamidated Ala-13. e. Unmodified Leu-16. f. Deamidated Leu-16. g. Unmodified Leu-27. h. Deamidated Leu-27. Red lines are included as a guide to the eye for the frequencies of the unlabeled amide I and isotope label peaks.

Figure 9

a. Aggregation kinetics of unseeded Val-17 amide I folding monitored by following the amide I peak intensity in unmodified amylin. b. Aggregation kinetics trace of seeded Val-17 amide I folding monitored by following the amide I peak intensity. c. Slice through the diagonal of a 2D IR spectrum of unmodified Val-17 monomers seeded with deamidated Ala-13 fibers, averaged over the first 8 minutes following mixing. d. Slice through the diagonal of a 2D IR spectrum averaged over 8 minutes 2.5 hours after mixing.