December 2002
Volume 43, Issue 13
ARVO Annual Meeting Abstract  |   December 2002
Interaction of the Molecular Chaperone -Crystallin, with Partially Unfolded States of ßB1-Crystallin
Author Affiliations & Notes
  • HA Sathish
    Molecular Physiology and Biophysics Vanderbilt University Nashville TN
  • HS Mchaourab
    Molecular Physiology and Biophysics Vanderbilt University Nashville TN
  • Footnotes
    Commercial Relationships   H.A. Sathish, None; H.S. Mchaourab, None. Grant Identification: NIH EY 12018, NIH EY 12683
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 4799. doi:
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      HA Sathish, HS Mchaourab; Interaction of the Molecular Chaperone -Crystallin, with Partially Unfolded States of ßB1-Crystallin . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4799.

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

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Abstract: : Purpose:α-crystallin,a major eye lens protein, is known to act as a chaperone in preventing aggregation of several proteins. Yet the states of the target proteins of α-crystallin in eye lens and the molecular interactions are not clearly understood. The main goal of this study is to identify the conformational states of ß-crystallins recognized by α-crystallin and the nature of interaction between the two proteins. Methods:For this purpose truncated mutants of ßB1-crystallin were prepared by shortening the coding sequence using appropriate 5' and 3' primers. The PCR products were sub cloned in a suitable cloning vector. Both the wild type and truncated mutants of ßB1-crystallin were overexpressed and purified by a two-step process. Expression and purification of α-crystallin were carried out using available standard procedure. Unfolding and aggregation studies were done using fluorescence spectroscopy and size exclusion chromatography(SEC ). Results:Fluorescence spectroscopy and SEC studies indicated that at neutral pH, under low concentrations of denaturants ßB1-crystallin aggregates, while ßB2-crystallin does not. Studies with the N and C-terminal truncated mutants of ßB1-crystallin showed no role of the flexible N and C-terminal arms in the formation of aggregates. This result indicates that overall partial unfolding of the molecule and the exposure of hydrophobic surfaces is responsible for aggregation. The partial unfolding of the protein molecule is further supported by the red shift observed in the λ maxvalue at low concentrations of denaturants. When the pH was lowered this aggregation is completely prevented, indicating that at lower pH charge-charge repulsion prevent the intermediate state from self associating. Both spectroscopic and SEC studies showed that α-crystallin is able to prevent this unspecific aggregation by interacting with the partially unfolded protein molecule. The interaction between ß-crystallins and α-crystallin is further characterized by Electron Paramagnetic Resonance(EPR) spectroscopic studies. Conclusion:The results suggest that GuHCl / Urea induced unfolding of ßB1-crystallin occurs through the formation of an intermediate. α-crystallin is able to interact with this intermediate and prevent its aggregation. Studies on the interaction between ß-crystallins and α-crystallin in the absence of denaturants are in progress.

Keywords: 378 crystallins • 343 chaperones • 338 cataract 

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