May 2005
Volume 46, Issue 13
ARVO Annual Meeting Abstract  |   May 2005
Role of Arg–163 (R163) in the Oligomerization of A–Crystallin
Author Affiliations & Notes
  • S. Rajan
    Biochemistry & Molecular Biology, Univ of Arkansas for Medicine, Little Rock, AR
  • E.C. Abraham
    Biochemistry & Molecular Biology, Univ of Arkansas for Medicine, Little Rock, AR
  • Footnotes
    Commercial Relationships  S. Rajan, None; E.C. Abraham, None.
  • Footnotes
    Support  EY11352
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 3885. doi:
  • Views
  • Share
  • Tools
    • Alerts
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      S. Rajan, E.C. Abraham; Role of Arg–163 (R163) in the Oligomerization of A–Crystallin . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3885.

      Download citation file:

      © ARVO (1962-2015); The Authors (2016-present)

  • Supplements

Abstract: : Purpose: Our recent studies on recombinant truncated αA–crystallins have shown about 60% decrease in the chaperone activity and 75% decrease in the oligomeric size in αA–crystallins lacking 11(αA1–162) , 16 (αA1–157), and 22 (αA1–151) C–terminal residues. Interestingly, truncated αA1–163 in which residue 163 is an Arg had nearly normal chaperone function and oligomeric size. So, the current study was aimed at exploring the possibility that Arg–163 is a participant in the oligomerization of αA–crystallin. Methods: The Quick–Change site–directed mutagenesis kit was used to generate truncated αA1–163 and αA1–162 and to mutate Arg–163 to Gly in both αA1–163 and in the full–length αA–wt (αA1–173). Expression of the αA–wt and all the mutants, all in the pET expression vector, was achieved in E. coli BL21 (DE3) pLysS cells and proteins purified by size–exclusion chromatography on Sephacryl S–300 HR columns. Chaperone activity was assayed at 37o C by measuring the ability of αA–wt and the mutants to prevent EDTA induced aggregation of alcohol dehydrogenase. Molecular masses were estimated by molecular sieve HPLC. Secondary and tertiary structures were assessed at room temperature by measuring far–UV and near–UV CD spectra, respectively with a Jasco 715 spectropolarimeter. Results: Molecular masses of αA–wt, αA1–163, and αA1–162 were about 600, 500, and 80 kDa, respectively, whereas the molecular mass of αA1–163/R163G mutant was similar to that of αA1–162 and that of αA1–173/R163G mutant was similar to that of αA–wt. Chaperone activities of αA1–162 and αA1–63/R163G were decreased about 60% whereas the chaperone activity of αA1–173/R163G was similar to that of αA–wt. Structural similarity was evident between αA1–163/R163G and αA1–162 and between αA1–173/R163G and αA–wt.Conclusions: Arg–163 does not appear to play a major role in the oligomerization of the full–length alphaA–crystallin. However, in the absence of all or part of the hydrophilic flexible tail, the Arg–163 positive charge seems to play a role in stabilizing the oligomeric structure of αA–crystallin. Additional studies are in progress to show the role of Arg–163 in the oligomerization of other C–terminal truncated alphaA–crystallins.

Keywords: protein structure/function • chaperones • protein modifications-post translational 

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.