Abstract

alpha-Crystallin is known to exhibit chaperone-like activity. We have studied its chaperone-like activity toward the aggregation of betaL-crystallin upon refolding of this protein from its unfolded state in guanidinium chloride. The chaperone-like activity of alpha-crystallin is less pronounced below 30 degrees C and is enhanced above this temperature. The plot of percentage protection as a function of temperature shows two transitions; one at 30 degrees C and another at around 55 degrees C. We have performed steady state fluorescence, fluorescence polarization, fluorescence quenching, circular dichroism, sedimentation analysis, and gel filtration chromatography to probe the temperature-induced structural changes of alpha-crystallin. Our results show that at above 50 degrees C, alpha-crystallin undergoes a transition to a multimeric molten globule-like state. Above 30 degrees C, a minor but detectable perturbation in its tertiary structure occurs that might lead to the observed exposure of its hydrophobic surfaces. These results support our earlier hypothesis that alpha-crystallin prevents the aggregation of other proteins by providing appropriately placed hydrophobic surfaces; a structural transition above 30 degrees C involving enhanced or reorganized hydrophobic surfaces of alpha-crystallin is important for its chaperone-like activity. It is possible that a structural alteration induced by temperature forms a part of the general mechanism of chaperone function, because they are required to function more effectively at nonpermissible temperatures.

Highlights

  • ␣-Crystallin is shown to prevent the heat-induced aggregation of other crystallins and enzymes like a molecular chaperone [14]

  • We concluded that ␣-crystallin prevents the aggregation of other proteins by providing properly placed hydrophobic surfaces; a structural transition above 30 °C might be important for its chaperone-like property [17]

  • We have investigated the aggregation of ␤L-crystallin upon refolding from its GdmCl-unfolded state and the effect of ␣-crystallin on this aggregation

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Summary

Introduction

␣-Crystallin is shown to prevent the heat-induced aggregation of other crystallins and enzymes like a molecular chaperone [14]. We concluded that ␣-crystallin prevents the aggregation of other proteins by providing properly placed hydrophobic surfaces; a structural transition above 30 °C might be important for its chaperone-like property [17]. We have used refolding-induced aggregation of ␤L-crystallin and investigated the structural aspects of ␣-crystallin in the context of its chaperone-like activity.

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