RESEARCH ARTICLE Interaction of βA3-Crystallin with Deamidated Mutants of αA- and αB-

Interaction among crystallins is required for the maintenance of lens transparency. Deamida-tion is one of the most common post-translational modifications in crystallins, which results in incorrect interaction and leads to aggregate formation. Various studies have established interaction among the α- and β-crystallins. Here, we investigated the effects of the deamida-tion of αA- and αB-crystallins on their interaction with βA3-crystallin using surface plasmon resonance (SPR) and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) methods. SPR analysis confirmed adherence of WT αA- and WT αB-crystallins and their deamidatedmutants with βA3-crystallin. The deamidated mutants of αA–crystallin (αA N101D and αA N123D) displayed lower adherence propensity for βA3-crystallin relative to the binding affinity shown byWT αA-crystallin. Among αB-crystal-lin mutants, αB N78D displayed higher adherence propensity whereas αB N146Dmutant showed slightly lower binding affinity for βA3-crystallin relative to that shown byWT αB-crys-tallin. Under the in vivo condition (FLIM-FRET), both αA-deamidated mutants (αA N101D and αA N123D) exhibited strong interaction with βA3-crystallin (32±4 % and 36±4%FRET effi