Abstract
Purpose::
Our recent studies on the role of the 163REEK166 motif in the oligomerization of rat αA-crystallin have shown that mutation of R163G did not cause any change in the oligomeric size of the full length αA-crystallin. However, R163G mutants which were truncated at E164, E165 and K166 showed significant decrease in molecular mass, pointing to the importance of these residues. The present study is aimed to explore the role of each amino acid residue of this motif in the oligomerization of the full length αA-crystallin.
Methods::
The mutation of R163, K166, R163K166, E165K166, E164E165K166 and R163E164E165K166 to glycine in the full length rat αA-crystallin was achieved by the Quick Change site-directed mutagenesis kit (Stratagene). Coding sequences were confirmed by automated DNA sequencing. Expression of the wild type and all the mutants (in pET expression vector) was done in E.coli BL21 (DE3) pLysS cells and proteins were double purified by size-exclusion chromatography on Sephacryl S-300 HR columns. Molecular masses were determined by molecular sieve HPLC using a 600 mm x 7.8 mm Biosep SEC 4000 column (Phenomenex). The mutants were then analyzed for their secondary & tertiary structure by CD measurements and chaperone function by using alcohol dehydrogenase (ADH) as the target protein.
Results::
αA-crystallin in which R163, K166 and R163K166 were mutated had molecular masses of 635, 620 and 640 kDa, respectively, thus showing no change as compared to wild type αA which is 625 kDa. However, mutation of E165K166, E164E165K166 and R163E164E165K166 resulted in molecular masses of 500, 475 and 520 kDa respectively, thus showing 17-24% decrease in the molecular mass. The chaperone activity when measured at αA: ADH ratio of 1:5 was not significantly affected. All the mutants showed alterations in the secondary and tertiary structures with increase in ß-sheet and decrease in α-helix as compared to wild type αA; E164E165K166, E165K166 and R163E164E165K166 mutations showing the largest changes in this order.
Conclusions::
The results suggest that E164 and E165 along with R163 and K166 play a role in the oligomerization of full length αA-crystallin. It is apparent that E164 and E165 are playing a dominant role. Further studies are in progress to show the specific role of E164 and E165 in the oligomerization of αA- crystallin.
Keywords: crystallins • protein structure/function • protein purification and characterization