Mutation of Tyr138 disrupts the structural coupling between the opposing domains in vertebrate calmodulin.
Mutation of Tyr138 disrupts the structural coupling between the opposing domains in vertebrate calmodulin.
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DOI：
10.1021/bi0104266
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发表时间：
2001-08
期刊：
影响因子：
2.9
通讯作者：
Hongye Sun;Danhong Yin;Laurel A. Coffeen;Madeline A. Shea;T. Squier
中科院分区：
文献类型：
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作者：
Hongye Sun;Danhong Yin;Laurel A. Coffeen;Madeline A. Shea;T. Squier
我们已经使用圆二色谱和频域荧光光谱，以确定如何与Phe 138或Gln 138的位点特异性取代Tyr 138影响钙调蛋白（CaM）的相对域之间的结构耦合。如前所述，构建涉及Tyr 99-> Trp 99和Leu 69-> Cys 69的保守取代的双突变体，以评估相对结构域之间的结构偶联[Sun，H.，Yin，D.，和Squier，T. C.（1999）Biochemistry 38，12266-12279]。Trp 99在距离测量中充当荧光共振能量转移（FRET）供体以探测中心螺旋的构象。Cys 69为5-（2-碘乙酰基）氨基）乙基）氨基）萘-1-磺酸（IAEDANS）的共价连接提供反应性基团，其作为FRET受体起作用并允许测量氨基末端结构域的旋转动力学。这些钙调素突变体表现出正常的钙依赖性凝胶迁移率的变化和近紫外CD光谱的变化，具有类似的二级结构，野生型钙调素钙激活后，并保留能力，完全激活质膜钙ATP酶。因此，在这些钙调素突变体的羧基和氨基末端结构域的全球折叠是类似的野生型钙调素。然而，与野生型CaM相比，Tyr 138被Phe 138或Gln 138取代导致（i）平均空间分离的改变和相对球状结构域之间构象异质性的增加，以及（ii）氨基末端结构域的独立旋转动力学。这些结果表明，无论是在Tyr 138和Glu 82之间的氢键或芳香族氨基酸侧链之间的接触相互作用的改变有可能启动通常与靶蛋白结合和激活的钙调素的结构崩溃。
We have used circular dichroism and frequency-domain fluorescence spectroscopy to determine how the site-specific substitution of Tyr138 with either Phe138 or Gln138 affects the structural coupling between the opposing domains of calmodulin (CaM). A double mutant was constructed involving conservative substitution of Tyr99 --> Trp99 and Leu69 --> Cys69 to assess the structural coupling between the opposing domains, as previously described [Sun, H., Yin, D., and Squier, T. C. (1999) Biochemistry 38, 12266-12279]. Trp99 acts as a fluorescence resonance energy transfer (FRET) donor in distance measurements to probe the conformation of the central helix. Cys69 provides a reactive group for the covalent attachment of 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS), which functions as a FRET acceptor and permits the measurement of the rotational dynamics of the amino-terminal domain. These CaM mutants demonstrate normal calcium-dependent gel-mobility shifts and changes in their near-UV CD spectra, have similar secondary structures to wild-type CaM following calcium activation, and retain the ability to fully activate the plasma membrane Ca-ATPase. The global folds, therefore, of both the carboxyl- and amino-terminal domains in these CaM mutants are similar to that of wild-type CaM. However, in comparison to wild-type CaM, the substitution of Tyr138 with either Phe138 or Gln138 results in (i) alterations in the average spatial separation and increases in the conformational heterogeneity between the opposing globular domains and (ii) the independent rotational dynamics of the amino-terminal domain. These results indicate that alterations in either the hydrogen bond between Tyr138 and Glu82 or contact interactions between aromatic amino acid side chains have the potential to initiate the structural collapse of CaM normally associated with target protein binding and activation.