Characterization of the basic amphiphilic alpha-helix calmodulin-binding domain of a 61.5 kDa tobacco calmodulin-binding protein
Characterization of the basic amphiphilic alpha-helix calmodulin-binding domain of a 61.5 kDa tobacco calmodulin-binding protein
复制标题
DOI：
10.1021/bi9621674
复制
发表时间：
1997-02-25
期刊：
影响因子：
2.9
通讯作者：
Harrington, HM
中科院分区：
文献类型：
--
作者：
Dash, S;Niemaczura, W;Harrington, HM
以烟草钙调素结合蛋白TCB 60为研究对象，设计合成了一个19个氨基酸残基的多肽序列：Gly-Trp-Leu-Lys-Ile-Lys-Ala-Ala-Met-Arg-Trp-Gly-Phe-Phe-Val-Arg-Lys-Lys-Ala。通过凝胶迁移率变动分析、磷酸二酯酶竞争分析、荧光、圆二色性和核磁共振光谱分析合成的结合结构域与钙调蛋白（CaM）的相互作用。迁移率变动分析表明，在合成肽和CaCl 2存在下，在4 M尿素聚丙烯酰胺凝胶电泳中CaM M（r）明显增加2 kDa。HPLC测量的环AMP的水解钙调素依赖性磷酸二酯酶表明，合成肽竞争性抑制（Ki = 15-20 nM）刺激磷酸二酯酶活性的钙调素。结合钙调素后，合成肽的荧光发射最大值，其中含有两个双羟基残基，移向蓝色和强度增加。圆二色性光谱表明，在208和222 nm处的椭圆度增加后，与合成肽的复合物形成的钙调素。1H-1 NMR研究表明，肽相互作用的芳香族残基在结构域I和III的钙调蛋白。两者合计，这些数据提供了直接的证据，结构保守的基本两亲性α-螺旋CaM结合结构域的重组烟草CaM结合蛋白与CaM相互作用，在生理上显着的纳摩尔浓度和CaM和合成的结合结构域的微环境被修改后，复合物的形成。
A 19-amino acid residue peptide, Gly-Trp-Leu-Lys-Ile-Lys-Ala-Ala-Met-Arg-Trp-Gly-Phe-Phe-Val-Arg-Lys-Lys-Ala, corresponding to the basic amphiphilic alpha-helix (BAA) motif at the C-terminus of a recombinant tobacco calmodulin-binding protein, TCB60, was synthesized. The interaction of the synthetic binding domain with calmodulin (CaM) was analyzed by gel mobility shift assays, phosphodiesterase competition assays, and fluorescence, circular dichroism, and nuclear magnetic resonance spectroscopy. Mobility shift assays showed an apparent 2 kDa increase in CaM M(r) in presence of synthetic peptide and CaCl2 in 4 M urea polyacrylamide gel electrophoresis. HPLC measurements of hydrolysis of cyclic AMP by CaM-dependent phosphodiesterase indicated the synthetic peptide competitively inhibits (K-i = 15-20 nM) stimulation of phosphodiesterase activity by CaM. Upon binding CaM, the fluorescence emission maximum of the synthetic peptide which contained two tryptophanyl residues, shifted toward blue and increased in intensity. The circular dichroism spectra indicated the ellipticity of CaM increased at 208 and 222 nm upon complex formation with the synthetic peptide. H-1 NMR studies showed that the peptide interacts with the aromatic residues in domains I and III of CaM. Taken together, these data provide direct evidence that the structurally conserved basic amphiphilic alpha-helix CaM-binding domain of the recombinant tobacco CaM-binding protein interacts with CaM at physiologically significant nanomolar concentrations and the microenvironments of both CaM and the synthetic binding domain are modified upon complex formation.