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Human γS-Crystallin Resists Unfolding Despite Extensive Chemical Modification from Exposure to Ionizing Radiation

  • Brenna Norton-Baker
    Brenna Norton-Baker
    Department of Chemistry, University of California, Irvine, California 92697-2025, United States
  • Megan A. Rocha
    Megan A. Rocha
    Department of Chemistry, University of California, Irvine, California 92697-2025, United States
  • Jessica Granger-Jones
    Jessica Granger-Jones
    Department of Chemistry, University of California, Irvine, California 92697-2025, United States
  • Dmitry A. Fishman
    Dmitry A. Fishman
    Department of Chemistry, University of California, Irvine, California 92697-2025, United States
  • , and 
  • Rachel W. Martin*
    Rachel W. Martin
    Department of Chemistry, University of California, Irvine, California 92697-2025, United States
    Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697-3900, United States
    *Email: [email protected]
Cite this: J. Phys. Chem. B 2022, 126, 3, 679–690
Publication Date (Web):January 13, 2022
https://doi.org/10.1021/acs.jpcb.1c08157
Copyright © 2022 American Chemical Society

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    Abstract

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    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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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpcb.1c08157.

    • Figure S1, experimental setup for γ irradiation in the 137Cs Irradiator and Fricke dosimetry results; Figure S2, comparison of the extent of protein modification <1 h, 4 h, and 1 week post γ irradiation; Figure S3, tandem mass spectrum and peak list for the pepsin digest peptide GSKTGTKITF, suggesting oxidation at K3; Figure S4, tandem mass spectrum and peak list for the trypsin digest peptide VEGGTWAVYERPNFAGYMYILPQGEYPEYQR, suggesting oxidation at M59; Figure S5, tandem mass spectrum and peak list for the trypsin digest peptide WMGLNDR, suggesting oxidation at M74, Figure S6, tandem mass spectrum and peak list for the trypsin digest peptide GDFSGQMYETTEDCPSIMEQFHMR, suggesting oxidation at M108; Figure S7, tandem mass spectrum and peak list for the trypsin digest peptide GDFSGQMYETTEDCPSIMEQFHMR, suggesting oxidation at M119; Figure S8, tandem mass spectrum and peak list for the trypsin digest peptide GDFSGQMYETTEDCPSIMEQFHMR, suggesting oxidation at M124; Figure S9, tandem mass spectrum and peak list for the trypsin digest peptide VLEGVWIFYELPNYR, suggesting oxidation at W137; Figure S10, tandem mass spectrum and peak list for the trypsin digest peptide KPIDWGAASPAVQSFR, suggesting oxidation at W163; Figure S11, tandem mass spectrum and peak list for the trypsin digest peptide RYDCDCDCADFHTYLSR, suggesting oxidation at C25; Figure S12, tandem mass spectrum and peak list for the trypsin digest peptide RYDCDCDCADFHTYLSR, suggesting oxidation at C23; Figure S13, tandem mass spectrum and peak list for the trypsin digest peptide RYDCDCDCADFHTYLSR, suggesting oxidation at C27; Figure S14, comparison of the extent of protein modification at low and high protein concentrations; Figure S15, evidence of HγS dimerization in deconvoluted intact mass spectra (PDF)

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    Cited By

    This article is cited by 2 publications.

    1. Meng’en Kang, Yuzhu Weng, Yi Liu, Haoke Wang, Ling Ye, Yanlin Gu, Xue Bai. A Review on the Toxicity Mechanisms and Potential Risks of Engineered Nanoparticles to Plants. Reviews of Environmental Contamination and Toxicology 2023, 261 (1) https://doi.org/10.1007/s44169-023-00029-x
    2. Stephen G. R. Barnard, Nobuyuki Hamada. Individual response of the ocular lens to ionizing radiation. International Journal of Radiation Biology 2023, 99 (2) , 138-154. https://doi.org/10.1080/09553002.2022.2074166

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