Radical Redesign of a Tandem Array of Four R67 Dihydrofolate Reductase Genes Yields a Functional, Folded Protein Possessing 45 Substitutions
Radical Redesign of a Tandem Array of Four R67 Dihydrofolate Reductase Genes Yields a Functional, Folded Protein Possessing 45 Substitutions
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DOI：
10.1021/bi1005943
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发表时间：
2010-08-31
期刊：
影响因子：
2.9
通讯作者：
Howell, Elizabeth E.
中科院分区：
文献类型：
--
作者：
Feng, Jian;Grubbs, Jordan;Howell, Elizabeth E.
R67 二氢叶酸还原酶 (DHFR) 是一种质粒编码的 II 型酶。四个单体（长 78 个氨基酸）组装成具有 222 个对称性的同源四聚体。在之前的研究中，四个 R67 DHFR 基因拷贝的串联阵列被融合在框内，生成名为 Quad l 的功能单体。该蛋白质具有必要的三级结构()I-R67“亲本”。为了促进诱变反应，在串联基因阵列中引入了限制性酶位点。还添加了 S59A 和 H362L 突变，以尽量减少可能的折叠拓扑；这种名为Quad3的蛋白质产品拥有10个取代基并且具有功能性。由于R67 DHFR拥有稳定的支架，因此通过进一步添加45个氨基酸取代实现了序列空间的大跳跃。突变设计利用了其他类型 If DHFR 的替代序列。此外，大多数突变位于蛋白质表面以及无序的 N 端序列中，该序列充当融合结构域之间的接头。由此产生的 Quad4 蛋白功能非常强大；然而，它不如 Quad I 稳定，在 pH 5 时 Delta Delta G 损失为 5 kcal/mol。一个意想不到的结果是在 pH 8 时形成 Quad4 二聚体和更高阶寡聚体。R67 DHFR 及其衍生的 Quad 蛋白拥有坚固的支架，能够承受 >= 55 个取代的引入。
R67 dihydrofolate reductase (DHFR) is a plasmid-encoded, type II enzyme. Four monomers (78 amino acids long) assemble into a homotetramer possessing 222 symmetry. In previous studies, a tandem array of four R67 DHFR gene copies was fused in frame to generate a functional monomer named Quad l. This protein possessed the essential tertiary structure ()I-the R67 "parent". To facilitate mutagenesis reactions, restriction enzyme sites were introduced in the tandem gene array. S59A and H362L mutations were also added to minimize possible folding topologies; this protein product, named Quad3, possesses 10 substitutions and is functional. Since R67 DHFR possesses a stable scaffold, a large jump in sequence space was performed by the further addition of 45 amino acid substitutions. The mutational design utilized alternate sequences from other type If DHFRs. In addition, most of the mutations were positioned on the surface of the protein as well as in the disordered N-terminal sequence, which serves as the linker between the fused domains. The resulting Quad4 protein is quite functional; however, it is less stable than Quad I, suffering a Delta Delta G loss of 5 kcal/mol at pH 5. One unexpected result was formation of Quad4 dimers and higher order oligomers at pH 8. R67 DHFR, and its derivative Quad proteins, possesses a robust scaffold, capable of withstanding introduction of >= 55 substitutions.