Mutations in human αA-crystallin/sHSP affect subunit exchange interaction with αB-crystallin.

PloS One
Ilangovan RajuEdathara C Abraham

Abstract

Mutation in αA-crystallin contributes to the development of congenital cataract in humans. Heterooligomerization of αA-crystallin and αB-crystallin is essential for maintaining transparency in the eye lens. The effect of congenital cataract causing mutants of αA-crystallin on subunit exchange and interaction with αB-crystallin is unknown. In the present study, interaction of the mutants of αA-crystallin with αB-crystallin was studied both in vitro and in situ by the fluorescence resonance energy transfer (FRET) technique. In vitro FRET technique was used to demonstrate the rates of subunit exchange of αB-wt with the following αA-crystallin mutants: R12C, R21L, R21W, R49C, R54C, and R116C. The subunit exchange rates (k values) of R21W and R116C with αB-wt decreased drastically as compared to αA-wt interacting with αB-wt. Moderately decreased k values were seen with R12C, R49C and R54C while R21L showed nearly normal k value. The interaction of αA- mutants with αB-wt was also assessed by in situ FRET. YFP-tagged αA mutants were co-expressed with CFP-tagged αB-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 αB-...Continue Reading

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Citations

Jul 5, 2015·Biochimica Et Biophysica Acta·Murugesan RajuK Krishna Sharma
Jun 17, 2015·Biochimica Et Biophysica Acta·Alok Kumar PandaAshis Biswas
Feb 28, 2015·International Journal of Biological Macromolecules·Srinivasagan RamkumarKumarasamy Anbarasu
May 10, 2020·Cell Stress & Chaperones·Junna Hayashi, John A Carver

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Methods Mentioned

BETA
dynamic light scattering
fluorescence
FRET
Fluorescence resonance
PCR
transfection
size column chromatography
subunit exchange
confocal microscopy

Software Mentioned

GraphPad Prism
5301PC
RF

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