Synthesis of proteins containing modified arginine residues
Synthesis of proteins containing modified arginine residues
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DOI：
10.1021/bi062042r
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发表时间：
2007-04-03
期刊：
影响因子：
2.9
通讯作者：
Hecht, Sidney M.
中科院分区：
文献类型：
--
作者：
Choudhury, Ambar K.;Golovine, Serguei Y.;Hecht, Sidney M.
非天然氨基酸的突变为深入研究蛋白质的功能提供了必要的手段。为了扩大可用于该技术研究的功能化氨基酸的谱系，我们制备了7个精氨酸结构类似物并用于激活抑制tRNA(CUA)。这些化合物包括N-甲基精氨酸、N-γ-硝基精氨酸、瓜氨酸、高精氨酸，以及三种构象受限的基于脯氨酸的类似物。这些错酰化的tRNA能够将精氨酸类似物引入二氢叶酸还原酶(第22位)和石楠荧光素酶(第218和437位)。大多数在第218位含有精氨酸类似物的修饰荧光素酶的发光效率低于野生型，且发射波长更长。这与该残基对于维持荧光素结合位点的极性和刚性所必需的假设作用是一致的。有趣的是，在第218位含有N-伽马-甲基精氨酸的荧光素酶发出的光波长与野生型相同，而且至少同样有效。改变437位的精氨酸残基对发光波长没有影响，但提供了类似物，所有这些类似物的发光效率都低于野生型。这与Arg437的假定作用完全一致，Arg437是包括吡喃假单胞菌荧光素酶在内的酶超家族中的一个不变残基。这种氨基酸是荧光素酶两个结构域之间的连接物的一部分，荧光素酶被认为是有效酶功能所必需的，但不是底物结合部位的一部分。
Unnatural amino acid mutagenesis provides the wherewithal to study protein function in great detail. To extend the repertoire of functionalized amino acids available for study by this technique, seven structural analogues of arginine were prepared and used to activate a suppressor tRNA(CUA). These included N-gamma-methylarginine, N-gamma-nitroarginine, citrulline, homoarginine, and three conformationally constrained analogues based on proline. These misacylated tRNAs were shown to be capable of introducing the arginine analogues into dihydrofolate reductase (position 22) and Photinus pyralis luciferase (positions 218 and 437). Most of the modified luciferases containing arginine analogues at position 218 emitted light with less efficiency and at longer wavelength than the wild type. This is consistent with the postulated role of this residue as essential for maintaining the polarity and rigidity of the luciferin-binding site. Interestingly, the luciferase containing N-gamma-methylarginine at position 218 emitted light at the same wavelength as the wild type and was at least as efficient. Alteration of the arginine residue at position 437 had no effect on the wavelength of emitted light but afforded analogues, all of which emitted light less efficiently than the wild type. This is altogether consistent with the putative role of Arg437, which is an invariant residue within the superfamily of enzymes that includes P. pyralis luciferase. This amino acid is part of the linker between the two structural domains of luciferase that is believed to be essential for efficient enzyme function but not part of the substrate-binding site.