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Cade B. Fox, Joshua P. Smith, Larry L. David, Kirsten J. Lampi; Sites of Interaction between βB2-crystallin or Deamidated βB2-crystallin with αA-crystallin. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1613.
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© ARVO (1962-2015); The Authors (2016-present)
To identify regions of interaction between deamidated or wild-type (WT) betaB2-crystallin and alphaA-crystallin in complex.
Deamidation was mimicked by replacing two glutamines located at the dimer interface of the betaB2 homodimer with glutamic acids. AlphaA and betaB2 were incubated at 50-55° C to form large, multimeric complexes. Complexes were isolated by gel filtration. Changes in solvent accessibility in regions of alphaA and betaB2 resulting from complex formation were identified using hydrogen/deuterium exchange with electrospray ionization source mass spectrometry (HDMS). These changes were then used to model the interaction of WT betaB2 or deamidated betaB2 with alphaA.
AlphaA and betaB2WT formed a polydisperse complex with the greatest number of species having a molar mass of about 2.1 MDa. Mimicking deamidation at both interface glutamines decreased the average molar mass of the complex to about 1.6 MDa. Based on the molecular weights of alphaA and betaB2 and the observed alphaA:betaB2 ratio of about 2:1, the alphaA/betaB2 WT complex and alphaA/betaB2 DM complex were found to contain about 100 and 70 total subunits, respectively. Increasing alphaA concentration in incubations with betaB2 resulted in a large fraction of betaB2 in complex and very little betaB2 not bound to alphaA. Furthermore, no significant amount of betaB2 in a monomeric state was detected. After forming a complex with alphaA, betaB2 WT showed a slight decrease in solvent exposure in the N-terminal domain and a significant increase in solvent exposure in the C-terminal domain.
Upon forming complex with alphaA, betaB2 undergoes changes in solvent accessibility suggesting that the binding induces structural changes. We hypothesize that the increased solvent accessibility of the N-terminal domain of betaB2 may be due to an interaction of this domain with alphaA, and the increased solvent accessibility of the C-terminal domain may be due to the disruption of the dimer interface, exposing the C-terminal domain. Because betaB2 monomers were not detected in our system, it is also possible that interaction with alphaA did not fully disrupt the betaB2 dimer. Future studies will determine the exact nature of wild-type and deamidated betaB2 when in complex with alphaA.
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