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Ekta Tiwary, Shylaja Hegde, Om P Srivastava; Analysis of βA3-Crystallin Interaction with αA- and αB-Crystallins. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5728.
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© ARVO (1962-2015); The Authors (2016-present)
The purpose was to determine the interaction of human βA3-crystallin with αA- and αB-crystallins, and identify interacting regions using Surface Plasmon Resonance (SPR) and Fluorescence Lifetime Imaging-Fluorescence Resonance Energy Transfer (FLIM-FRET) microscopic methods.
The wild type (WT) αA- and αB-crystallins, their deamidated mutants (αAN101D, αAN123D, αBN78D and αBN146D) and WT βA3-crystallin were cloned into pET-28b, expressed in E.coli and purified by Ni+2, affinity chromatography, and used for SPR analysis. Similarly, the above proteins were also cloned in pZsYellow-N1 vector (αA- and αB-clones) and in pA Cyan- N1 vector (βA3-clone), transfected into HeLa cells and analyzed by FLIM-FRET method.
SPR kinetic studies revealed that affinities of WT αA- and WT αB-crystallins to βA3-crystallin were very high with the binding constant (KA) of 4.66e8M and 1.44e8M, respectively. Although both WT αA- and WT αB-crystallins exhibited high affinity (KA) for βA3-crystallin, their dissociation from βA3-crystallin was very fast, with the KD of 2.15e-9 and 6.95e-9 for WT αA and WT αB, respectively. Surprisingly, no significant changes were observed in KA and KD values during interactions of deamidated αA mutants (αAN101D and αAN123D) and αB mutants (αBN78D and αBN146D) with βA3-crystallin. Similar interactions were observed between these crystallins during FLIM-FRET analysis.
WT αA and WT αB-crystallins and their deamidated mutants (αAN101D, αBN123D, αBN78D and αBN146D) showed strong binding affinity to βA3-crystallin. However, under in vitro conditions, their interactions seem to be transient.
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