May 2006
Volume 47, Issue 13
ARVO Annual Meeting Abstract  |   May 2006
Effect of Crosslinking at the Subunit Interface on the Structure–Function of Human B–Crystallin
Author Affiliations & Notes
  • K. Sharma
    University of Missouri, Columbia, MO
    Ophthalmology and Biochemistry,
  • P. Santhoshkumar
    University of Missouri, Columbia, MO
  • J. Wang
    University of Missouri, Columbia, MO
  • Footnotes
    Commercial Relationships  K. Sharma, None; P. Santhoshkumar, None; J. Wang, None.
  • Footnotes
    Support  NIH Grants EY11981, EY14795 and Research to Prevent Blindness
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 1518. doi:
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      K. Sharma, P. Santhoshkumar, J. Wang; Effect of Crosslinking at the Subunit Interface on the Structure–Function of Human B–Crystallin . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1518.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Previous studies have shown that several sequences in αB–crystallin participate during subunit interaction, oligomerization and chaperone–like activity of the protein. The purpose of this study was to confirm the role of specific regions in αB–crystallin in subunit interactions and to determine the necessity of dynamic state of the protein in its chaperone–like function.

Methods: : Site–directed mutagenesis was used to replace S19, S59, and K90 in αB–crystallin with Cysteine to generate single Cys mutants. The mutant proteins were expressed in E. coliBL21(DE3)pLysS cells, purified and analyzed by SDS–PAGE, and mass spectrometry. The mutants were characterized by Dynamic Light Scattering (DLS) method and their chaperone–like activities were estimated in oxidized and reduced form. The subunit exchange rate of reduced and oxidized αB–S59C was measured by FRET assay of labeled αB–WT and αB–S59C with Alexa 488 and Alexa 350 respectively.

Results: : αB–S19C and αB–S59C spontaneously formed dimers due to oxidation of Cys residues whereas αB–K90C required oxidation with 1,10–phenanthroline to form dimers. The average molar mass of αB–K90C in reduced form was comparable to that of αB–WT (4.83e5 and 4.63e5) whereas the molar masses of αB–S19C and αB–S59C in reduced form were 6.82e5 and 7.55e5 respectively. Further, a higher molar mass and decreased polydispersity were observed for oxidized forms of αB–S19C and αB–S59C. The chaperone–like activity of αB–S19C, αB–S59C and αB–K90C were comparable to that of the wild type whereas the oxidized αB–S19C and αB–S59C showed about 50 percent decrease in chaperone–like activity when assayed with alcoholdehydrogenase. The FRET studies with oxidized αB–S59C revealed a decreased subunit exchange rate (1.5x10–4/sec) with wild type αB– compared to the reduced αB–S59C (2.6x10–4/sec).

Conclusions: : The S19 and S59 are located at the subunit interface in αB–crystallin. The dynamic state of αB–crystallin determines its chaperone–like activity. Crosslinking events in part may be responsible for the diminished chaperone–like activity of α–crystallin in aged and cataract lenses.

Keywords: chaperones • protein structure/function • oxidation/oxidative or free radical damage 

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