Abstract
Purpose:
To investigate the effect of a point mutation in the N terminal domain of αA- crystallin (αAR21Q), implicated in congenital cataract.
Methods:
αA-wild-type (αAWT) and αAR21Q were expressed in BL21(DE3)pLysS cells and purified. The molecular mass of αAR21Q was compared to that of wild type αA crystallin (αAWT) using a TSKG5000PWXL size exclusion column connected to an HPLC system with a Refractive Index Detector-coupled with a Quasi Elastic Light Scattering detection technology. Intrinsic Tryptophan fluorescence and Bis-ANS binding measurements of αAWT and αAR21Q were performed to assess the tryptophan microenvironment and the overall surface hydrophobicity of the protein. Near and far UV-CD spectra of αAWT and αAR21Q were compared to see if the mutation affected the secondary and quaternary structure of the proteins. The ability of both αAWT and αAR21Q to act as molecular chaperones was investigated using model protein substrates such as Alcohol dehydrogenase and Insulin. The proteins were subjected to urea unfolding studies to test their stability, and susceptibility to cleavage by trypsin.
Results:
The point mutation did not significantly alter the overall molar mass of αAR21Q (4.5×105g/mol) as compared to that of αAWT (5.18×105g/mol). Intrinsic Tryptophan fluorescence and Bis-ANS binding studies showed an increase in the tryptophan fluorescence as well as an increase in the surface hydrophobicity. The near and far UV-CD profiles of αAWT and αAR21Q showed similar characteristics, hinting subtler effects of the point mutation on protein structure. However, αAR21Q showed greater chaperone-like activity (CLA) compared to αAWT with both client proteins used. Urea unfolding studies on αAR21Q indicate that the mutant is less stable in urea (C1/2 = 1.67M) than αAWT (C1/2 = 2.64M). αAR21Q was found to be more susceptible to digestion by trypsin than αAWT.
Conclusions:
αAR21Q exhibits increased Bis-ANS binding compared to αAWT, indicating greater surface hydrophobicity and an increased chaperone-like activity. An enhanced chaperone function to prevent aggregation of unfolding proteins in the eye lens can virtually predispose the chaperone-substrate protein complex to precipitate out of solution. Interaction of multiple large aggregates in the lens can thus advance opacities and eventually impair vision.