SITE-SPECIFIC INCORPORATION OF BIOPHYSICAL PROBES INTO PROTEINS
SITE-SPECIFIC INCORPORATION OF BIOPHYSICAL PROBES INTO PROTEINS
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DOI：
10.1073/pnas.91.8.2910
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发表时间：
1994-04-12
影响因子：
11.1
通讯作者：
SCHULTZ, PG
中科院分区：
文献类型：
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作者：
CORNISH, VW;BENSON, DR;SCHULTZ, PG
生物物理探针可以检测蛋白质的结构变化和蛋白质与其他大分子的相互作用，是研究蛋白质功能的重要工具。然而，在将探针引入特定位点的蛋白质中仍然存在许多困难。在这里，我们报道了通过使用非自然氨基酸诱变，将自旋标记的、荧光的和光活化的氨基酸成功地结合到噬菌体T4溶菌酶的各种表面和内部位点。此外，我们报道了含有自旋标记氨基酸和荧光氨基酸的T4溶菌酶突变体的纯化和光谱表征。结合这些探针特异位点的能力允许对蛋白质结构和动力学进行新的研究。此外，这项工作表明，大肠杆菌蛋白生物合成机制可以耐受与天然氨基酸几乎没有相似之处的非天然氨基酸。
Biophysical probes which can detect structural changes in proteins and the interaction of proteins with other macromolecules are important tools in studying protein function. Many difficulties remain, however, in introducing probes into proteins site-specifically. Here we report the successful site-specific incorporation of a spin-labeled, a fluorescent, and a photoactivatible amino acid into a variety of surface and internal sites in bacteriophage T4 lysozyme by using unnatural amino acid mutagenesis. In addition, we report the purification and spectral characterization of T4 lysozyme mutants containing the spin-labeled amino acid and the fluorescent amino acid. The ability to incorporate these probes site-specifically allows for novel studies of protein structure and dynamics. Moreover, this work demonstrates that the Escherichia coli protein biosynthetic machinery can tolerate unnatural amino acids with little resemblance to the natural amino acids.