Figure 1.
Dynamic light scattering and Thioflavin T fluorescence.
(A) Dynamic light scattering and Thioflavin T comparison Venn diagram. The items within each circle summarize aspects of fibril formation which each technique is capable of measuring. (B) Schematic of DLS and ThT apparatus.
Figure 2.
TEM micrograph of the products formed after TEV cleavage of the MBP from the polyQ-fusion protein complex. (A) Q45 proto-fibrilar precursors. (B) Mature Q45 fibrils.
Figure 3.
Repeat length dependent polyQ aggregation.
Aggregation of Q15, Q25, Q35, and Q45 measured by ThT fluorescence (A) ([ThT] = 700 μM) and DLS (B). The data are average values of mean radius and ThT fluorescence compiled from three independent experiments. The dotted lines represent standard deviation. Both ThT fluorescence and mean hydrodynamic radius data for all glutamine repeat lengths were normalized to the steady state value of the Q45 growth curve, respectively. DLS growth curves were smoothed with a moving average filter.
Figure 4.
Simultaneous measurement of aggregation by DLS and ThT fluorescence.
Repeat length and concentration dependent aggregation measured simultaneously by DLS and ThT fluorescence for (A) 28 µM Q45 (B) 70 µM Q45 and (C)70 µM Q35. Columns i, ii, and iii display repeat experiments. The DLS data are smoothed with a moving average filter. (D) Final ThT fluorescence normalized to 70uM Q45. (E) Calculation of Tlag and K1/2 from sigmoidal fit. (F) Tlag and (G) K1/2 extracted from sigmoidal fits to DLS and ThT data.
Figure 5.
Size distribution evolution during aggregation.
CONTIN size distributions of aggregating polyQ. (A) 70 uM Q45 (B) 28 uM Q45 (C) 70 uM Q35 (D) 28 uM Q35.
Figure 6.
Model for fibril and bundle formation.
(A) Fibril nucleation and growth. n* is the number of polyQ monomers needed to form the critical nucleus for fibril nucleation. Elongation proceeds by monomer addition. When the length of the fibril reaches a critical length Lc, which occurs when the number of monomers which make up the fibril reaches Nc, the fibril can begin to assemble into fibril bundles. (B) Bundle nucleation and growth. Fibrils of length L≥Lc are the minimal unit for bundle assembly. A critical bundle nucleus is formed when m* bundles associate. Bundle growth ensues as individual bundles of L≥Lc attach to the bundle nucleus.