title: Influence of the C-terminal residues on oligomerization of alpha A-crystallin.
abstract: Earlier studies have shown that the chaperone activity of alpha-crystallin is significantly affected in diabetic rat and human lenses. Subsequently, mass spectrometric analysis showed diabetic lenses having high levels of the alphaA-crystallins in which different numbers of C-terminal residues were deleted. The present study was aimed to show whether cleavage of these residues influences protein structure, oligomerization, and chaperone function. For generation of various mutants, a stop codon was introduced at the positions of interest, proteins were expressed in BL21(DE3)pLys S E. coli, and the truncated alphaA-crystallins were purified by size-exclusion chromatography. The molecular masses, as determined by molecular sieve HPLC, of mutants with deletions of 1, 5, and 10 C-terminal residues (group-1) were 519-602 kDa, and those of mutants with deletions of 11, 16, and 22 C-terminal residues (group-2) were 148-152 kDa, as compared to 607 kDa for alphaA-wild type. On the basis of circular dichroism measurements, the alpha helix content was 2-fold higher and the tertiary structure was significantly altered in the group-2 mutants. Chaperoning abilities, as determined by the ADH assay and the betaL-crystallin heat denaturation assay, of the group-1 mutants, with the exception of alphaA(1-163), were slightly improved or unchanged, that of alphaA(1-163) was moderately affected, and those of the group-2 mutants were severely affected. Most strikingly, cleavage of 11 C-terminal residues including Arg-163 showed a substantial decrease in oligomeric size and chaperone function and significant changes in protein structure whereas cleavage of 10 residues had either a small effect or no effect at all. This points to an important role for the C-terminal extension, Arg-163 in particular, and no significant role for the C-terminal flexible tail in the oligomer assembly of alphaA-crystallin.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
authors.forename: Prajitha
authors.initials: P
authors.lastname: Thampi
authors.forename: Edathara C
authors.initials: EC
authors.lastname: Abraham
mesh_terms: D000818:Animals; D006801:Humans; D008027:Light; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation / Q000235:genetics; D017434:Protein Structure, Tertiary; D051381:Rats; D012542:Scattering, Radiation; D017384:Sequence Deletion; D013696:Temperature; D013816:Thermodynamics; D038202:alpha-Crystallin A Chain / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D018832:Molecular Chaperones; D010446:Peptide Fragments; D038202:alpha-Crystallin A Chain