Photo-cross-linkers incorporated into G-protein-coupled receptors in mammalian cells: a ligand comparison.
Photo-cross-linkers incorporated into G-protein-coupled receptors in mammalian cells: a ligand comparison.
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DOI：
10.1002/anie.201102646
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发表时间：
2011-08-22
影响因子：
16.6
通讯作者：
Wang, Lei
中科院分区：
文献类型：
--
作者：
Coin, Irene;Perrin, Marilyn H.;Vale, Wylie W.;Wang, Lei
尽管G蛋白偶联受体（GPCR）是当今几乎一半药物的分子靶点，但由于难以获得整合在细胞膜中的这种高度灵活和复杂系统的光谱数据，因此对其与配体特异性相互作用的分子基础知之甚少。研究天然环境中配体-受体相互作用的一种强有力的实验方法是使用光亲和交联，其通常涉及安装到化学可实现的配体中的可光活化的交联剂，以在配体-受体复合物中建立空间约束。[1]然而，配体内只有少数位置可以用光交联剂修饰而不影响结合或信号传导行为。[2]这一限制阻碍了配体-受体相互作用的全面作图，也阻碍了具有不同药理学特性的配体的比较。非经典氨基酸可以通过遗传密码的扩增在活细胞中被遗传地整合到蛋白质中。[3]简言之，将外源正交tRNA/氨酰-tRNA合成酶（阿尔斯）对引入宿主细胞。阿尔斯被工程化以将所需的非天然氨基酸（Uaa）加载到其同源tRNA上，其响应于独特密码子（通常为琥珀终止密码子UAG）而掺入Uaa。使用这种方法，光活化氨基酸已被引入到细菌，酵母和哺乳动物细胞中的蛋白质中，[4]并且已经报道了一些交联的例子，这些例子大多限于胞质蛋白或验证已知的相互作用。[5]用可光活化的Uaas交联
Although G-protein coupled receptors (GPCRs) are the molecular target of almost half of today s pharmaceuticals, little is known about the molecular basis of the specific interaction with their ligands, owing to the difficulty of obtaining spectroscopic data for such highly flexible and complex systems integrated in the cell membrane. A powerful experimental approach to investigate ligand–receptor interactions in a native environment is the use of photoaffinity cross-linking, which usually involves photo-activatable crosslinkers installed into chemically achievable ligands to establish spatial constraints in the ligand–receptor complex.[1] However, only a few positions within the ligand can be modified with the photo-cross-linker without affecting binding or signaling behavior.[2] This limitation prevents comprehensive mapping of the ligand–receptor interaction and prevents comparison of ligands with different pharmacological properties.Noncanonical amino acids can be genetically incorporated into proteins in live cells through the expansion of the genetic code.[3] Briefly, an exogenous orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pair is introduced into the host cell. The aaRS is engineered to charge a desired unnnatural amino acid (Uaa) onto its cognate tRNA, which incorporates the Uaa in response to a unique codon, usually the amber stop codon UAG. Using this approach, photoactivatable amino acids have been introduced into proteins in bacteria, yeast, and mammalian cells,[4] and a few crosslinking examples have been reported, which are mostly limited either to cytosolic proteins or to validate a known interaction.[5] Cross-linking with photoactivatable Uaas
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影响因子：
2.9
作者：
Grunbeck, Amy;Huber, Thomas;Sakmar, Thomas P.
通讯作者：
Sakmar, Thomas P.
影响因子：
2.9
作者：
Huang, Li-Yin;Umanah, George;Becker, Jeffrey M.
通讯作者：
Becker, Jeffrey M.
影响因子：
14.9
作者：
Tate, JJ;Persinger, J;Bartholomew, B
通讯作者：
Bartholomew, B
DOI：
10.1073/pnas.121165198
发表时间：
2001-06-19
影响因子：
11.1
作者：
Lewis, K;Li, C;Vale, WW
通讯作者：
Vale, WW
影响因子：
7.3
作者：
Beyermann, M.;Heinrich, N.;Berger, H.
通讯作者：
Berger, H.