Interaction among crystallins is required for the maintenance of lens transparency. Deamidation 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 deamidation 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 ofWT 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 by WT αA-crystallin. Among αB-crystallin mutants, αB N78D displayed higher adherence propensity whereas αB N146D mutant showed slightly lower binding affinity for βA3-crystallin relative to that shown by WT αB-crystallin. Under the in vivo condition (FLIM-FRET), both αA-deamidated mutants (αAN101D and αA N123D) exhibited strong interaction with βA3-crystallin (32±4% and 36±4% FRET efficiencies, respectively) compared to WT αA-crystallin (18±4%). Similarly, the αBN78Dand αB N146D mutants showed strong interaction (36±4% and 22±4% FRET efficiencies, respectively) with βA3-crystallin compared to 18±4% FRET efficiency of WT αB-crystallin. Further, FLIM-FRET analysis of the C-Terminal domain (CTE), N-Terminal domain (NTD), and core domain (CD) of αA-and αB-crystallins with βA3-crystallin suggested that interaction sites most likely reside in the αA CTE and αB NTD regions, respectively, as these domains showed the highest FRET efficiencies. Overall, results suggest that similar to WT αA-andWTαB-crystallins, the deamidatedmutants showed strong interactionfor βA3-crystallin. Variable in vitro and in vivo interactions are most likely due to the mutant's large size oligomers, reduced hydrophobicity, and altered structures. Together, the results suggest that deamidation of α-crystallin may facilitate greater interaction and the formation of large oligomers with other crystallins, and this may contribute to the cataractogenic mechanism.