Structural and functional consequences of the mutation of a conserved arginine residue in alphaA and alphaB crystallins.
A point mutation of a highly conserved arginine residue in alphaA and alphaB 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 alphaA and alphaB crystallin genes were cloned and the conserved arginine residue (Arg-116 in alphaA crystallin and Arg-120 in alphaB 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 alphaA crystallin and R120G in alphaB 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 alphaA crystallin is also observed. However, the change in oligomer size of the alphaB mutant is less significant. These results suggest that the conserved arginine of the alpha-crystallin domain of the small heat shock proteins is essential for their structural integrity and subsequent in vivo function.
Kumar
L V
LV
Centre for Cellular and Molecular Biology, Hyderabad 500 007, India.
Ramakrishna
T
T
Rao
C M
CM
D001120:Arginine; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003459:Crystallins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D006801:Humans; D018832:Molecular Chaperones / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship
D003459:Crystallins; D018832:Molecular Chaperones; D001120:Arginine