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Several mutations associated with congenital cataracts in human beings target conserved arginine residues in αA-crystallin. The N-terminal region of αA-crystallin is a “mutational hotspot,” with multiple cataract-related mutations reported in this region. Two mutations at arginine 21 in the N-terminal domain of αA-crystallin — αA–R21L and αA–R21W have been associated with congenital cataract. A third mutant of R21, αA–R21Q, was recently identified to be associated with congenital cataract in a South Australian family. The point mutation was reported to compromise the quaternary structure of αA-crystallin by preventing its assembly into higher ordered oligomers. To assess the effect of the αA–R21Q mutation on αA-crystallin function, recombinant αA–R21Q was expressed, purified and characterized in vitro. Compared to wild-type αA-crystallin, the recombinant αA–R21Q exhibits enhanced chaperone-like activity, increased surface hydrophobicity, lesser stability in urea and increased susceptibility to digestion by trypsin. αA–R21Q demonstrated increased binding affinity towards unfolding ADH and bovine lens fiber cell membranes. αA–R21Q homo-oligomers and hetero-oligomers also prevented H2O2–induced apoptosis in ARPE–19cells. Taken together, αA–R21Q exhibited a gain of function despite subtle structural differences as compared to wild-type αA-crystallin. This study further validates the involvement of arginine 21 in regulating αA-crystallin structure and function.Mutant αA-R21Q exhibits an increase in its surface hydrophobicity.αA-R21Q displays better chaperone-like activity and increased lens membrane binding.Transduced αA-R21Q prevents apoptosis of ARPE-19cells under oxidative stress.Gain of function in αA–R21Q mutant may affect its interaction with other lens proteins resulting in loss of transparency.