Expanding the repertoire of amyloid polymorphs by co-polymerization of related protein precursors

Abstract

Amyloid fibrils can be generated from proteins with diverse sequences and folds. Although amyloid fibrils assembled in vitro commonly involve a single protein precursor, fibrils formed in vivo can contain more than one protein sequence. How fibril structure and stability differ in fibrils composed of single proteins (homopolymeric fibrils) from those generated by co-polymerization of more than one protein sequence (heteropolymeric fibrils) is poorly understood. Here we compare the structure and stability of homo and heteropolymeric fibrils formed from human β2-microglobulin and its truncated variant ΔN6. We use an array of approaches (limited proteolysis, magic angle spinning NMR, Fourier transform infrared spectroscopy, and fluorescence) combined with measurements of thermodynamic stability to characterize the different fibril types. The results reveal fibrils with different structural properties, different side-chain packing, and strikingly different stabilities. These findings demonstrate how co-polymerization of related precursor sequences can expand the repertoire of structural and thermodynamic polymorphism in amyloid fibrils to an extent that is greater than that obtained by polymerization of a single precursor alone.

FIGURE 1.

Fibrils formed from hβ₂m and ΔN6. A, shown is a ¹H,¹⁵N HSQC spectrum of ΔN6 at pH 6.2. B, shown is fibril formation of 0.5 mg/ml ΔN6 (black lines) and hβ₂m (gray lines, no growth) at pH 6.2 measured using thioflavin T fluorescence (relative fluorescence units (rfu)). Three replicates for each protein are shown. The inset shows an SDS-polyacrylamide gel of the supernatant of the ΔN6 sample after an incubation time of 120 h (lane i) and before fibril growth (lane ii). C, shown are negative stain EM images of ΔN6 fibrils. Inset i shows an expanded view, and inset ii shows the absence of hβ₂m fibrils under the same conditions (scale bar = 100 nm). D–F are as in A–C, but for hβ₂m at pH 2.0 in 10 mm sodium phosphate, 50 mm sodium chloride.

FIGURE 2.

ESI-MS spectra reveal that the disulfide bond is intact in ΔN6 fibrils. A, shown are monomers of ΔN6 released from fibrils formed at pH 6.2 by treatment with HFIP. B is as A, but the sample was treated with a 20-fold molar excess of iodoacetamide. *, results from derivatization of methionine (11,194 Da) and a subsequent loss of the carboxyamido group (marked with $) (62). C is as B, but the sample was treated with DTT followed by the addition of a ∼20-fold molar excess (over the total thiol concentration) of iodoacetamide.

FIGURE 3.

MAS NMR spectra of uniformly ¹³C,¹⁵N-labeled hβ₂m (blue) and ΔN6 (red) fibrils. A, shown are one-bond ¹³C-¹³C correlations from a RFDR experiment. B, shown are backbone Nα-Cα correlations obtained with ZF TEDOR.

FIGURE 4.

Characterization of fibrils formed from mixtures of ΔN6 and hβ₂m at pH 6.2. A, shown are ThT fluorescence traces of ΔN6 at 60 μm (solid black), 120 μm (dashed black), and a 60:60 μm mixture of ΔN6 and hβ₂m (dashed gray). Note hβ₂m incubated alone (60 and 120 μm) does not form fibrils under these conditions (solid gray). The kinetic traces of three different replicates are shown for each sample. Shown are negative stain EM images of the end point of incubation of hβ₂m alone (B) and the ΔN6:hβ₂m mixed fibril sample (the inset shows a single fibril; C) both at pH 6.2; scale bars are 100 nm. rfu, relative fluorescence units. D, shown is an ESI mass spectrum of depolymerized fibrils formed from a 1:1 (mol/mol) mixture of hβ₂m (11,859 Da) and ΔN6 (11,136 Da). E, shown are fluorescence microscopy images of TAMRA-labeled hβ₂m at pH 6.2 (no fibrils). F, shown are fibrils of TAMRA-hβ₂m formed at pH 2. FITC-labeled ΔN6 fibrils formed at pH 6.2 (G) and fibrils formed from a 1:1 (mol/mol) mixture of TAMRA-hβ₂m monomers and FITC-ΔN6 (H) are shown. The yellow color shows the superposition of red and green fluorescence. Scale bar = 5 μm. The scattergraphs depict co-localization plots of the contribution from the green (FITC fluorescence, x axis) and red (TAMRA fluorescence, y axis) channels for each pixel location. The y2 axis is the intensity of the signal. The images are 8 bit, thus the x and y axes are from 0–256 pixels.

FIGURE 5.

Limited proteolysis of ΔN6 monomer, hβ₂m, and ΔN6 homopolymeric fibrils and heteropolymeric fibrils. Limited proteolysis was performed using aspergillopepsin I at pH 6 and chymotrypsin at pH 8 (ΔN6 fibrils, mixed fibrils, and ΔN6 monomer) or aspergillopepsin I only at pH 2 (hβ₂m fibrils) at a 1:100 (w/w) proteinase:protein ratio, mapped using ESI-MS and ESI-MS/MS. Potential chymotrypsin cleavage sites are found throughout the sequence of hβ₂m (gray bar). Cleavage sites in the fibrils and ΔN6 monomer are shown using vertical bars. The horizontal filled bars represent the peptide fragments observed. Cleavage of ΔN6 monomers with chymotrypsin at pH 8 is also shown (pink).

FIGURE 6.

Spectroscopic analysis of the fibrils formed from ΔN6, hβ₂m, and a mixture of the two monomers. A, shown are FTIR absorbance spectra of hβ₂m monomer at pH 2 (blue) and ΔN6 (red) monomer at pH 6.2 (dotted lines) and fibrils formed from hβ₂m pH 2 (blue solid line), ΔN6 at pH 6.2 (red solid line) and a 1:1 mixture of ΔN6:hβ₂m at pH 6.2 (green). For clarity the spectrum of ΔN6 fibrils has been vertically offset. Its spectrum is otherwise very similar to that of the heteropolymeric fibrils. au, normalized absorbence units. B, dot blots of different samples incubated with the antibody WO1 and a polyclonal anti-β₂m antibody are shown. C, shown are fluorescence emission spectra of ANS in the presence of fibrils formed from ΔN6 (red), hβ₂m (blue), and the mixed fibrils (green). Spectra of monomeric ΔN6 and hβ₂m are also shown at ∼0 relative fluorescence units (rfu). D, shown are intrinsic fluorescence emission spectra of ΔN6 monomer at pH 6.2 (red dotted line) and hβ₂m monomer at pH 2 (blue dotted line) and hβ₂m fibrils (blue solid line), ΔN6 fibrils (red solid line) and the mixed fibrils (green).

FIGURE 7.

Thermodynamic stability of fibrils formed from ΔN6 (red), hβ₂m (blue), and heteropolymeric fibrils (green). The release of soluble material was measured using absorbance at 280 nm after incubation in GuHCl for 1.5 h. The results are presented as the proportion of soluble material by dividing the concentration of the soluble monomer by the total starting monomer concentration. The fit is to guide the eye.

FIGURE 8.

Co-polymerization of hβ₂m and ΔN6 can occur by a variety of different possible mechanisms, involving oligomer formation, initial heterodimer formation, or cross-seeding. See Discussion for details.