Nucleation of protein fibrillation by nanoparticles

1. 

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   Fig. 1.

   Size comparison of monomeric β₂m (see Upper Left for enlargement) with nanoparticles. One protein molecule is placed on each particle in scale for size comparison with nanoparticles of 70 nm (Lower Left) and 200 nm (Right). This figure was prepared with Molmol (58).

   
   
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   Fig. 2.

   β₂m fibrillation in the presence of nanoparticles. (A) Kinetics of β₂m fibrillation. Thioflavin T fluorescence as a function of time for 80 μM (1 mg/ml) β₂m at 37°C in 10 mM sodium phosphate buffer, pH 2.5, with 0.02% NaN₃, without (black) or with 0.01 mg/ml nanoparticles with 85:15 (blue) or 50:50 (red) NIPAM/BAM ratio is shown. Smaller symbols are used for 70-nm particles. (B) Negative stain electron microscopy image of fibers grown in the presence of 70-nm 85:15 NIPAM/BAM copolymer nanoparticles. (Scale bar: 100 nm.)

   
   
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   Fig. 3.

   Kinetics of β₂m fibrillation. (A and E) Histograms of observed lag times. (B, D, and F) Thioflavin T fluorescence as a function of time showing kinetic traces for all samples in each group. (C) The average of all samples of each kind shown in D. (A and B) Forty micromolar β₂m at 37°C in 20 mM sodium phosphate buffer, pH 2.5, with 50 mM NaCl and 0.02% NaN₃. (C and D) Forty micromolar β₂m at 37°C in 20 mM sodium phosphate buffer, pH 2.5, with 40 mM NaCl and 0.02% NaN₃. (E and F) Forty micromolar β2m at 37°C in 20 mM sodium phosphate buffer, pH 2.5, with 50 mM NaCl and 0.02% NaN₃. Color coding of kinetic traces and histograms: blue (0.01 mg/ml 70-nm 85:15 NIPAM/BAM particles), cyan (0.01 mg/ml 200-nm 85:15 NIPAM/BAM particles), red (0.01 mg/ml 70-nm 50:50 NIPAM/BAM particles), pink (0.01 mg/ml 200-nm 50:50 NIPAM/BAM particles), orange (100 nM 16-nm quantum dots), green (≤0.01 mg/ml 6-nm-diameter multiwalled carbon nanotubes), yellow (≤0.01 mg/ml 16-nm cerium oxide particles), and black (samples without particles).

   
   
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   Fig. 4.

   SPR data for β₂m associating to and dissociating from 200-nm 15:85 NIPAM/ BAM nanoparticles (blue) or 200-nm 50:50 NIPAM/BAM nanoparticles (red) linked to gold via a thiol group. The protein was injected between 0 and 30 min at constant concentration of 40 μM and followed by a constant buffer flow. The black curves were fitted to the association and dissociation data by using Eqs. 1 and 2 in SI Text. (Inset) An expansion of the dissociation data (blue) with fitted curve (black) for the 200-nm 15:85 NIPAM/BAM nanoparticles.

   
   
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   Fig. 5.

   Change in β₂m conformation in the presence of nanoparticles. (A) Trp fluorescence spectra of β₂m titrated into buffer (dashed lines) and into a solution with 70-nm 50:50 NIPAM/BAM nanoparticles (solid lines). (B) Fluorescence intensity at 335 nm versus β₂m concentration. β₂m titrated into buffer (black triangles) fitted by a straight line, and β₂m titrated into 70 nm 50:50 nanoparticles (red filled circles) fitted by a 1:1 binding curve is shown.

   
   
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   Fig. 6.

   Isothermal titration calorimetry data at 5°C from titration of β₂m into a solution containing 70-nm 50:50 NIPAM/BAM nanoparticles. The protein concentration was 160 μM, and the particle concentration was 1 mg/ml. Each injection was 15 μl with a total of 19 injections. (Upper) Raw data. (Lower) integrated data. The black line shows the fitted curve assuming a simple 1:1 binding model with one kind of sites (Eq. 3 in SI Text), with the parameter values ΔH = 45 kJ/mol and K_A = 4 × 10⁵ M⁻¹, n = 1,040.