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H. S. Mchaourab, H. A. Koteiche, M. Kumar, G. Yang; Thermodynamic Analysis Of -crystallin Binding to Destabilized Mutants of ßB1-crystallin. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1525.
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The formation of complexes between α-crystallin and various ß-crystallins has been proposed to be a key event in the aging of the lens. The interaction is most likely related to the chaperone activity of α-crystallin and is triggered by changes in the thermodynamic stability of the ß-crystallins. Therefore, we have investigated the structural and energetic determinants of complex formation between α-crystallin and destabilized mutants of ßB1-crystallin.
We constructed three site-directed mutants of ßB1-crystallin targeting the packing interface between the N-and C-terminal domains within a monomer. The mutants were introduced in the wild type (WT) background or a K117C/C79V background. The latter allows the attachment of a bimane probe at site 117 to monitor the dimer interface. The stabilities of the mutants were assessed via denaturant unfolding coupled to tryptophan and/or bimane fluorescence. Binding to α-crystallin is detected through changes in the emission properties of the bimane introduced at either site C79 or K117C.
The trends of stability changes correlate with those previously observed in ßB2-crystallin in response to the equivalent substitutions. Clear evidence of an intermediate are observed in the bimane unfolding curves. Introduction of the mutations in the K117C background where the lys117/trp174 interaction at the dimer inerface is disrupted, results in concentration-dependent stability curves. The concentration dependence arises from the transition of the intermediate to the unfolded state. For mutants in the WT background, bindingt o α-crystallin correlates with the stability ranking and the concentration dependence is determined by the dissociation constants of binding. In contrast, the mutants in the K117C background display anomalous concentration dependence that reflects the stabilization of the intermediate.
The results show that α-crystallin binding occurs to the unfolded monomeric ßB1-crystallin. Thus, the stabilization of the ßB1-crystallin folding intermediate reduces the favorable free energy of association with α-crystallin. This may reflect a design element to delay this terminal interaction in the lens.
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