Zhang M, Vogel H J
Department of Biological Sciences, University of Calgary, Alberta, Canada.
J Mol Biol. 1994 Jun 17;239(4):545-54. doi: 10.1006/jmbi.1994.1393.
Calmodulin (CaM) is a ubiquitous calcium regulatory protein that can interact with almost 30 different target proteins. The majority of the CaM-binding domains of the target proteins are believed to interact with two hydrophobic surfaces on Ca(2+)-CaM; these two regions are very rich in Met residues. To obtain more information about the role of these residues, we have biosynthetically incorporated selenomethionine (SeMet) in place of the nine Met residues of CaM. Amino acid analysis shows that the SeMet-CaM contains 15% Met and 85% SeMet. SeMet-CaM retains many of the properties of the wild-type protein; it activates the enzyme cyclic nucleotide phosphodiesterase, it binds to phenyl-Sepharose and myosin light chain kinase (MLCK) in a calcium-dependent manner, and it experiences a calcium-dependent band shift during SDS-gel electrophoresis. Moreover, by comparing the natural abundance (1H,13C)-heteronuclear multiple quantum coherence (HMQC) spectra of the calcium, apo and target peptide-bound forms of wild-type CaM and SeMet-CaM, we have found that the two proteins have very similar, if not identical, structures. Thus, the substitution of SeMet for Met does not cause a change in the conformation and function of CaM, in agreement with the results obtained for other proteins. The apo, calcium and target peptide-bound forms of SeMet-CaM were subsequently studied by natural abundance (1H,77Se)-heteronuclear multiple bond correlation (HMBC) and (1H,13C)-HMQC NMR. Nine well-resolved 77Se resonances could be observed. Substitution of SeMet for Met gave rise to the same 1H and 13C chemical shift changes for each individual Met residue, this facilitated making the assignments from known 1H,13C assignments of the Met residues. Some of these assignments were confirmed by studying Met-->Leu mutants of CaM. With the exception of Met76, which always remains solvent exposed, all resonances experienced large 77Se chemical shift changes upon the addition of Ca2+ and the MLCK peptide. The large shift changes indicate that the electron distribution in the SeMet side-chain can be adjusted for the different states of CaM, suggesting that the polarizability of sulfur or selenium may be important for the proper functioning of CaM. This study also shows that the natural abundance (1H,77Se)-HMBC experiment provides a sensitive approach for the study of SeMet proteins.
钙调蛋白（CaM）是一种普遍存在的钙调节蛋白，它能与近30种不同的靶蛋白相互作用。据信，靶蛋白的大多数CaM结合结构域与Ca(2+)-CaM上的两个疏水表面相互作用；这两个区域富含甲硫氨酸（Met）残基。为了获得更多关于这些残基作用的信息，我们通过生物合成方法用硒代甲硫氨酸（SeMet）取代了CaM的九个Met残基。氨基酸分析表明，SeMet-CaM含有15%的Met和85%的SeMet。SeMet-CaM保留了野生型蛋白的许多特性；它能激活环核苷酸磷酸二酯酶，以钙依赖的方式与苯基琼脂糖和肌球蛋白轻链激酶（MLCK）结合，并且在SDS凝胶电泳过程中会发生钙依赖的条带迁移。此外，通过比较野生型CaM和SeMet-CaM的钙结合、脱钙和与靶肽结合形式的天然丰度（1H,13C）-异核多量子相干（HMQC）谱，我们发现这两种蛋白的结构即使不完全相同也非常相似。因此，用SeMet取代Met不会导致CaM的构象和功能发生变化，这与其他蛋白的研究结果一致。随后，通过天然丰度（1H,77Se）-异核多键相关（HMBC）和（1H,13C）-HMQC核磁共振研究了SeMet-CaM的脱钙、钙结合和与靶肽结合形式。可以观察到九个分辨率良好的77Se共振峰。用SeMet取代Met会使每个单独的Met残基产生相同的1H和13C化学位移变化，这有助于根据已知的Met残基的1H,13C归属进行归属。通过研究CaM的Met→Leu突变体，证实了其中一些归属。除了始终暴露于溶剂中的Met76外，所有共振峰在加入Ca2+和MLCK肽后都经历了较大的77Se化学位移变化。大的位移变化表明，SeMet侧链中的电子分布可以根据CaM的不同状态进行调整，这表明硫或硒的极化率可能对CaM的正常功能很重要。这项研究还表明，天然丰度（1H,77Se）-HMBC实验为研究SeMet蛋白提供了一种灵敏的方法。
