Biodesign Center for Bioenergetics, and School of Molecular Sciences, Arizona State University, Tempe, Arizona 85287, United States.
J Am Chem Soc. 2023 Nov 1;145(43):23600-23608. doi: 10.1021/jacs.3c07524. Epub 2023 Oct 23.
The introduction of noncanonical amino acids into proteins and peptides has been of great interest for many years and has facilitated the detailed study of peptide/protein structure and mechanism. In addition to numerous nonproteinogenic α-l-amino acids, bacterial ribosome modification has provided the wherewithal to enable the synthesis of peptides and proteins with a much greater range of structural diversity, as has the use of endogenous bacterial proteins in reconstituted protein synthesizing systems. In a recent report, elongation factor P (EF-P), putatively essential for enabling the incorporation of contiguous proline residues into proteins, was shown to facilitate the introduction of an N-methylated amino acid in addition to proline. This finding prompted us to investigate the properties of this protein factor with a broad variety of structurally diverse amino acid analogues using an optimized suppressor tRNA that we designed. While these analogues can generally be incorporated into proteins only in systems containing modified ribosomes specifically selected for their incorporation, we found that EF-P could significantly enhance their incorporation into model protein dihydrofolate reductase using wild-type ribosomes. Plausibly, the increased yields observed in the presence of structurally diverse amino acid analogues may result from the formation of a stabilized ribosomal complex in the presence of EF-P that provides more favorable conditions for peptide bond formation. This finding should enable the facile incorporation of a much broader structural variety of amino acid analogues into proteins and peptides using native ribosomes.
将非天然氨基酸引入蛋白质和肽中多年来一直受到广泛关注，这为研究肽/蛋白质的结构和机制提供了便利。除了许多非蛋白源的α-L-氨基酸外，细菌核糖体的修饰为合成具有更多结构多样性的肽和蛋白质提供了手段，而内源性细菌蛋白在重组蛋白合成系统中的应用也是如此。在最近的一份报告中，伸长因子 P（EF-P）被认为对于允许连续脯氨酸残基掺入蛋白质是必需的，它被证明除了脯氨酸之外还能促进引入 N-甲基氨基酸。这一发现促使我们使用我们设计的优化的抑制 tRNA 来研究这种蛋白因子与各种结构多样的氨基酸类似物的性质。虽然这些类似物通常只能在专门选择用于掺入的修饰核糖体的系统中掺入蛋白质，但我们发现 EF-P 可以在用野生型核糖体的情况下显著增强它们在模型蛋白二氢叶酸还原酶中的掺入。合理的是，在存在 EF-P 的情况下观察到的结构多样的氨基酸类似物的产量增加可能是由于形成了稳定的核糖体复合物，为肽键形成提供了更有利的条件。这一发现应该能够使用天然核糖体轻松地将更广泛的结构多样的氨基酸类似物掺入蛋白质和肽中。
