alexa Subunit exchange of alphaA-crystallin.


Biochemistry & Analytical Biochemistry

Author(s): Bova MP, Ding LL, Horwitz J, Fung BK

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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.
This article was published in J Biol Chem and referenced in Biochemistry & Analytical Biochemistry

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