Ribosome-Mediated Incorporation of Dipeptides and Dipeptide Analogues into Proteins in Vitro.
Ribosome-Mediated Incorporation of Dipeptides and Dipeptide Analogues into Proteins in Vitro.
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DOI：
10.1021/jacs.5b03135
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发表时间：
2015-09-09
影响因子：
15
通讯作者：
Hecht SM
中科院分区：
文献类型：
--
作者：
Maini R;Dedkova LM;Paul R;Madathil MM;Chowdhury SR;Chen S;Hecht SM
将含有随机化在2057 - 2063和2502 - 2507位置的23 S rRNA的质粒导入大肠杆菌中，提供了一个克隆文库，该文库除了先前存在的野生型核糖体之外，还产生了修饰的核糖体。用嘌呤霉素衍生物筛选这些克隆，嘌呤霉素是一种天然产物，作为氨酰-tRNA 3′-末端的类似物，通过接受生长的多肽链终止蛋白质合成，从而杀死细菌细胞。在这项研究中的嘌呤霉素衍生物含有二肽p-甲氧基苯丙氨酰甘氨酸，这意味着在克隆的修饰的核糖体敏感的嘌呤霉素类似物识别二肽的能力。使用嘌呤霉素衍生物抑制的几个克隆制备S-30制剂，其中一些显示支持二肽掺入蛋白质。四种掺入的物质包括两种二肽（Gly-Phe（2）和Phe-Gly（3）），以及硫醇化二肽类似物（4）和荧光恶唑（5），所述荧光恶唑具有与正常二肽中大致相同的距离的胺和羧基。发现含有巯基化二肽4和7-甲氧基香豆素荧光团的蛋白质进行荧光猝灭。将恶唑荧光团5引入二氢叶酸还原酶或绿色荧光蛋白中，使其荧光发射得到很强的增强，并研究了这种增强的基础。聚集体结果证明了将二肽作为单个核糖体事件并入的可行性，并说明了二肽中中心肽键的缺乏识别，从而可能使各种各样的结构类似物的并入成为可能。
Plasmids containing 23S rRNA randomized at positions 2057−2063 and 2502−2507 were introduced into Escherichia coli, affording a library of clones which produced modified ribosomes in addition to the preexisting wild-type ribosomes. These clones were screened with a derivative of puromycin, a natural product which acts as an analogue of the 3′-end of aminoacyl-tRNA and terminates protein synthesis by accepting the growing polypeptide chain, thereby killing bacterial cells. The puromycin derivative in this study contained the dipeptide p-methoxyphenylalanylglycine, implying the ability of the modified ribosomes in clones sensitive to this puromycin analogue to recognize dipeptides. Several clones inhibited by the puromycin derivative were used to make S-30 preparations, and some of these were shown to support the incorporation of dipeptides into proteins. The four incorporated species included two dipeptides (Gly-Phe (2) and Phe-Gly (3)), as well as a thiolated dipeptide analogue (4) and a fluorescent oxazole (5) having amine and carboxyl groups approximately the same distance apart as in a normal dipeptide. A protein containing both thiolated dipeptide 4 and a 7-methoxycoumarin fluorophore was found to undergo fluorescence quenching. Introduction of the oxazole fluorophore 5 into dihydrofolate reductase or green fluorescent protein resulted in quite strong enhancement of its fluorescence emission, and the basis for this enhancement was studied. The aggregate results demonstrate the feasibility of incorporating dipeptides as a single ribosomal event, and illustrate the lack of recognition of the central peptide bond in the dipeptide, potentially enabling the incorporation of a broad variety of structural analogues.
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影响因子：
3.4
作者：
Patterson, GH;Knobel, SM;Piston, DW
通讯作者：
Piston, DW
影响因子：
14.9
作者：
ROBERTSON, SA;NOREN, CJ;SCHULTZ, PG
通讯作者：
SCHULTZ, PG
影响因子：
2.9
作者：
Dedkova, Larisa M.;Fahmi, Nour Eddine;Hecht, Sidney M.
通讯作者：
Hecht, Sidney M.
影响因子：
15
作者：
Rothman, DM;Petersson, EJ;Imperiali, B
通讯作者：
Imperiali, B
影响因子：
2.8
作者：
Kamionka M
通讯作者：
Kamionka M