Unravelling γD-crystallin aggregation pathway to understand cataract formation using fluorescence correlation spectroscopy

Abstract

Purpose: To characterize the aggregation behavior of the γD-crystallin protein in an acidic environment with a focus on the formation of intermediate species. The research employs fluorescence correlation spectroscopy to unravel the intricate molecular events leading to aggregation, contributing to a comprehensive understanding of cataract formation.

Methods: The kinetics of γD-crystallin protein aggregation were studied with a reversed-phase high-performance liquid chromatography sedimentation assay, a ThT binding assay, and light scattering. We used fluorescence correlation spectroscopy (FCS) to recognize intermediate aggregate species and characterized them with Fourier transform infrared spectroscopy (FTIR). Further, the morphologic characterization of aggregates was done by transmission electron microscopy (TEM), and their hydrophobic characteristics were analyzed using the 8-anilino-1-naphthalenesulfonic acid binding assay.

Results: A negligible lag phase was observed in the aggregation kinetic experiments of the γD-crystallin protein. Pentamer, 25-mer, and higher oligomer intermediates were formed on the aggregation pathway. Conformation studies by FCS and FTIR have shown that oligomers are rich in cross-β sheet and random coil structure; however, they constitute more α-helix and less cross-β sheet structure than fibrils. TEM analysis revealed the approximate size of oligomers (diameter ~10 nm), protofibrils (~15 nm), and fibrils (~15 to ~35 nm).

Conclusions: In this study, we reported the presence of various intermediate aggregate species formed on the aggregation pathway of γD-crystallin protein at low pH. This will open new areas of research in understanding the detailed aggregation mechanism and aggregation hotspot within unfolded γD-crystallin monomers. The insights gained will also pave the way for future research in the realm of amyloid formation in cataract.

Figure 1

Aggregation kinetics of γD-crystallin protein at pH 2.5. The black line represents the RP-HPLC sedimentation assay, the red line represents the Thioflavin-T (ThT) binding assay, and the blue line represents the light scattering of γD-crystallin. In the RP-HPLC sedimentation assay, the percentage of monomers was converted into the percentage of aggregates, while the intensity values of light scattering and the ThT binding assay were normalized, considering the intensity value at 18 h as 100%.

Figure 2

Fluorescence correlation spectroscopy (FCS) analysis of γD-crystallin protein aggregation at pH 2.5. A: The radius of the aggregation intermediates formed during the aggregation pathway at pH 2.5, calculated using diffusion time with equations 4 and 5. B: Best-fit lines (black) of autocorrelation curves (circles) representing aggregate species at different time points. To avoid overcrowding of data, the FCS traces for 6 min, 27 min, and 35 min are not shown.

Figure 3

Transmission electron microscopy (TEM) images of γD-crystallin aggregation at different time points during the aggregation reaction. Oligomers were observed at 1.5 h (indicated by the black arrow), which grew to form protofibrils at 3 hours and fibrils at 5 h and 30 h. The scale bar represents 100 nm.

Figure 4

Biophysical characterization of γD-Crystallin aggregation. A: Aggregation kinetics of γD-crystallin at different temperatures in pH 2.5. B: Tryptophan fluorescence assay of monomers, oligomers, and fibrils. C) ANS binding assay of monomers, oligomers, and fibrils.

Figure 6

Proposed aggregation pathway for γD-crystallin protein at pH 2.5. The monomer PDB (Protein Data Bank) ID is 1HK0, while all other images are for representative purposes only.

Figure 5

Curve fit fourier-transform infrared spectroscopy (FTIR) spectra of different aggregate species. A: Curve-fitted primary spectrum of monomers. B: Curve-fitted primary spectrum of oligomers. C: Curve-fitted primary spectrum of fibrils. The spectra of all three species differ from one another.