Detection and characterization of aggregates, prefibrillar amyloidogenic oligomers, and protofibrils using fluorescence spectroscopy

Abstract

Transthyretin (TTR) is a protein linked to a number of different amyloid diseases including senile systemic amyloidosis and familial amyloidotic polyneuropathy. The transient nature of oligomeric intermediates of misfolded TTR that later mature into fibrillar aggregates makes them hard to study, and methods to study these species are sparse. In this work we explore a novel pathway for generation of prefibrillar aggregates of TTR, which provides important insight into TTR misfolding. Prefibrillar amyloidogenic oligomers and protofibrils of misfolded TTR were generated in vitro through induction of the molten globule type A-state from acid unfolded TTR through the addition of NaCl. The aggregation process produced fairly monodisperse oligomers (300-500 kD) within 2 h that matured after 20 h into larger spherical clusters (30-50 nm in diameter) and protofibrils as shown by transmission electron microscopy. Further maturation of the aggregates showed shrinkage of the spheres as the fibrils grew in length, suggesting a conformational change of the spheres into more rigid structures. The structural and physicochemical characteristics of the aggregates were investigated using fluorescence, circular dichroism, chemical cross-linking, and transmission electron microscopy. The fluorescent dyes 1-anilinonaphthalene-8-sulfonate (ANS), 4-4-bis-1-phenylamino-8-naphthalene sulfonate (Bis-ANS), 4-(dicyanovinyl)-julolidine (DCVJ), and thioflavin T (ThT) were employed in both static and kinetic assays to characterize these oligomeric and protofibrillar states using both steady-state and time-resolved fluorescence techniques. DCVJ, a molecular rotor, was employed for the first time for studies of an amyloidogenic process and is shown useful for detection of the early steps of the oligomerization process. DCVJ bound to the early prefibrillar oligomers (300-500 kD) with an apparent dissociation constant of 1.6 muM, which was slightly better than for ThT (6.8 muM). Time-resolved fluorescence anisotropy decay of ANS was shown to be a useful tool for giving further structural and kinetic information of the oligomeric aggregates. ThT dramatically increases its fluorescence quantum yield when bound to amyloid fibrils; however, the mechanism behind this property is unknown. Data from this work suggest that unbound ThT is also intrinsically quenched and functions similarly to a molecular rotor, which in combination with its environmental dependence provides a blue shift to the characteristic 482 nm wavelength when bound to amyloid fibrils.

FIGURE 1

Far-UV CD spectra of native, unfolded, and aggregated misfolded A-state TTR into soluble aggregates over time. All spectra are recorded from 14 μM (monomer concentration) TTR samples. Color code: Native tetrameric TTR 10 mM phosphate buffer (pH 7.5; blue). Acid unfolded monomeric TTR, 10 mM HCl (pH 2.0; red). Oligomeric aggregated and protofibrillar TTR generated from acid unfolded TTR incubated for various times in A-state conditions 10 mM HCl, 100 mM NaCl (pH 2.0) after 30 min (black), 70 min (green), and 48 h (magenta).

FIGURE 2

Formation of soluble oligomeric aggregates over time from incubation of acid unfolded TTR under A-state conditions. Chemical cross-linking using glutaraldehyde of soluble aggregates (14 μM monomer concentration) was analyzed by SDS-PAGE, and the gel is shown in the upper part of the figure. The arrow indicates the ∼300–500 kD oligomeric band that increases in abundance over time. A relative quantitative densiometric analysis is shown in the lower part of the figure, where the most dense oligomer band was set to 100%.

FIGURE 3

Transmission electron microscopy micrographs of TTR aggregates. The aggregates were generated after incubation of acid unfolded TTR under A-state conditions for 2 h, 24 h, and 430 h (14 μM monomer concentration). The scale bar indicates 100 nm. All micrographs are taken at 100,000 × magnification.

FIGURE 4

Molecules and processes in the study. (A) Schematic representation of the misfolding and aggregation reaction in the study. From left to right, acid unfolded TTR monomers from which the A-state was induced by addition of NaCl. The A-state forms oligomers which mature into large spherical clusters and protofibrils. (B) Structure of the different fluorescence probes used in the study. ANS and Bis-ANS binds to both native and misfolded TTR, ThT and DCVJ binds specifically to misfolded and aggregated TTR. (C) The native TTR tetramer structure with small molecule binding site indicated in yellow at the dimer-dimer interface. The Protein Data Bank file 1THA was used to generate the structure showing the bound small molecule metabolite 3, 3, diiodo-l-thyronine (Wojtczak et al., 1992).

FIGURE 5

Fluorescence spectra of the different probes binding to native and misfolded aggregated TTR. Aggregates were generated from A-state incubation at 14 μM monomer concentration. (A) Native TTR binding, direct excitation (370 nm) of ANS (blue) and Bis-ANS (black; 2 μM probe + 2 μM TTR tetramer). The red curve shows TTR in the absence of probe. (B) ANS and Bis-ANS fluorescence, after direct excitation (370 nm), bound to protofibrillar and spherical aggregates of TTR (2 μM probe + 2 μM TTR monomer concentration; A-state incubation for 24 h). Same color code as in A. (C) Emission spectra after Trp excitation at 290 nm of native TTR. Same color code as in A. (D) Emission spectra after Trp excitation at 290 nm of protofibrillar and spherical aggregates of TTR. Same color code as in A. (E) DCVJ spectra in the presence of native TTR (red) and misfolded oligomers of TTR (A-state incubation for 2 h; blue; 5 μM DCVJ + 5 μM TTR monomer concentration). (F) ThT spectra in the presence of native TTR (red) and misfolded oligomers of TTR (A-state incubation for 2 h; blue; 5 μM ThT + 5 μM TTR monomer concentration).

FIGURE 6

Kinetics of TTR misfolding and aggregation followed by different fluorescent probes. Aliquots of the aggregation reaction (A-state incubation at 14 μM monomer concentration) was withdrawn and assayed at 2 μM probe + 2 μM TTR. The corrected and integrated fluorescence spectra from the different probes were normalized by the maximum intensity for each probe. Symbols: ANS (red circles), Bis-ANS (green triangles), DCVJ (blue triangles), and ThT (black squares). The fluorescence intensity of the different probes in the presence of the unfolded monomer state and the burst amplitude from the fit is indicated with horizontal lines labeled with the letter U and “burst” in colors corresponding to the probe. The kinetic trace over the initial 6 h is shown in the main figure, the complete kinetic trace (>300 h) is shown in the inset, and the rate parameters are summarized in Table 1.

FIGURE 7

Binding curve of different probes to TTR A-state oligomers incubated at 14 μM (monomer concentration) for 2 h. Probe binding to 1 μM TTR A-state monomers that has formed misfolded oligomers corresponding to ∼30 nM oligomers. Symbols: ANS (red circles), DCVJ (blue triangles), and ThT (black squares).

FIGURE 8

Time-resolved fluorescence of the different probes. ANS and Bis-ANS were bound to native TTR, excitation at 403 nm, and emission at 490 nm. Native tetrameric TTR (1 μM) in 50 mM Tris-HCl (pH 7.5) binding 2 μM ANS (red circles) or 2 μM Bis-ANS (green triangles). DCVJ and ThT were bound to misfolded aggregated TTR after 1 h incubation under A-state conditions. 2 μM DCVJ (blue triangles) or 2 μM ThT (black squares) were bound to 2 μM TTR (monomer concentration), excitation at 443 nm, emission at 500 nm. The ThT curve is hidden behind the DCVJ curve in the figure due to very similar decay profiles. The lamp response is indicated with open black circles. Fluorescence lifetimes are listed in Table 3.

FIGURE 9

Anisotropy decay of ANS bound to TTR in different states. Excitation at 403 nm, emission detected at 475 nm. (A) Native tetrameric TTR (6 μM) in 50 mM Tris-HCl (pH 7.5) with added 2 μM ANS (blue). Unfolded monomeric TTR (2 μM) in 10 mM HCl no salt (pH 2.0) with added 2 μM ANS (black). Aggregated TTR generated from acid unfolded monomeric TTR during A-state incubation for 24 h (8 μM monomer) in 10 mM HCl, 100 mM NaCl (pH 2.0) with added 2 μM ANS (red). (B) ANS anisotropy of aggregates of A-state TTR incubated for 24 h at different monomer concentrations: 2 μM (black), 4 μM (green), and 8 μM (red). As a comparison, the trace for native tetrameric TTR is shown in blue. (C) Kinetics of TTR aggregation. Average times for anisotropy decay traces: 1 min (blue), 11 min (black), 33 min (green), and 125 min (red). Aggregation was induced through addition of a final concentration of 50 mM Na-acetate (final pH 2.5) and 100 mM NaCl to acid unfolded monomeric TTR (pH 2.0), inducing A-state formation that aggregate at a final TTR monomer concentration of 8 μM. The solid lines are fits to single exponential decays and are included merely to guide the eye.

FIGURE 10

DCVJ and ThT fluorescence in water-glycerol mixtures. (A) DCVJ fluorescence. (B) ThT fluorescence. The concentration of glycerol (v/v) was varied between 0% (lower spectrum) and 82.5% (upper spectrum). Increased viscosity will decrease tumbling and self-quenching. (C) Fluorescence intensity of DCVJ (○) and ThT (•) as a function of viscosity.

FIGURE 11

pH sensitivity on fluorescence of DCVJ and ThT. TTR aggregates were generated from acid unfolded TTR, which was incubated at A-state conditions for 24 h (14 μM monomer concentration). Spectra were recorded from mixing 5 μM TTR (monomer concentration) and 5 μM dye in different buffers. (A) TTR aggregates + DCVJ in 50 mM Tris-HCl (pH 7.5; solid line) and in 40 mM Na-acetate (pH 3.0; dashed line) (B) TTR aggregates + ThT in 50 mM Tris-HCl (pH 7.5; solid line) and in 40 mM Na-acetate (pH 3.0; dashed line). (C) DCVJ (5 μM) without protein in 80% glycerol buffered with 50 mM Tris-HCl (pH 7.5; solid line); 40 mM Na-acetate (pH 3.0; dashed line); 25 mM HCl (pH 1.6; dotted line). (D) ThT (5 μM) without protein, same buffers as in C.