Abstract
Purpose: :
An earlier study has suggested that Arg–163 (R163) may play a key role in the formation of high molecular mass aggregates in αA–crystallin. However, when R163 was mutated to R163G in the full length αA–crystallin, oligomer size and chaperone function were unaffected. The present study is aimed to test whether R163 exerts its influence only when critical number of C–terminal amino acid residues are cleaved.
Methods: :
A site–directed mutagenesis kit from Stratagene was used to generate αA–crystallins lacking 5, 6, 7, 8, 9, 10, or 11 C–terminal residues and to mutate R163 to G (R163G) in every truncated αA and to create R163K only in αA1–163. Expression was done in E. coli and proteins were purified by chromatography on Sephacryl S–300 HR columns followed by molecular sieve HPLC which was also used for molecular mass determination. Chaperone activity was assayed with alcohol dehydrogenase as the target protein and structural changes were ascertained by CD measurements.
Results: :
A gradual decrease in the oligomeric size which averaged about 3% per residue was seen in αA1–168, αA1–167, αA1–166, αA1–165, αA1–164, and αA1–163. Mutation of R163 to G in all of the truncated αA–crystallins showed consistently larger decrease in the oligomeric size than due to truncation alone. R163G mutants of αA1–168, αA1–167, and αA1–166 had 13–20% decrease in the oligomeric size in contrast to 70–80% decrease in the R163G mutants of αA1–165, αA1–164, and αA1–163. Substitution of R163 with a lysine (R163K) had only a marginal effect on the oligomeric size which suggests that the major function of R163 is to provide a positive charge. The chaperone activities also showed similar changes due to truncation alone and with R163G and R163K mutations, although the percentage changes were smaller. Noteworthy is the observation that the chaperone activity of the R163K mutant of αA1–163 was not different from that of αA1–163 without mutation of R163.
Conclusions: :
The data support the C–terminal flexible tail of 8 or 9 amino acids having a dominant influence on the oligomeric structure of αA–crystallin. A role for R163 in the full length αA is not apparent. When 5–7 amino acids are deleted, the presence or absence of R163 shows some effect on the oligomeric size. However, the most dramatic influence of R163 is seen only after the flexible tail is cleaved. Here, the R163 positive charge becomes the major factor which strengthens the interactions needed to form high molecular mass oligomers. When the R163 residue is deleted, as in αA1–162, the oligomeric structure collapses.
Keywords: crystalline lens • chaperones • protein structure/function