Guiding protein aggregation with macromolecular crowding

Abstract

Macromolecular crowding is expected to have a significant effect on protein aggregation. In the present study we analyzed the effect of macromolecular crowding on fibrillation of four proteins, bovine S-carboxymethyl-alpha-lactalbumin (a disordered form of the protein with reduced three out of four disulfide bridges), human insulin, bovine core histones, and human alpha-synuclein. These proteins are structurally different, varying from natively unfolded (alpha-synuclein and core histones) to folded proteins with rigid tertiary and quaternary structures (monomeric and hexameric forms of insulin). All these proteins are known to fibrillate in diluted solutions, however their aggregation mechanisms are very divers and some of them are able to form different aggregates in addition to fibrils. We studied how macromolecular crowding guides protein between different aggregation pathways by analyzing the effect of crowding agents on the aggregation patterns under the variety of conditions favoring different aggregated end products in diluted solutions.

Figure 1

FTIR amide I spectra of bovine S-carboxymethyl-α-lactalbumin (A), human insulin (B), bovine core histones (C) and human α-synuclein (D) in non-fibrillar (black circles) and fibrillar forms after the incubation at appropriate conditions at 37°C with stirring (open reversed triangles).

Figure 2

Effect of crowding agents on fibrillation and structural properties of bovine S-carboxymethyl-α-lactalbumin. (A) Fibrillation of bovine S-carboxymethyl-α-lactalbumin (1 mg/ml) monitored by the enhancement of thioflavin T fluorescence intensity at 37°C with stirring in the absence (circles) or presence of high concentrations of model macromolecular crowding agents: PEG-3500 (100 mg/ml, reversed triangles), Ficoll-70000 (150 mg/ml, diamonds) and bovine serum albumin (30 mg/ml, squares). Conditions were 20 mM Tris-HCl, 100 mM NaCl, pH 7.5. (B) Near-UV CD spectra of the native α-lactalbumin (solid black line 1) and its S-carboxymethylated form (solid black line 2) in the absence of crowding agents, as well as in the presence of PEG-3500 (100 mg/ml, solid gray line) and Ficoll-70000 (150 mg/ml, dashed gray line). (C) Far-UV CD spectra of the native (solid black line 1) and its S-carboxymethyl-α-lactalbumin (solid black line 2) in the absence of crowding agents, as well as in the presence of PEG-3500 (100 mg/ml, solid gray line) and Ficoll-70000 (150 mg/ml, dashed gray line).

Figure 3

Fibrillation of human insulin in 20% acetic acid, pH 2.0 (A, A’) or in 25 mM HEPES buffer, pH 7.5 (B. B’) detected by changes in the ThT fluorescence intensity (plots A and B) and by the increase in the b-structure-related peack in the FTIR amide I spectrum (plots A’ and B’). Protein (2 mg/ml) was incubated at 37°C with stirring alone (circles) or in the presence of crowding agents: PEG-3500 (100 mg/ml, reversed triangles), and Ficoll-70000 (150 mg/ml, squares). At appropriate time points, small samples were taken for ThT or FTIR measurements.

Figure 4

Effect of macromolecular crowding agent Ficoll-70000 (150 mg/ml, dashed lines) on near-UV CD (A) and far-UV CD spectra of human insulin (B) in different conformational states: native hexameric protein in 25 mM HEPES buffer, pH 7.5 (black lines) and in 20% acetic acid, pH 2.0 (gray lines). Solid lines in both plots correspond to spectra measured in the absence of the crowder.

Figure 5

EM of insulin, α-synuclein, and histone aggregates. A) Fibrils of α-synuclein grown in the presence of Ficoll-70000 and 1 M TMAO (5 hours of incubation); B) oligomers of α-synuclein formed in the presence of Ficoll-70000 and 2.5 M TMAO (5 hours of incubation); C) fibrils of histones grown at pH 2.0 and PEG 10,000 (48 hours of incubation); D) oligomers and amorphous aggregates of histones formed at pH 7.5 and PEG 10,000 (48 hours of incubation); E) insulin fibrils grown at pH 2 and PEG 10,000 (20 hours of incubation); F) oligomers and amorphous aggregates of insulin formed at pH 7.5 and PEG 10,000 (20 hours of incubation). Scale bar is 100 nm.

Figure 6

Fibrillation of bovine core histones at pH 7.0, 200 mM NaCl (reversed triangles) or pH 2.0, 200 mM NaCl (circles) in the absence (black symbols) or presence of 100 mg/ml PEG-3500 (open symbols). Protein at concentration of 0.5 mg/ml was incubated at 37°C with agitation.

Figure 7

Fibrillation of human recombinant α-synuclein in the absence (black symbols) or presence of 100 mg/ml PEG-3500 (open symbols). Protein was incubated alone (circles) or in the presence of 1 (reversed triangles), 2 (squares) or 2.5 M TMAO (diamonds). Panel A represents a set of normalized data, whereas Panel B shows the same data in the non-normalized form. In Panel B numbers correspond to: 1, control, no additives; 2, α-synuclein + PEG; 3, 1 M TMAO; 4, 1 M TMAO + PEG; 5, 2 M TMAO; 6, 2 M TMAO + PEG; 7, 2.5 M TMAO; 8, 2.5 M TMAO + PEG. Protein at concentration of 0.5 mg/ml was incubated in 20 mM Tris-HCl, 100 mM NaCl buffer pH 7.5 at 37°C with agitation and fibrillation was monitored by the enhancement of thioflavin T fluorescence intensity.

Figure 8

Fibrillation of human recombinant α-synuclein in the absence (black circles) and the presence of 150 mg/ml Ficoll-70000 (open reversed triangles). Fibrillation was assessed by the increase in the intensity of the FTIR peak at 1630 cm^-1 (plots A, B, and C) or via the characteristic increase in the ThT fluorescence (plots A’, B’, and C’). Protein (0.5 mg/ml) was incubated at 37°C with stirring alone (circles) or in the presence of Ficoll-70000 (150 mg/ml, squares). Panels A and A’ represent data at 0 M TMAO, Panels B and B’ were obtained in the presence of 1 M TMAO, whereas Panels C and C’ show data for 2 M TMAO. At appropriate time points, small samples were taken for ThT or FTIR measurements.

Figure 9

Effect of TMAO and Ficoll-70000 on structural properties of human α-synuclein. (A) Far-UV CD spectra measured for 0.5/ml α-synuclein in the absence (dark lines) and presence (gray lines) of 150 mg/ml Ficoll-70000. Solid lines correspond to the protein in the absence of TMAO, whereas dotted, short dashed and dash-dotted lines represent data obtained in the presence of 1.0 M, 2.0 M and 2.5 M TMAO, respectively. Black long dashed line corresponds to a-synuclein in the presence of 4.5 M TMAO. Spectrum of the amyloidogenic partially folded conformation induced in α-synucelin at acidic pH is shown as a line with open circles. (B) TMAO-induced changes in the [θ]₂₂₀ measured in the absence (black circles and black line) or in the presence of 150 mg/ml Ficoll-70000 (gray circles and gray line).

Figure 10

Effects of different alcohols on the fibrillation of α-synuclein in the absence (black symbols) or presence of 100 mg/ml PEG-3500 (open symbols). Fibrillation was monitored by Thioflavin T. α-Synuclein (0.5 mg/ml) was incubated at 37°C with agitation in the various aqueous-organic solvents. (A) EtOH; (B) TFE; (C) HFiP. EtOH concentrations were 0% ○, 10% (▽), and 20% (□). TFE concentrations were 0% ○, 5% (▽), and 10% (□). HFiP concentrations were 0% ○, 1% (▽), 2.5% (□) and 5% (◇).