Site-Specific Mutagenesis of Rat Oncomodulin

寡核苷酸定向突变将被用来研究两种不同生理功能的小钙结合蛋白中金属结合部位的结构和性质。这种名为螺调蛋白的蛋白质与肌肉蛋白小白蛋白有惊人的同源性。肌肉中的小白蛋白可能参与缓冲细胞内的钙离子，但缺乏触发收缩蛋白的钙依赖相互作用的能力。另一方面，Oncomodrin能够激活环核苷酸磷酸二酯酶，其方式类似于钙调蛋白。这两种蛋白质之间的功能差异反映在它们与金属结合的特性上。螺调蛋白具有一个钙/镁结合部位和一个钙专一性结合部位，而小白蛋白含有两个钙/镁结合部位。这个项目的目标是：1.确定是什么因素区分了钙专一性位点(“触发”位点)和钙/镁位点(“松弛”位点)，以及2.确定在螺调蛋白中对蛋白质的调节能力至关重要的特定氨基酸残基。现已构建了肿瘤素表达载体，并将单突变引入到肿瘤素编码序列中。然后将研究这些突变对蛋白质的金属结合特性和调节能力的影响。发光的稀土离子探针，结合传统的金属结合分析，将被用来研究改变后的蛋白质的金属结合性能。肿瘤素刺激心脏环核苷酸磷酸二酯酶的能力将被用作调节能力的探针。细胞内钙结合蛋白家族包括肌钙蛋白C、钙调蛋白、小白蛋白和维生素D依赖的钙结合蛋白，此外还有肿瘤调节蛋白。这些蛋白质在它们的金属结合域中显示出高度的同源性，并被认为是从共同的前体进化而来的。然而，它们的金属结合性能有相当大的变化。某些结合部位是钙离子的专一性结合部位，在生理条件下不与镁离子结合，而另一些结合部位对钙和镁离子都有亲和力。金属结合的特异性与生理功能有关。钙特定部位参与启动(“触发”)生物过程，而钙/镁部位则不参与。这种功能差异的分子基础尚不清楚。对具有“触发”活性的肿瘤素和不具有“触发”活性的小白蛋白的详细结构进行的拟议比较，应该会为这个有趣的、具有重要生理意义的问题提供重要的见解。

Oligonucleotide-directed mutagenesis will be used to study the structure and properties of the metal binding sites in two, small, calcium-binding proteins that differ in their physiological functions. The protein, oncomodulin, exhibits striking homology to the muscle protein, parvalbumin. Parvalbumin in muscle may be involved in buffering intracellular calcium, but is devoid of the ability to trigger calcium- dependent interactions of contractile proteins. Oncomodulin, on the other hand, is capable of activating cyclic nucleotide phosphodiesterase in a manner reminiscent of calmodulin. This functional difference between the two proteins is mirrored in their metal-binding properties. Oncomodulin possesses one Ca/Mg binding site and one Ca-specific site, while parvalbumin contains two Ca/Mg sites. The objectives of this project are: 1. To determine what factors differentiate a Ca-specific site (a "triggering" site) from a Ca/Mg site (a "relaxing" site), and 2. to identify specific amino acid residues in oncomodulin that are essential to the protein's regulatory capability. An oncomodulin expression plasmid has been constructed, and now single mutations will be introduced into the oncomodulin coding sequence. The effects of these mutations on the protein's metal-binding properties and regulatory ability will then be investigated. Luminescent lanthanide ion probes, together with conventional metal-binding assays, will be used to study the metal-binding properties of the altered proteins. The ability of oncomodulin to stimulate heart cyclic nucleotide phosphodiesterase will be used as a probe of regulatory capability. The family of intracellular calcium-binding proteins includes troponin C, calmodulin, parvalbumin, and vitamin D- dependent calcium-binding protein, in addition to oncomodulin. These proteins display a high degree of homology in their metal- binding domains and are believed to have evolved from a common precursor. There is, however, considerable variation in their metal binding properties. Certain binding sites are quite specific for calcium and do not bind magnesium ion under physiological conditions, while other sites have affinity for both calcium and magnesium. The metal-binding specificity is correlated with physiological function. Calcium specific sites are involved in initiating ("triggering") biological processes, while Ca/Mg sites are not. The molecular basis for the difference in function is not understood. The proposed comparison of the detailed structure of oncomodulin, which has "triggering" activity, and parvalbumin, which does not, should provide important insight into this interesting and physiologically important question.