IN-VITRO AGING OF CALMODULIN GENERATES ISOASPARTATE AT MULTIPLE ASN-GLY AND ASP-GLY SITES IN CALCIUM-BINDING DOMAIN-II, DOMAIN-III, AND DOMAIN-IV
by POTTER-SM HENZEL-WJ ASWAD-DW (*R)
UNIV CALIF IRVINE,SCH BIOL SCI/IRVINE//CA/92717 (*R)
PROTEIN SCIENCE
VOL: 2 (10):1648-1663(1993)
LOC: CHEM
(INCLUDES MAGE MODEL ON DISKETTE)
Abstract:
We have determined the major sites responsible for isoaspartate
formation during in vitro aging of bovine brain calmodulin under mild
conditions. Protein L-isoaspartyl methyltransferase (EC 2.1.1.77) was used
to quantify isoaspartate by the transfer of methyl-H-3 from
S-adenoSyl-L-[methyl-H-3]methionine to the isoaspartyl (alpha-carboxyl)
side chain. More than 1.2 mol of methyl-acceptor sites per mol of
calmodulin accumulated during a 2-week incubation without calcium at pH
7.4, 37-degrees-C. Analysis of proteolytic peptides of aged calmodulin
revealed that >95% of the methylation capacity is restricted to residues in
the four calcium-binding domains, which are predicted to be highly flexible
in the absence of calcium. We estimate that domains III, IV, and II
accumulated 0.72, 0.60, and 0. 13 mol of isoaspartate per mol of
calmodulin, respectively. The Asn-97-Gly-98 sequence (domain III) is the
greatest contributor to isoaspartate formation. Other major sites of
isoaspartate formation are Asp-131-Gly-132 and Asp-133-Gly-134 in domain
IV, and Asn-60-Gly-61 in domain II. Significant isoaspartate formation was
also localized to Asp-20, Asp-22, and/or Asp-24 in domain I, to Asp-56
and/or Asp-58 in domain II, and to Asp-93 and/or Asp-95 in domain III. All
of these residues are calcium ligands in the highly conserved EF-hand
calcium-binding motif. Thus, other EF-hand proteins may also be subject to
isoaspartate formation at these ligands. The results support the idea that
isoaspartate formation in structured proteins is strongly influenced by
both the C-flanking residue and by local flexibility.