科研之友
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摘要
Background: Mutation in alpha A-crystallin contributes to the development of congenital cataract in humans. Heterooligomerization of alpha A-crystallin and alpha B-crystallin is essential for maintaining transparency in the eye lens. The effect of congenital cataract causing mutants of alpha A-crystallin on subunit exchange and interaction with alpha B-crystallin is unknown. In the present study, interaction of the mutants of alpha A-crystallin with alpha B-crystallin was studied both in vitro and in situ by the fluorescence resonance energy transfer (FRET) technique. %26lt;br%26gt;Methodology/Principal Findings: In vitro FRET technique was used to demonstrate the rates of subunit exchange of alpha B-wt with the following alpha A-crystallin mutants: R12C, R21L, R21W, R49C, R54C, and R116C. The subunit exchange rates (k values) of R21W and R116C with alpha B-wt decreased drastically as compared to alpha A-wt interacting with alpha B-wt. Moderately decreased k values were seen with R12C, R49C and R54C while R21L showed nearly normal k value. The interaction of alpha A-mutants with alpha B-wt was also assessed by in situ FRET. YFP-tagged alpha A mutants were co-expressed with CFP-tagged alpha B-wt in HeLa cells and the spectral signals were captured with a confocal microscope before and after acceptor laser photobleaching. The interaction of R21W and R116C with alpha B-wt was decreased nearly 50% as compared to alpha A-wt while the rest of the mutants showed slightly decreased interaction. Thus, there is good agreement between the in vitro and in situ FRET data. %26lt;br%26gt;Conclusions/Significance: Structural changes occurring in these mutants, as reported earlier, could be the underlying cause for the decreased interaction with alpha B may contribute to development of congenital cataract.
- 出版日期2012-2-8
