December 2002
Volume 43, Issue 13
ARVO Annual Meeting Abstract  |   December 2002
Effect of Phosphorylation on the Chaperone Function of Alpha B Crystallin
Author Affiliations & Notes
  • HA Koteiche
    Molecular Physiology & Biophysics Vanderbilt Univ Med Ctr Nashville TN
  • HS Mchaourab
    Molecular Physiology and Biophysics Vanderbilt University Medical Center Nashville TN
  • Footnotes
    Commercial Relationships   H.A. Koteiche, None; H.S. Mchaourab, None. Grant Identification: EY12018, EY12683
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 3567. doi:
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      HA Koteiche, HS Mchaourab; Effect of Phosphorylation on the Chaperone Function of Alpha B Crystallin . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3567.

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

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Abstract: : Purpose: Mammalian small heat-shock proteins (sHSPs) are phosphorylated in vivo in response to cellular signals and stress. In this study, we explore the effect of phosphorylation on the chaperone function of αB-crystallin (αB). For this purpose, we compared the binding of αB and various forms of phosphorylated αB to destabilized T4 Lysozyme (T4L) mutants. The experiments conducted also probed the effects of temperature and pH on the binding affinity and stoichiometry. Methods:The three phosphorylation sites of αB: S19, S45 and S59 were substituted with aspartate using site directed mutagenesis to generate the mutant αB-D3 as well as various single and double aspartate mutants. The binding to T4L was detected with a spin-label at a non-destabilizing site on the surface of the protein. For each binding experiment, samples were prepared with varying ratios of T4L mutant to αB/αB-D3 and incubated under different conditions until the endpoint is reached. The bound fraction was measured using Electron Paramagnetic Resonance (EPR) spectroscopy and the appropriate binding isotherms determined. Results:Three T4L mutants were used: 99A/130S, 99A and 70N having increased destabilization with free energy of unfolding of 7, 8, 10 Kcal/mol respectively. All three mutants bind to αB-D3 with higher affinity than to αB. Furthermore, αB-D3 binds mutants of decreasing stability with increasing affinity. Increasing the pH from 7 to 8 increases the number of binding sites but leaves the affinity unchanged for all mutants. Binding affinity of T4L-99A to αB-D3 at 37C increases by at least 10 fold. Preliminary results indicate that the effect of phosphorylation is not additive, with the mutant S45D producing the higher activation. Conclusion:These results suggest that the phosphorylation of αB-crystallin leads in fact to an activated state with increased binding affinity to destabilized mutants compared to WT-αB both at room temperature and at 37°C. This result contradicts the conclusion of previous aggregation-based chaperone assays and illustrates the use of direct detection of binding as a tool for characterizing the chaperone function of sHSPs.

Keywords: 378 crystallins • 343 chaperones • 338 cataract 

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