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.

␣A and ␣B crystallins, members of the small heat shock protein family, prevent aggregation of proteins by their chaperone-like activity. These two proteins, although very homologous, particularly in the C-terminal region, which contains the highly conserved "␣-crystallin domain," show differences in their protective ability toward aggregation-prone target proteins. In order to investigate the differences between ␣A and ␣B crystallins, we engineered two chimeric proteins, ␣ANBC and ␣BNAC, by swapping the N-terminal domains of ␣A and ␣B crystallins. The chimeras were cloned and expressed in Escherichia coli. The purified recombinant wild-type and chimeric proteins were characterized by fluorescence and circular dichroism spectroscopy and gel permeation chromatography to study the changes in secondary, tertiary, and quaternary structure. Circular dichroism studies show structural changes in the chimeric proteins. ␣BNAC binds more 8-anilinonaphthalene-1-sulfonic acid than the ␣ANBC and the wild-type proteins, indicating increased accessible hydrophobic regions. The oligomeric state of ␣ANBC is comparable to wild-type ␣B homoaggregate. However, there is a large increase in the oligomer size of the ␣BNAC chimera. Interestingly, swapping domains results in complete loss of chaperone-like activity of ␣ANBC, whereas ␣BNAC shows severalfold increase in its protective ability. Our findings show the importance of the N-and C-terminal domains of ␣A and ␣B crystallins in subunit oligomerization and chaperone-like activity. Domain swapping results in an engineered protein with significantly enhanced chaperone-like activity.␣-Crystallin, a major lens protein having homology with small heat shock proteins (1-3), prevents aggregation of other proteins like a molecular chaperone (4). We had earlier shown that ␣-crystallin can prevent photo-aggregation of ␥-crystallin, which may have relevance in cataractogenesis (5). By using various non-thermal modes of aggregation, it was shown that chaperone-like activity of ␣-crystallin is temperature-dependent. A structural perturbation above 30°C enhances this activity severalfold (6, 7). In order to probe the molecular mechanism of the chaperone-like activity and its enhancement upon structural perturbation, we have been studying ␣-crystallin and its constituent subunits. Our recent study on the ␣A and ␣B homoaggregates showed that, despite high sequence homology, these proteins differ in their stability, chaperone-like activity, and the temperature dependence of this activity (8). This study also indicated different roles for the two proteins in the ␣-crystallin heteroaggregate in the eye lens and as separate proteins in non-lenticular tissues. Several investigators have introduced mutations in ␣A and ␣B crystallins to gain an insight into the structure-function relation (9 -12). Derham and Harding in their recent review (13) list about 30 sitedirected mutations from different laboratories. These mutations either result in some decrease or no change in the protective ability. It is interes...

We have studied the chaperone activity and conformation of Escherichia coli heat shock protein (Hsp)33, whose activity is known to be switched on by oxidative conditions. While oxidized Hsp33 completely prevents the heatinduced aggregation of j j-crystallin at 42³C at a ratio of 1:1 (w/w), the reduced form exhibits only a marginal effect on the aggregation. Far UV^circular dichroism (CD) spectra show that reduced Hsp33 contains a significant K K-helical component. Oxidation results in significant changes in the far UV^CD spectrum. Near UV^CD spectra show changes in tertiary structural packing upon oxidation. Polarity-sensitive fluorescent probes report enhanced hydrophobic surfaces in the oxidized Hsp33. Our studies show that the oxidative activation of the chaperone function of Hsp33 involves observable conformational changes accompanying increased exposure of hydrophobic pockets. ß

In the present study a combination of BLAST mediated homology search and 3′ RACE was utilized to isolate the full-length gene of Eleusine coracana alpha prolamin (Ec-α-prolamin) from finger millet. Phylogenetic analysis of Ec-α-prolamin along with related prolamin genes of different cereals and millets shows the clustering of Ec-α-prolamin in a separate group. Secondary structure prediction reveals 59.4% alpha helix structure, a structural hallmark of Ec-α-prolamin. Besides this, the protein also possesses a balanced proportion of all essential amino acids. Expression analysis based on qPCR shows increased accumulation of α-prolamin transcripts in developing grains of finger millet until attainment of seed maturity. Western blotting, using a monospecific anti-α-prolamin antibody, further confirmed the expression of a 22 kDa band in the S3 and S4 stages of developing spikes. The heterologous expression of isolated full-length Ec-α-prolamin could be potentially harnessed for making nutritionally enhanced functional food products and value-added industrial products.