Unnatural amino acid replacement in a yeast G protein-coupled receptor in its native environment
Unnatural amino acid replacement in a yeast G protein-coupled receptor in its native environment
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DOI：
10.1021/bi701866e
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发表时间：
2008-05-20
期刊：
影响因子：
2.9
通讯作者：
Becker, Jeffrey M.
中科院分区：
文献类型：
--
作者：
Huang, Li-Yin;Umanah, George;Becker, Jeffrey M.
Ste 2 p是酿酒酵母的十三肽信息素CC因子的G蛋白偶联受体（GPCR）。这种受体-信息素对已被广泛用作研究GPCR结构和功能的范例。在含有同源tRNA/氨酰-tRNA合成酶对的酵母中表达，该合成酶对特异性进化为响应琥珀密码子而掺入对苯甲酰基-L-苯丙氨酸（Bpa），从而允许Bpa取代的Step 2 p在其天然细胞中生物合成。我们通过将琥珀色TAG终止密码子工程化到质粒上编码的STE 2中，用Bpa替换了STE 2 p中的天然氨基酸残基。几个表达的BPA取代的Ste 2 p受体表现出高亲和力的配体结合，并纳入BPA到Ste 2 p影响的生物活性作为测量响应CC因子的整个细胞的生长停滞。我们发现，在0.1-0.5 mM的浓度下，含有Bpa的二肽可用于增强Bpa向细胞中的递送，而在2 mM时，二肽和Bpa同样有效。非天然氨基酸的肽递送系统的应用将非天然氨基酸置换方法的使用扩展到酵母不可渗透的氨基酸。通过质谱分析验证了Bpa掺入到Step2 p中，并且两个Bpa-Step2 p突变体能够在光活化后选择性地捕获因子到配体结合位点中。据我们所知，这是第一个实验证据，记录了在其天然环境中表达的GPCR中的非天然氨基酸置换以及使用突变的受体来光捕获肽配体。
Ste2p is the G protein-coupled receptor (GPCR) for the tridecapeptide pheromone cc factor of Saccharomyces cerevisiae. This receptor- pheromone pair has been used extensively as a paradigm for investigating GPCR structure and function. Expression in yeast harboring a cognate tRNA/aminoacyl-tRNA synthetase pair specifically evolved to incorporate p-benzoyl-L-phenylalanine (Bpa) in response to the amber codon allowed the biosynthesis of Bpa-substituted Ste2p in its native cell. We replaced natural amino acid residues in Ste2p with Bpa by engineering amber TAG stop codons into STE2 encoded on a plasmid. Several of the expressed Bpa-substituted Ste2p receptors exhibited high-affinity ligand binding, and incorporation of Bpa into Ste2p influenced biological activity as measured by growth arrest of whole cells in response to cc factor. We found that, at concentrations of 0.1-0.5 mM, a dipeptide containing Bpa could be used to enhance delivery of Bpa into the cell, while at 2 mM, both dipeptide and Bpa were equally effective. The application of a peptide delivery system for unnatural amino acids will extend the use of the unnatural amino acid replacement methodology to amino acids that are impermeable to yeast. Incorporation of Bpa into Ste2p was verified by mass spectrometric analysis, and two Bpa-Ste2p mutants were able to selectively capture a factor into the ligand-binding site after photoactivation. To our knowledge, this is the first experimental evidence documenting an unnatural amino acid replacement in a GPCR expressed in its native environment and the use of a mutated receptor to photocapture a peptide ligand.