Peptide conformation and supramolecular organization in amylin fibrils: constraints from solid-state NMR

Abstract

The 37-residue amylin peptide, also known as islet amyloid polypeptide, forms fibrils that are the main peptide or protein component of amyloid that develops in the pancreas of type 2 diabetes patients. Amylin also readily forms amyloid fibrils in vitro that are highly polymorphic under typical experimental conditions. We describe a protocol for the preparation of synthetic amylin fibrils that exhibit a single predominant morphology, which we call a striated ribbon, in electron microscopy and atomic force microscopy images. Solid-state nuclear magnetic resonance (NMR) measurements on a series of isotopically labeled samples indicate a single molecular structure within the striated ribbons. We use scanning transmission electron microscopy and several types of one- and two-dimensional solid-state NMR techniques to obtain constraints on the peptide conformation and supramolecular structure in these amylin fibrils and to derive molecular structural models that are consistent with the experimental data. The basic structural unit in amylin striated ribbons, which we call the protofilament, contains four layers of parallel beta-sheets, formed by two symmetric layers of amylin molecules. The molecular structure of amylin protofilaments in striated ribbons closely resembles the protofilament in amyloid fibrils with a similar morphology formed by the 40-residue beta-amyloid peptide that is associated with Alzheimer's disease.

Figure 1

TEM images of negatively stained amylin fibrils from polymorphic (A,B) and morphologically homogeneous (C,D) preparations. Red and blue arrows indicate fibrils with apparent periodic twists. Yellow arrows indicate striated ribbons. Double-headed yellow arrow indicates a single protofilament that appears to be the building block of striated ribbons. Homogeneous samples prepared by the gel filtration protocol described in the text are primarily striated ribbons.

Figure 2

Topographic AFM images of amylin fibrils absorbed to mica from (A) polymorphic and (B) morphologically homogeneous preparations. Image areas are 2 µm X 2 µm. Height profiles for selected fibrils (indicated by arrows of corresponding colors) are shown to the right.

Figure 3

(A) Histogram of MPL values extracted from STEM images of amylin fibrils. Vertical dashed lines indicate the ideal MPL values of integral numbers of molecular layers with cross-β structures. (B) Representative STEM image (50,000 X magnification). Approximate MPL values of individual fibrils are indicated in units of the MPL of one molecular layer. Double-headed arrows indicate tobacco mosaic virus particles used to determine the ratio of MPL to image intensity.

Figure 4

Selected regions from 2D ¹³C fpRFDR spectra of polymorphic (A) and morphologically homogenous (B,C) amylin fibrils with the AMY05 labeling pattern. Fibrils are rehydrated after lyophilization in A and B, but lyophilized without rehydration in C. Chemical shift assignment paths for uniformly ¹⁵N,¹³C-labeled residues are shown in B, with dashed lines extending to A and C to permit comparisons of ¹³C chemical shifts in the three samples. 1D slices from each spectrum at the indicated chemical shift positions are shown directly below. Measurements were performed at 150.7 MHz ¹³C NMR frequency, with a 2.2 ms fpRFDR exchange period and a 25.00 kHz MAS frequency. Gaussian line broadening of 150 Hz was applied in both dimensions. In the 2D plots, the lowest contour levels are slightly above the noise level and increase by successive factors of 1.7.

Figure 5

Same as in Figure 3B, for morphologically homogeneous amylin fibrils with the AMY06, AMY07, AMY08 labeling patterns (A, B, and C, respectively). Measurements were performed at 100.4 MHz ¹³C NMR frequency, with a 1.6 ms fpRFDR exchange period and a 20.00 kHz MAS frequency.

Figure 6

Same as in Figure 3B, for morphologically homogeneous amylin fibrils with the AMY09, AMY10, and AMY11 labeling patterns (A, B, and C, respectively). Measurements were performed at 150.7 MHz ¹³C NMR frequency, with a 2.2 ms fpRFDR exchange period and MAS frequencies of 25.00 kHz, 30.00 kHz, and 15.00 kHz (A, B, and C, respectively).

Figure 7

¹³C NMR secondary chemical shifts for carbonyl, C_α, and C_β sites in amylin fibrils with the morphology in Figs. 1C and 1D, determined from spectra in Figs. 4, 5, and 6. Light blue arrows indicate likely β-strand segments. Amino acid sequences of human amylin (with likely β-strands in light blue), rodent amylin (with amino acid differences in red), and β-amyloid (with previously determined β-strands (20-22) in light blue) are shown.

Figure 8

(A) Experimental (symbols) and simulated (solid and dashed lines) ¹⁵N fpRFDR-CT data for amylin fibrils with AMY07, AMY08, AMY09, and AMY11 isotopic labeling patterns, detected through ¹³C NMR signals of C_α sites in the indicated residues. The experimental data serve as constraints on the backbone ψ torsion angles of residues in parentheses. Simulations are for ψ values ranging from ±30° (most rapidly decreasing solid line) to ±170° (least rapidly decreasing dashed line), in ±20° steps. Data for AMY07, AMY08, and AMY09 were obtained at 100.8 MHz ¹³C NMR frequency and 20.00 kHz MAS frequency. Data for AMY11 were obtained at 150.7 MHz ¹³C NMR frequency and 15.00 kHz MAS frequency. (B) ¹³C NMR spectra from which AMY08 (left) and AMY11 (right) data were obtained, with ¹⁵N dipolar evolution periods and assignments of ¹³C_α lines indicated.

Figure 9

(A) 2D RAD spectrum of AMY10 fibrils, obtained at 150.7 MHz ¹³C NMR frequency with MAS at 21.00 kHz and a 1000 ms exchange period. Arrows indicate crosspeaks between ¹³C NMR signals of Phe15 aromatic carbons and Ile26/Leu27 methyl carbons (solid lines), Asn14 C_α carbons (dashed lines), and Phe15 C_α and C_β carbons (dotted lines). (B) 1D RAD spectrum of AMY10 fibrils, obtained at 150.7 MHz ¹³C NMR frequency with MAS at 25.00 kHz and a 1000 ms exchange period after selective preparation of longitudinal spin polarization on Phe15 aromatic carbons. Assignments of aliphatic ¹³C NMR signals that arise from polarization transfer from the Phe15 aromatic carbons are shown. (C) 2D RAD spectrum of AMY08 fibrils, obtained at 150.7 MHz ¹³C NMR frequency with MAS at 18.00 kHz and a 500 ms exchange period. Arrows indicate crosspeaks between ¹³C NMR signals of Phe23 aromatic carbons and Gly24 C_α carbons (dashed lines) and Phe23 C_α and C_β carbons (dotted lines). Short dotted arrows indicate crosspeaks that arise from MAS sidebands of Phe23 carbonyl signals. Contour levels in 2D plots increase by successive factors of 1.5.

Figure 10

Experimental and simulated ¹³C fpRFDR-CT data for amylin fibrils with the AMY04 labeling pattern. Separate measurements were performed on the labeled Phe23 carbonyl (triangles) and Ala13 methyl (circles) sites. Experimental data are corrected for signal contributions from natural-abundance ¹³C nuclei by subtraction of a constant baseline equal to 16.5% and 13.2% of the initial signal (based on the numbers of carbonyl and methyl sites). Simulations are for linear chains of ¹³C nuclei with the indicated nearest-neighbor spacings.

Figure 11

Molecular structural models for the protofilament in amylin fibrils with striated ribbon morphologies, generated using restrained Langevin dynamics simulations as described in the text. (A) Ribbon representation of one cross-β molecular layer, with N-terminal and C-terminal β-strand segments colored red and blue, respectively. The black arrow indicates the fibril axis. (B) Cross-sectional view of two amylin molecules in the protofilament. This configuration, with approximate C₂ symmetry about the fibril axis, is supported by the STEM data in Figure 3 and the observation of a single set of chemical shifts in ¹³C NMR spectra. The overall dimensions are consistent with TEM and AFM images. (C, D) All-atom representations of two possible models, with hydrophobic residues in green, polar residues in magenta, positively charged residues in blue and disulfide-linked cysteine residues in yellow.