Purchase this article with an account.
K.R. Schmitz, J.F. Koretz; Site-Directed Mutagenesis of a Conserved Region Along the C-Terminal Arm of Alpha-Crystallin . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2371.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: The small heat shock protein (sHSP) superfamily is characterized by a highly conserved beta-sandwich core region, with N- and C-terminal arms of generally more variable composition. However, sequence alignments reveal a small hydrophobic patch with sequence IXI or IXV on the C-terminal arm that is well conserved across the superfamily, and, in the X-ray crystal structures of wheat HSP16.9 and M. jannaschii HSP16.5, is involved in stabilizing dimer-dimer interactions. Additionally, mammalian alpha crystallins possess a conserved proline residue in the center of this patch, which restricts the orientation of the C-terminal arm. The present study examines how this conserved patch might contribute to quaternary structure and function in alphaA crystallin. Methods: A series of site-directed mutants was constructed in both wild-type alphaA and in an alphaA deletion mutant lacking the entire N-terminal arm. Analysis of these mutants includes size determination by gel exclusion chromatography, evaluation of chaperone activity by the insulin aggregation assay, and structural investigation by far-UV CD spectroscopy. Results: Preliminary structural and functional results have been obtained for some of the constructs. Using the alphaA deletion mutant lacking the N-terminal arm, the small hydrophobic patch sequence was changed from IPV to TPT or GPG. The resultant constructs appear to aggregate into octamers, in contrast to the alphaA deletion mutant with the unaltered IPV sequence, which exhibits a dimer-tetramer equilibrium. Chaperone-like activity is also altered by these substitutions, but differently for GPG and TPT. Conclusion: This highly conserved motif appears to influence the association of the alphaA deletion mutants lacking the N-terminal region and to affect, either directly or indirectly, chaperone-like activity. It is expected to have a role in stabilization of the quaternary structure of aggregates of the intact alpha crystallin.
This PDF is available to Subscribers Only