Human γS-Crystallin Resists Unfolding Despite Extensive Chemical Modification from Exposure to Ionizing Radiation
- Brenna Norton-BakerBrenna Norton-BakerDepartment of Chemistry, University of California, Irvine, California 92697-2025, United StatesMore by Brenna Norton-Baker,
- Megan A. RochaMegan A. RochaDepartment of Chemistry, University of California, Irvine, California 92697-2025, United StatesMore by Megan A. Rocha,
- Jessica Granger-JonesJessica Granger-JonesDepartment of Chemistry, University of California, Irvine, California 92697-2025, United StatesMore by Jessica Granger-Jones,
- Dmitry A. Fishman , and
- Rachel W. Martin*Rachel W. Martin*Email: [email protected]Department of Chemistry, University of California, Irvine, California 92697-2025, United StatesDepartment of Molecular Biology and Biochemistry, University of California, Irvine, California 92697-3900, United StatesMore by Rachel W. Martin
Ionizing radiation has dramatic effects on living organisms, causing damage to proteins, DNA, and other cellular components. γ radiation produces reactive oxygen species (ROS) that damage biological macromolecules. Protein modification due to interactions with hydroxyl radical is one of the most common deleterious effects of radiation. The human eye lens is particularly vulnerable to the effects of ionizing radiation, as it is metabolically inactive and its proteins are not recycled after early development. Therefore, radiation damage accumulates and eventually can lead to cataract formation. Here we explore the impact of γ radiation on a long-lived structural protein. We exposed the human eye lens protein γS-crystallin (HγS) to high doses of γ radiation and investigated the chemical and structural effects. HγS accumulated many post-translational modifications (PTMs), appearing to gain significant oxidative damage. Biochemical assays suggested that cysteines were affected, with the concentration of free thiol reduced with increasing γ radiation exposure. SDS-PAGE analysis showed that irradiated samples form protein–protein cross-links, including nondisulfide covalent bonds. Tandem mass spectrometry on proteolytic digests of irradiated samples revealed that lysine, methionine, tryptophan, leucine, and cysteine were oxidized. Despite these chemical modifications, HγS remained folded past 10.8 kGy of γ irradiation as evidenced by circular dichroism and intrinsic tryptophan fluorescence spectroscopy.
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