Subunit Exchange of αA-Crystallin

Michael P Bova,Lin-Lin Ding,Joseph Horwitz,Bernard K.-K Fung

doi:10.1074/jbc.272.47.29511

Abstract

alpha-Crystallin, the major protein in the mammalian lens, is a molecular chaperone that can bind denaturing proteins and prevent their aggregation. Like other structurally related small heat shock proteins, each alpha-crystallin molecule is composed of an average of 40 subunits that can undergo extensive reorganization. In this study we used fluorescence resonance energy transfer to monitor the rapid exchange of recombinant alpha-crystallin subunits. We labeled alphaA-crystallin with stilbene iodoacetamide (4-acetamido-4'-((iodoacetyl)amino)stilbene-2,2'-disulfonic acid), which serves as an energy donor and with lucifer yellow iodoacetamide, which serves as an energy acceptor. Upon mixing the two populations of labeled alphaA-crystallin, we observed a reversible, time-dependent decrease in stilbene iodoacetamide emission intensity and a concomitant increase in lucifer yellow iodoacetamide fluorescence. This result is indicative of an exchange reaction that brings the fluorescent alphaA-crystallin subunits close to each other. We further showed that the exchange reaction is strongly dependent on temperature, with a rate constant of 0.075 min-1 at 37 degrees C and an activation energy of 60 kcal/mol. The subunit exchange is independent of pH and calcium concentration but decreases at low and high ionic strength, suggesting the involvement of both ionic and hydrophobic interactions. It is also markedly reduced by the binding of large denatured proteins. The degree of inhibition is directly proportional to the molecular mass and the amount of bound polypeptide, suggesting an interaction of several alphaA-crystallin subunits with multiple binding sites of the denaturing protein. Our findings reveal a dynamic organization of alphaA-crystallin subunits, which may be a key factor in preventing protein aggregation during denaturation.

Highlights

In the past, ␣-crystallin was considered to play only a structural role in maintaining the transparency of the lens
In this study we developed a fluorescence resonance energy transfer method to monitor the exchange of recombinant ␣Acrystallin subunits
Bound ␣-lactalbumin (14 kDa) was found to be more effective than insulin but less effective than ovotransferrin in inhibiting subunit exchange. These results suggest the association of larger polypeptides with several ␣A-crystallin subunits, effectively cross-linking them together and preventing them from exchanging

Summary

Introduction

In the past, ␣-crystallin was considered to play only a structural role in maintaining the transparency of the lens. Binding of large denatured proteins to ␣A-crystallin markedly reduced the exchange rate, indicating an association of the polypeptides with several ␣A-crystallin subunits. The fluorescence intensity of the sample was determined immediately and at 15 min after mixing an equal volume of 0.4 mg/ml AIAS-labeled ␣A-crystallin and 0.4 mg/ml LYI-labeled ␣A-crystallin at 37 °C.

Results

Conclusion