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. 2017 Aug 8;113(3):580-596.
doi: 10.1016/j.bpj.2017.06.030.

Critical Influence of Cosolutes and Surfaces on the Assembly of Serpin-Derived Amyloid Fibrils

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Free PMC article

Critical Influence of Cosolutes and Surfaces on the Assembly of Serpin-Derived Amyloid Fibrils

Michael W Risør et al. Biophys J. .
Free PMC article

Abstract

Many proteins and peptides self-associate into highly ordered and structurally similar amyloid cross-β aggregates. This fibrillation is critically dependent on properties of the protein and the surrounding environment that alter kinetic and thermodynamic equilibria. Here, we report on dominating surface and solution effects on the fibrillogenic behavior and amyloid assembly of the C-36 peptide, a circulating bioactive peptide from the α1-antitrypsin serine protease inhibitor. C-36 converts from an unstructured peptide to mature amyloid twisted-ribbon fibrils over a few hours when incubated on polystyrene plates under physiological conditions through a pathway dominated by surface-enhanced nucleation. In contrast, in plates with nonbinding surfaces, slow bulk nucleation takes precedence over surface catalysis and leads to fibrillar polymorphism. Fibrillation is strongly ion-sensitive, underlining the interplay between hydrophilic and hydrophobic forces in molecular self-assembly. The addition of exogenous surfaces in the form of silica glass beads and polyanionic heparin molecules potently seeds the amyloid conversion process. In particular, heparin acts as an interacting template that rapidly forces β-sheet aggregation of C-36 to distinct amyloid species within minutes and leads to a more homogeneous fibril population according to solid-state NMR analysis. Heparin's template effect highlights its role in amyloid seeding and homogeneous self-assembly, which applies both in vitro and in vivo, where glycosaminoglycans are strongly associated with amyloid deposits. Our study illustrates the versatile thermodynamic landscape of amyloid formation and highlights how different experimental conditions direct C-36 into distinct macromolecular structures.

Figures

Figure 1
Figure 1
Surface type profoundly affects C-36 fibrillation kinetics and alters the balance between primary and secondary nucleation. (a) Given here is an overview of the C-36 peptide with indication of the secondary structure in the context of α1AT and the weak β-strand secondary structural propensity (SSP) in solution determined by liquid-state NMR (adapted from (42)). The aggregation-prone FVFLM is boxed in gray. (b) Kinetic traces are displayed in triplicates for C-36 under quiescent conditions carried out in NBS. (c) Double logarithmic plots show the time to half-completion (t1/2) ± SD under quiescent or shaking conditions (300 RPM) as a function of C-36 concentration. The scaling relationship for indicated linear fits with SE is γ = −1.45 ± 0.09 for quiescent conditions and γ = −0.89 ± 0.04 for shaking. (d) Kinetic traces are displayed in triplicates for C-36 under quiescent conditions carried out in polystyrene surface plates (PS). (e) Double logarithmic plots of the time to half completion (t1/2) ± SD under quiescent or shaking conditions (300 RPM) are given as a function of C-36 concentration. The scaling relationship for indicated linear fits with SE is γ = −0.88 ± 0.03 for quiescent conditions and γ = −0.91 ± 0.03 for shaking. Insets in (c) and (e) compare kinetic traces for 16 μM C-36 under shaking (S) and quiescent (Q) conditions. To see this figure in color, go online.
Figure 2
Figure 2
Secondary nucleation pathway models describe C-36 fibrillation behavior. Global fits to averaged C-36 time-course profiles were performed with the Amylofit online tool (4). (a) The PP model with primary nucleation alone failed to represent the C-36 NBS quiescent data. The nucleus size (nc) was set to 2.9, derived from the γNBS-Q scaling exponent of −1.45 (γ = nc/2). (b) The SP model with primary and secondary nucleation terms fit the data adequately for lower m0. The nucleus size for secondary nucleation (n2) was set to 1.9 (γNBS-Q = (n2−1)/2 = −1.45) and nc was fitted to 1.85. The extracted values for the secondary and primary nucleation contribution were k+k2 = 1.0⋅1015 and k+kn = 93, respectively. Fcrit at which the two nucleation pathways contributed equally to new aggregate formation was 10−8 (for m0 = 8 μM). (c) The PP model failed to represent the C-36 PS quiescent data with nc = 1.76, derived from γPS−Q scaling exponent of −0.88. (d) The SP model with n2 set to 0.76 and nc = 1.425 (fitted) represented the data up to t1/2 adequately. The fit estimates for the secondary and primary nucleation contribution were k+k2 = 2.0⋅109 and k+kn = 2.1⋅105, respectively. Fcrit at which the two nucleation pathways contributed equally to new aggregate formation was 0.005 (for m0 = 8 μM). To see this figure in color, go online.
Figure 3
Figure 3
The PS surface bypasses a pre-ThT hydrophobic species observed by the DCVJ tracer. ThT and DCVJ fluorescence was measured for 16 μM C-36 fibrillation reactions in PS and NBS. (a) The DCVJ signal (gray) preceded the ThT signal (black) in NBS plates, potentially probing a pre-ThT oligomeric population. (b) The DCVJ and ThT signals displayed highly similar traces in PS plates with no apparent DCVJ increase before the ThT signal. The decrease in the DCVJ signal after reaching its maximal value is interpreted as consolidation of the mature fibrils. Gray error bars indicate the triplicate SD for each point.
Figure 4
Figure 4
Fibrillation is highly affected by the solution composition. C-36 fibrillation kinetics (8 μM) and ThT endpoint signal showed a large effect of the buffer ion identity and the ionic strength for reactions carried out in NBS (a) or PS (b). All buffers were prepared to pH 7.42 and had approximate ionic strengths of IPB = 49 mM, IPBS = 200 mM, IABC = 20 mM, ITB = 16 mM, ITBS = 166 mM, IHEPES = 7 mM, and IMOPS = 11 mM. Colored error bars indicate the triplicate SD for each point. TBS is Tris-buffered saline, TB is Tris buffer, PBS is phosphate-buffered saline, PB is phosphate buffer, and ABC is ammonium bicarbonate buffer. See Materials and Methods for exact buffer compositions. To see this figure in color, go online.
Figure 5
Figure 5
An exogenous glass bead surface stimulates nucleation pathways of C-36. (a) Shown here are ThT traces of 8 μM C-36 in PBS with the addition of the indicated number of silica glass beads. A semilog relationship between the number of glass beads and t1/2 is observed (right). (b) Given here are normalized aggregate mass curves using the highest ThT level for each reaction. Global fits to the SP model are displayed with best fit values: nc = 2, n2 = 5, k+kn = 5.7⋅107. The secondary nucleation term, k+k2, was individually fit to each curve and displayed a linear relationship to the number of glass beads (right). Fcrit at which the two nucleation pathways contributed equally to new aggregate formation in the presence of one glass bead was 0.0038 (for m0 = 8 μM). To see this figure in color, go online.
Figure 6
Figure 6
Heparin induces rapid C-36 fibrillation and β-sheet aggregates with lower ThT fluorescence. (a) ThT traces of 8 μM C-36 in PBS show potent alteration of C-36 fibrillation by additions of heparin with lag phase and endpoint ThT reductions. Colored error bars indicate triplicate SD for each point. (b) A semilogarithmic plot of the of t1/2 as a function of heparinDS/peptide reveals a scaling relationship for ratios ranging from 0.02:1 to 1:1, correlating with the heparin-induced ThT fluorescence reduction. (c) A clear conversion to β-sheet is observed by CD (25 μM peptide) as a function of the heparinDS/peptide. Inset shows the mean residue ellipticity change at 222 nm. (d) Time-resolved heparin-induced C-36 aggregation is followed by ThT and CD at 222 nm with 25 μM C-36 and heparin addition to ∼5:1 heparinDS/peptide. Single exponential fits are shown for each curve with exclusion of the first three data points for the ThT signal (crosses). Inset illustrates increasing aggregation speed (normalized 222 nm CD signal) at higher heparin concentrations (29, 58, 115, 231, and 461 μM). The maximal extrapolated C-36 aggregation rate was 0.42 ± μM/s (Fig. S9e). (e) FITC-labeled heparin binds C-36 and is removed from solution. FITC-heparin concentration was 50 μg/mL, corresponding to 83 μM heparinDS. The mean value for heparinDS per C-36 peptide was 1.26 ± 0.04 (SE) based on the linear regression of two combined independent experiments. To see this figure in color, go online.
Figure 7
Figure 7
Surface-guided fibrillation of C-36 to distinct amyloid arrangements is shown. C-36 is intrinsically disordered in solution and has an overall positive charge. The distribution of charged residues is shown by blue (negative) and red (positive) balls. On nonbinding surfaces, several pathways coexist and lead to a large array of fibrillar structures (bottom) with protofibrillar organization into helical and ribbon twist fibrils of various sizes. Polystyrene (blue), silica glass (green), and heparin (dark blue) all bypass a slow primary nucleation to enhance fibrillation rates, likely through a favorable charge compensation by their negative surface. The presence of these stimulating surfaces selectively enhances the formation of distinct fibril morphologies. To see this figure in color, go online.
Figure 8
Figure 8
Nonheparin and heparin-induced fibrils display atomic level differences by solid-state NMR. (a) Given here is the fibril morphology of recombinant C-36 fibrils formed on polystyrene in the absence (N-fibrils) or in the presence (H-fibrils) of heparin. (b) Shown here is an overlay of 13C-13C correlation DARR spectra of N-fibrils (green) and H-fibrils (blue) recorded by MAS ssNMR on a 700 MHz magnet. (c) Excerpts from selected chemical shift regions illustrate the clear differences between N- and H-fibrils. To see this figure in color, go online.

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