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
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
复制标题
DOI：
10.1002/pro.5560021011
复制
发表时间：
1993-10-01
期刊：
影响因子：
8
通讯作者：
ASWAD, DW
中科院分区：
文献类型：
--
作者：
POTTER, SM;HENZEL, WJ;ASWAD, DW
我们已经确定了负责在温和条件下的牛脑钙调素在体外老化过程中形成异天冬氨酸的主要网站。使用蛋白L-异戊酰基甲基转移酶（EC 2.1.1.77）通过将甲基-H-3从S-腺苷-L-[甲基-H-3]甲硫氨酸转移至异戊酰基（α-羧基）侧链来定量异天冬氨酸。超过1.2摩尔的甲基受体位点每摩尔的钙调素积累在2周的孵育过程中，在pH值7.4，37 ℃无钙。对老化钙调蛋白的蛋白水解肽的分析显示，>95%的甲基化能力限于四个钙结合结构域中的残基，预测这些残基在不存在钙的情况下是高度柔性的。我们估计，域III，IV和II累积0.72，0.60和0。13摩尔异天冬氨酸/摩尔钙调蛋白。Asn-97-Gly-98序列（结构域III）是异天冬氨酸形成的最大贡献者。异天冬氨酸形成的其他主要位点是结构域IV中的Asp-131-Gly-132和Asp-133-Gly-134，以及结构域II中的Asn-60-Gly-61。显著的异天冬氨酸形成也定位于结构域I中的Asp-20、Asp-22和/或Asp-24，结构域II中的Asp-56和/或Asp-58，以及结构域III中的Asp-93和/或Asp-95。所有这些残基都是高度保守的EF-手型钙结合基序中的钙配体。因此，其他EF-手蛋白质也可以在这些配体处形成异天冬氨酸。结果支持的想法，在结构蛋白质中的异天冬氨酸形成的强烈影响的C-侧翼残基和本地的灵活性。
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.