September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
LOSS OF FUNCTION IN αA-CRYSTALLIN MUTANT G98R IS RESCUED BY A COMPENSATORY MUTATION IN THE N-TERMINAL OF THE PROTEIN.
Author Affiliations & Notes
  • Ashutosh Phadte
    Biochemistry, University of Missouri, Columbia, Missouri, United States
  • Puttur Santhoshkumar
    Ophthalmology, University of Missouri, Columbia, Missouri, United States
  • K Krishna Sharma
    Ophthalmology, University of Missouri, Columbia, Missouri, United States
    Biochemistry, University of Missouri, Columbia, Missouri, United States
  • Footnotes
    Commercial Relationships   Ashutosh Phadte, None; Puttur Santhoshkumar, None; K Krishna Sharma, None
  • Footnotes
    Support  NIH(NEI) EY023219
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 728. doi:
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      Ashutosh Phadte, Puttur Santhoshkumar, K Krishna Sharma; LOSS OF FUNCTION IN αA-CRYSTALLIN MUTANT G98R IS RESCUED BY A COMPENSATORY MUTATION IN THE N-TERMINAL OF THE PROTEIN.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):728.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : αA-crystallin mutant G98R is associated with congenital cataract and is characterized by an increased oligomeric mass, reduced chaperone function and loss in stability over time. Another congenital cataract mutant in the N-terminal of αA-crystallin, αAR21Q exhibits structural characteristics similar to wild-type αA-crystallin, but exhibits enhanced chaperone function. The purpose of the study is to determine whether the inherent instability in αAG98R could be rescued by another compensatory mutation at a different location in the protein. Hence,a double mutant αA-crystallin αAR21Q/G98R was generated and characterized.

Methods : Wild-type αA-crystallin (αAWT) and mutants αAR21Q, αAG98R and αAR21Q/G98R were expressed in BL21(DE3)pLysS cells and purified. Effect of the point mutations on protein structure was assessed by Far UV CD, Intrinsic Tryptophan fluorescence and Bis-ANS binding studies.The molar masses of the proteins were estimated by MALS/DLS.The proteins were also tested for their stability as a function of time by incubating protein aliquots at 37°C for defined time periods, followed by MALS/DLS. The ability of both wild type and mutant αA-crystallins to act as molecular chaperones was investigated using chemically induced denaturation of model protein substrates such as ADH, Lysozyme and Insulin.

Results : Far-UV CD studies reveal an increased α-helical content in the double mutant in comparison to αAWT, αAR21Q and αAG98R. Compared to αAWT, αAR21Q and αAG98R the double mutant shows an increased intrinsic tryptophan fluorescence and Bis-ANS binding, suggesting an altered tryptophan microenvironment and an increased surface hydrophobicity. αAR21Q/G98R shows an increased molar mass in comparison to αAWT and αAR21Q. However, time dependent aggregation studies reveal that unlike αAG98R, αAR21Q/G98R did not self-aggregate on incubation at 37°C, exhibiting a behavior similar to αAWT and αAR21Q. αAR21Q/G98R also shows better chaperone like activity against ADH, Lysozyme and Insulin compared to αAG98R.

Conclusions : The loss of structure and function in αAG98R was overcome by a compensatory mutation αAR21Q, as the double mutant protein showed restoration in its chaperone function as well as its oligomeric mass. The study also highlights an important role played by the N-terminal region of αA-crystallin to contribute towards its oligomeric stability and function.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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