Frech M, Schlichting I, Wittinghofer A, Chardin P
Max-Planck-Institut für medizinische Forschung, Abteilung Biophysik, Heidelberg, Federal Republic of Germany.
J Biol Chem. 1990 Apr 15;265(11):6353-9.
The simian ralA cDNA was inserted in a ptac expression vector, and high amounts of soluble ral protein were expressed in Escherichia coli. The purified p24ral contains 1 mol of bound nucleotide/mol of protein that can be exchanged against external nucleotide. The ral protein exchanges GDP with a t 1/2 of 90 min at 37 degrees C in the presence of Mg2+, and has a low GTPase activity (0.07 min-1 at 37 degrees C). We have also studied its affinity for various guanine nucleotides and analogs. NMR measurements show that the three-dimensional environment around the nucleotide is similar in p21ras and p24ral. In addition to these studies on the wild-type ral protein, we used in vitro mutagenesis to introduce substitutions corresponding to the Val12, Val12 + Thr59, and Leu61 substitutions of p21ras. These mutant ral proteins display altered nucleotide exchange kinetics and GTPase activities, however, the effects of the substitutions are less pronounced than in the ras proteins. p24ralVal12 + Thr59 autophosphorylates on the substituted Thr, as a side reaction of the GTP hydrolysis, but the rate is much lower than those of the Thr59 mutants of p21ras. These results show that ras and ral proteins have similar structures and biochemical properties. Significant differences are found, however, in the contribution of the Mg2+ ion to GDP binding, in the rate of the GTPase reaction and in the sensitivity of these two proteins to substitutions around the phosphate-binding site, suggesting that the various "small G-proteins" of the ras family perform different functions.
将猿猴ralA互补DNA插入ptac表达载体中，在大肠杆菌中表达出大量可溶性ral蛋白。纯化后的p24ral每摩尔蛋白含有1摩尔结合核苷酸，且该核苷酸可与外部核苷酸进行交换。ral蛋白在Mg2+存在的情况下，于37℃时以90分钟的半衰期交换GDP，并且具有较低的GTP酶活性（37℃时为0.07分钟-1）。我们还研究了它对各种鸟嘌呤核苷酸及其类似物的亲和力。核磁共振测量结果表明，p21ras和p24ral中核苷酸周围的三维环境相似。除了对野生型ral蛋白的这些研究外，我们还利用体外诱变技术引入了与p21ras的Val12、Val12 + Thr59和Leu61替代相对应的替代物。这些突变ral蛋白表现出改变的核苷酸交换动力学和GTP酶活性，然而，这些替代的影响不如在ras蛋白中那么明显。p24ralVal12 + Thr59在被替代的苏氨酸上发生自磷酸化，这是GTP水解的副反应，但速率远低于p21ras的Thr59突变体。这些结果表明，ras和ral蛋白具有相似的结构和生化特性。然而，在Mg2+离子对GDP结合的贡献、GTP酶反应的速率以及这两种蛋白对磷酸结合位点周围替代的敏感性方面发现了显著差异，这表明ras家族的各种“小G蛋白”执行不同的功能。
