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K.J. Lampi, P. Ahmann; The Differing Effects of Deamidations on the Structures of Lens Beta–Crystallns . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3888.
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Purpose: BetaB1– and betaB2–crystallins are major crystallins within the human lens and both are deamidated during aging. We have previously characterized five different in vivo sites of deamidation in betaB1 and betaB2–crystallins and found two of these sites resulted in aggregation and/ or decreased stability. The aim of the present study was to determine the effects of deamidation on the highly conserved, interdomain–binding interface of beta B1 (Q226) and its homologous sequence in beta B2 (Q184E). This interface is between dimers of the betaB2 tetramer, but between monomers of the betaB1 dimer due to the connecting peptide being bent in betaB1. Methods: The present study investigated the stability of the recombinant mutants, betaB1 Q226E and betaB2 Q184E, containing in vivo deamidations at the proposed interfaces, during urea denaturation by tryptophan fluorescence measurement. The association of these recombinantly expressed proteins was further determined by multiangle laser light scattering. Results: It was found that adding a negative charge either in betaB1 or betaB2 close to the hypothetical intrerdomain–binding interface did not alter the protein stability. For example, the amount of urea needed to denature 50% of betaB1 Q226E was near 5 M urea, similar to that needed for wildtype betaB1. Conclusions: Deamidations close to the highly conserved interdomain–binding interfaces did not alter the protein stability compared to the wild type. Deamidations at this interface may not be as detrimental as deamidation at the intradomain interface as found with betaB1 Q204E.
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