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Mechanism of human γD-crystallin protein aggregation in UV-C light.

ABSTRACT:

Purpose

To characterize intermediate aggregate species on the aggregation pathway of γD-crystallin protein in ultraviolet (UV)-C light.

Methods

The kinetics of γD-crystallin protein aggregation was studied with reversed-phase high-performance liquid chromatography (RP-HPLC) sedimentation assay, ThT binding assay, and light scattering. We used analytical ultracentrifugation to recognize intermediate aggregate species and characterized them with Fourier transform infrared spectroscopy (FTIR). Quantification of free sulfhydryl groups in an ongoing aggregation reaction was achieved by using Ellman's assay.

Results

Negligible lag phase was found in the aggregation kinetic experiments of the γD-crystallin protein. Dimer, tetramer, octamer, and higher oligomer intermediates were formed on the aggregation pathway. The protein changes its conformation to form intermediate aggregate species. FTIR and trypsin digestion indicated structural differences between the protein monomer, intermediate aggregate species, and fibrils. Ellman's assay revealed that disulfide bonds were formed in the protein monomers and aggregates during the aggregation process.

Conclusions

This study showed that various intermediate and structurally different aggregate species are formed on the aggregation pathway of γD-crystallin protein in UV-C light.

SUBMITTER: Bawankar M

PROVIDER: S-EPMC8254662 | biostudies-literature |

REPOSITORIES: biostudies-literature

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