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G. B. Reddy, S. G. Bhagyalaxmi, P. B. Srinivas, T. Padma, K. A. Barton, J. M. Petrash, K. R. Kumar, M. Vidyavathi; A Novel Mutation (f71l) in aA-Crystallin Associated With Age-Related Cataract Results in Defective Chaperone-Like Function Despite Unaltered Structure. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1637.
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We identified a genetic variation (F71L) in the exon-2 of CRYAA gene in three unrelated sporadic cases among 450 age-related cataracts patients but not in 200 normal non-cataractous controls. Therefore, the purpose of this study was to investigate the impact of F71L mutation on structural and functional properties of A-crystallin.
We have constructed, expressed in E.coli, and purified human recombinant wild-type and F71L A-crystallin. We have characterized the chaperone function of F71L A-crystallin by light scattering method using a battery of target proteins. In addition, we have examined the hydrophobicity, secondary, tertiary and the quaternary structure by biophysical methods.
Size exclusion chromatography studies revealed that F71L mutation had no significant effect on the apparent molecular mass of A-crystallin oligomeric complexes. Intrinsic tryptophan fluorescence and far- and near-UV CD spectra indicated that F71L missense mutation did not significantly affect the secondary and tertiary structures of A-crystallin. The ANS fluorescence emission spectra suggested no changes in surface hydrophobicity due to the F71L conversion. While the mutant A displayed almost complete loss (90%) of chaperone-like activity (CLA) by measuring protection against heat-induced aggregation of carbonic anhydrase, it showed approximately 50% less protection in heat-induced aggregation of βL-crystallin. The CLA of the F71L mutant was decreased approximately 35% in heat-induced aggregation of γ-crystallin, and varied with other client proteins.
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