Abstract
alphaA and alphaB 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 "alpha-crystallin domain," show differences in their protective ability toward aggregation-prone target proteins. In order to investigate the differences between alphaA and alphaB crystallins, we engineered two chimeric proteins, alphaANBC and alphaBNAC, by swapping the N-terminal domains of alphaA and alphaB 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. alphaBNAC binds more 8-anilinonaphthalene-1-sulfonic acid than the alphaANBC and the wild-type proteins, indicating increased accessible hydrophobic regions. The oligomeric state of alphaANBC is comparable to wild-type alphaB homoaggregate. However, there is a large increase in the oligomer size of the alphaBNAC chimera. Interestingly, swapping domains results in complete loss of chaperone-like activity of alphaANBC, whereas alphaBNAC shows severalfold increase in its protective ability. Our findings show the importance of the N- and C-terminal domains of alphaA and alphaB crystallins in subunit oligomerization and chaperone-like activity. Domain swapping results in an engineered protein with significantly enhanced chaperone-like activity.
Highlights
␣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]
Our findings show the importance of the N- and C-terminal domains of ␣A and ␣B crystallins in subunit oligomerization and chaperone-like activity
This study indicated different roles for the two proteins in the ␣-crystallin heteroaggregate in the eye lens and as separate proteins in non-lenticular tissues
Summary
␣ANBC Chimera—The 235-base pair NdeI-XmnI fragment of pCR2.1-␣A plasmid [16] was ligated to the 384-base pair XmnI-HindIII fragment of pCR2.1-␣B plasmid [16] to generate chimeric coding region of ␣ANBC. ␣BNAC Chimera—The 247-base pair NdeI-XmnI fragment of pCR2.1-␣B was ligated to the 446-base pair XmnI-HindIII fragment of pCR2.1-␣A to generate the chimeric coding region of ␣BNAC. The ␣BNAC chimera with NdeI-HindIII overhangs was ligated to NdeIHindIII-linearized pET21a to produce pET21a-␣BNAC. Overexpression and Purification of Human Wild-type and Chimeric ␣A and ␣B Crystallins. Induction, lysis of cells, and purification of chimeric proteins was done as described for recombinant wild-type ␣A and ␣B crystallins [26]. Multimeric sizes of the wild-type and chimeric proteins were evaluated on Superose-6 HR 10/30 prepacked column (dimensions: 10 ϫ 300 mm, bed volume: 24 ml) with reference to high molecular mass standards (Sigma). Standards used were thyroglobulin (669 kDa), ferritin (440 kDa), and catalase (232 kDa)
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