Structural and functional consequences of the mutation of a conserved arginine residue in αA and αB crystallins

Abstract

A point mutation of a highly conserved arginine residue in αA and αB crystallins was shown to cause autosomal dominant congenital cataract and desmin-related myopathy, respectively, in humans. To study the structural and functional consequences of this mutation, human αA and αB crystallin genes were cloned and the conserved arginine residue (Arg-116 in αA crystallin and Arg-120 in αB crystallin) mutated to Cys and Gly, respectively, by site-directed mutagenesis. The recombinant wild-type and mutant proteins were expressed in Escherichia coli and purified. The mutant and wild-type proteins were characterized by SDS-polyacrylamide gel electrophoresis, Western immunoblotting, gel permeation chromatography, fluorescence, and circular dichroism spectroscopy. Biophysical studies reveal significant differences between the wild-type and mutant proteins. The chaperone-like activity was studied by analyzing the ability of the recombinant proteins to prevent dithiothreitol-induced aggregation of insulin. The mutations R116C in αA crystallin and R120G in αB crystallin reduce the chaperone-like activity of these proteins significantly. Near UV circular dichroism and intrinsic fluorescence spectra indicate a change in tertiary structure of the mutants. Far UV circular dichroism spectra suggest altered packing of the secondary structural elements. Gel permeation chromatography reveals polydispersity for both of the mutant proteins. An appreciable increase in the molecular mass of the mutant αA crystallin is also observed. However, the change in oligomer size of the αB mutant is less significant. These results suggest that the conserved arginine of the α-crystallin domain of the small heat shock proteins is essential for their structural integrity and subsequent in vivofunction.