Hecht M H, Sauer R T
J Mol Biol. 1985 Nov 5;186(1):53-63. doi: 10.1016/0022-2836(85)90256-6.
We have isolated same-site and second-site revertants that restore partial activity, wild-type activity, or greater than wild-type activity, to lambda repressor proteins bearing different mutations in the DNA binding domain. In some cases the revertant repressors contain same-site substitutions that are similar to the wild-type side-chain (e.g. Tyr22----Phe, Ser77----Thr). The activity of these revertants makes it possible to assess the role of specific hydrogen bonds and/or packing interactions in repressor structure and function. In other same-site revertants, a very different type of residue is introduced (e.g. Ser35----Leu, Gly48----Asn). This indicates that the chemical and steric requirements at these side-chain positions are relaxed. Two of the second-site revertants, Glu34----Lys and Gly48----Ser, restore activity to more than one primary mutant. Both substitutions apparently increase the affinity of the repressor-operator interaction by introducing new contacts with operator DNA. These results suggest that reversion may be a generally applicable method for identifying sequence changes that increase the activity of a protein to greater than wild-type levels.
我们已经分离出了同一位点和第二位点回复突变体，它们能使在DNA结合结构域带有不同突变的λ阻遏蛋白恢复部分活性、野生型活性或高于野生型的活性。在某些情况下，回复突变的阻遏蛋白含有与野生型侧链相似的同一位点取代（例如，Tyr22→Phe，Ser77→Thr）。这些回复突变体的活性使得评估特定氢键和/或堆积相互作用在阻遏蛋白结构和功能中的作用成为可能。在其他同一位点回复突变体中，引入了一种非常不同类型的残基（例如，Ser35→Leu，Gly48→Asn）。这表明这些侧链位置的化学和空间要求有所放宽。两个第二位点回复突变体，Glu34→Lys和Gly48→Ser，能使不止一种初级突变体恢复活性。这两种取代显然通过与操纵基因DNA形成新的接触来增加阻遏蛋白-操纵基因相互作用的亲和力。这些结果表明，回复突变可能是一种普遍适用的方法，用于鉴定能使蛋白质活性增加到高于野生型水平的序列变化。
