Vorherr T, James P, Krebs J, Enyedi A, McCormick D J, Penniston J T, Carafoli E
Laboratory of Biochemistry, Swiss Federal Institute of Technology, Zurich.
Biochemistry. 1990 Jan 16;29(2):355-65. doi: 10.1021/bi00454a008.
Peptides corresponding to the calmodulin binding domain of the plasma membrane Ca2+ pump (James et al., 1988) were synthesized, and their interaction with calmodulin was studied with circular dichroism, infrared spectroscopy, nuclear magnetic resonance, and fluorescence techniques. They corresponded to the complete calmodulin binding domain (28 residues), to its first 15 or 20 amino acids, and to its C-terminal 14 amino acids. The first three peptides interacted with calmodulin. The K value was similar to that of the intact enzyme in the 28 and 20 amino acid peptides, but increased substantially in the shorter 15 amino acid peptide. The 14 amino acid peptide corresponding to the C-terminal portion of the domain failed to bind calmodulin. 2D NMR experiments on the 20 amino acid peptides have indicated that the interaction occurred with the C-terminal half of calmodulin. A tryptophan that is conserved in most calmodulin binding domains of proteins was replaced by other amino acids, giving rise to modified peptides which had lower affinity for calmodulin. An 18 amino acid peptide corresponding to an acidic sequence immediately N-terminal to the calmodulin binding domain which is likely to be a Ca2+ binding site in the pump was also synthesized. Circular dichroism experiments have shown that it interacted with the calmodulin binding domain, supporting the suggestion (Benaim et al., 1984) that the latter, or a portion of it, may act as a natural inhibitor of the pump.
合成了与质膜Ca2+泵钙调蛋白结合结构域相对应的肽段（詹姆斯等人，1988年），并利用圆二色性、红外光谱、核磁共振和荧光技术研究了它们与钙调蛋白的相互作用。它们分别对应完整的钙调蛋白结合结构域（28个残基）、其前15或20个氨基酸以及其C端的14个氨基酸。前三个肽段与钙调蛋白相互作用。在28个氨基酸和20个氨基酸的肽段中，K值与完整酶的K值相似，但在较短的15个氨基酸的肽段中大幅增加。对应于该结构域C端部分的14个氨基酸的肽段未能结合钙调蛋白。对20个氨基酸的肽段进行的二维核磁共振实验表明，相互作用发生在钙调蛋白的C端一半区域。在大多数蛋白质的钙调蛋白结合结构域中保守的一个色氨酸被其他氨基酸取代，产生了对钙调蛋白亲和力较低的修饰肽段。还合成了一个18个氨基酸的肽段，它对应于钙调蛋白结合结构域紧邻N端的一个酸性序列，该序列可能是泵中的一个Ca2+结合位点。圆二色性实验表明，它与钙调蛋白结合结构域相互作用，支持了后者或其一部分可能作为泵的天然抑制剂的观点（贝纳姆等人，1984年）。
