April 2009
Volume 50, Issue 13
ARVO Annual Meeting Abstract  |   April 2009
The Cu2+ Binding Site of -Crystallin
Author Affiliations & Notes
  • H. S. Mchaourab
    Molec Physiol&Biophys-Med Ctr, Vanderbilt University, Nashville, Tennessee
  • W. E. Antholine
    Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin
  • H. A. Koteiche
    Molec Physiol&Biophys-Med Ctr, Vanderbilt University, Nashville, Tennessee
  • M. Bortolus
    Molec Physiol&Biophys-Med Ctr, Vanderbilt University, Nashville, Tennessee
  • J. McCracken
    Chemistry, Michigan State University, East Lansing, Michigan
  • Footnotes
    Commercial Relationships  H.S. Mchaourab, None; W.E. Antholine, None; H.A. Koteiche, None; M. Bortolus, None; J. McCracken, None.
  • Footnotes
    Support  NEI Grant EY12683
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 2109. doi:
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      H. S. Mchaourab, W. E. Antholine, H. A. Koteiche, M. Bortolus, J. McCracken; The Cu2+ Binding Site of -Crystallin. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2109.

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

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Purpose: : A number of studies have reported that lens proteins bind metal ions including redox active copper (Cu2+). Analysis of human lenses indicates the presence of copper at significant concentrations.Cu2+ catalyzes the production of oxygen reactive species and has been implicated as a catalyst for the autoxidation of SH groups in the lens. Here, we have used electron paramagnetic resonance spectroscopy (EPR) to characterize the Cu2+ binding site of A-crystallin and determine the identity of atoms coordinated to Cu2+.

Methods: : We prepared samples of recombinant rat A-crystallin mixed with varying concentrations of Cu2+ at pH 7.2. Continuous -wave EPR analysis was carried out at multiple microwave frequencies at 77 K. Pulse Electron Spin Echo Envelope Modulation spectroscopy (ESEEM) was carried out at 10 K.

Results: : The EPR spectrum of A-crystallin bound Cu2+ has with g=2.27 and A=172 G and is invariant with the amount of added Cu2+. These EPR parameters are consistent with three nitrogens and one oxygen donor atoms according to the Peisach-Blumberg plots, but no nitrogen superhyperfine lines were resolved at X-band (9.15 GHz). At a lower microwave frequency (S-band, 3.425 GHz), nitrogen superhyperfine lines were resolved in the g1 region. Simulation of the EPR spectrum gave a best fit with three nitrogens and 1 oxygen donor atoms. In comparison, addition of Cu2+ to Hsp16.5 leads to an EPR spectrum characteristic of non specific binding. The ESEEM spectrum for A-bound Cu2+ is typical of one strongly bound, equatorial nitrogen donor atom from the imidazole of a histidine amino acid. No resolved peaks are observed between 2.3 and 3.2 MHz where combination lines would indicate that more than one imidazole is bound to the same Cu2+ ion.

Conclusions: : The binding of Cu2+ appears to be an evolved property of mammalian sHSP. Its likely function is to protect cells from oxidative damage. The age-related truncation of -crystallin in the lens may lead to loss of Cu2+ chelation and thus exposes the lens to oxidative damage.

Keywords: crystallins • chaperones • oxidation/oxidative or free radical damage 

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