Structure and properties of Max protein which regulates the function of a proto-oncogene product, c-Myc

Max蛋白通过与Myc形成异源二聚体或与自身形成同源二聚体并与DNA结合来调节原癌基因产物Myc的功能。建立了利用基因工程技术制备Max及其两种DNA结合结构域的方法，并对其结构和性质进行了研究。Max蛋白质在溶液中处于单体和二聚体之间的平衡。二聚体含量随蛋白质浓度的增加和温度的降低而增加。蛋白质α-螺旋含量的增加同时伴随着二聚体的增加。加入含有特异性识别碱基序列的双链DNA后，α-螺旋含量随着DNA浓度的增加而进一步增加。当使用在中间两个碱基对中含有不同序列的DNA双链体时，α-螺旋含量增加较小。在高温下观察到同源序列的严格特异性识别。Max的生物活性通过N-末端区域中Ser残基的磷酸化来调节。我们制备了突变蛋白，其中一个或两个Ser残基被替换为Asp残基作为磷酸化Max的模拟物，并检查了突变蛋白的DNA结合能力。突变蛋白-DNA复合物的稳定性较低，双突变蛋白复合物的稳定性最低。我们还开发了一种合成方法，通过化学连接化学合成的磷酸化肽和基因工程制备的蛋白质的剩余部分来获得磷酸化Max。

Max Protein regulates the function of a proto-oncogene product, Myc, by formation of a heterodimer with Myc or a homodimer with itself and binding to DNA. We established a procedure for preparation of Max and two types of its DNA binding domains by genetic engineering techniques and examined their structure and properties. Max proteins were in equilibrium between a monomer and a dimer in solution. The dimer content increasesd with increasing protein concentration and decreasing temperature. Increase in α-helical content of the protein simultaneously accompanied the dimer increase. Upon addition of a duplex DNA containing the specific recognition base sequence, the α-helical content increased further with increasing DNA concentration. When a DNA duplex containing a sequence different in the central two base pairs was used, the α-helical content increase was smaller. A strictly specific recognition of the cognate sequence was observed at high temperature. The biological activity of Max is modulated by phosphorylation of Ser residues in the N-terminal region. We prepared mutant proteins where one or two of the Ser residues were replaced with Asp residue(s) as a mimic of the phosophorylated Max and examined DNA binding ability of the mutant proteins. The mutant protein-DNA complexes showed lower stability and the complex of the mutant protein with double mutation showed the lowest stability. We also developed a synthetic method for obtaining phosphorylated Max by chemically joining a phosphorylated peptied synthesized chemically and the remaining part of protein prepared by genetic engineering.