Department of Integrative Structural and Computational Biology , The Scripps Research Institute , 10550 North Torrey Pines Road , La Jolla , California 92037 , United States.
Bridge Institute, Departments of Biological Sciences and Chemistry, Michelson Center , University of Southern California , Los Angeles , California 90089 , United States.
J Am Chem Soc. 2018 Jul 5;140(26):8228-8235. doi: 10.1021/jacs.8b03805. Epub 2018 Jun 20.
Tryptophan indole N-H signals are well separated in nuclear magnetic resonance (NMR) spectra of proteins. Assignment of the indole N-H signals therefore enables one to obtain site-specific information on complex proteins in supramacromolecular systems, even when extensive assignment of backbone N-H resonances is challenging. Here we exploit the unique indole N-H chemical shift by introducing extrinsic tryptophan reporter residues at judiciously chosen locations in a membrane protein for increased coverage of structure and function by NMR. We demonstrate this approach with three variants of the human A adenosine receptor (AAR), a class A G protein-coupled receptor, each containing a single extrinsic tryptophan near the receptor intracellular surface, in helix V, VI, or VII, respectively. We show that the native AAR global protein fold and ligand binding activity are preserved in these AAR variants. The indole N-H signals from the extrinsic tryptophan reporter residues show different responses to variable efficacy of drugs bound to the receptor orthosteric cavity, and the indole N-H chemical shift of the tryptophan introduced at the intracellular end of helix VI is sensitive to conformational changes resulting from interactions with a polypeptide from the carboxy terminus of the Gα intracellular partner protein. Introducing extrinsic tryptophans into proteins in complex supramolecular systems thus opens new avenues for NMR investigations in solution.
色氨酸吲哚 N-H 信号在蛋白质的核磁共振（NMR）谱中很好地分离。因此，吲哚 N-H 信号的分配使人们能够获得超分子系统中复杂蛋白质的位点特异性信息，即使在广泛分配骨干 N-H 共振时具有挑战性。在这里，我们通过在膜蛋白中选择的位置引入外源性色氨酸报告残基，利用独特的吲哚 N-H 化学位移，来增加 NMR 对结构和功能的覆盖范围。我们使用三种人类 A 腺苷受体（AAR）变体来证明这种方法，AAR 是一种 A 类 G 蛋白偶联受体，每个变体在受体细胞内表面的螺旋 V、VI 或 VII 附近分别含有一个外源性色氨酸。我们表明，这些 AAR 变体保留了天然 AAR 的全局蛋白质折叠和配体结合活性。外源性色氨酸报告残基的吲哚 N-H 信号对与受体正位腔结合的药物的变构效力表现出不同的响应，并且引入到螺旋 VI 细胞内末端的色氨酸的吲哚 N-H 化学位移对由于与 Gα 细胞内伴侣蛋白的羧基末端多肽相互作用而导致的构象变化敏感。在复杂的超分子系统中引入外源性色氨酸为溶液中的 NMR 研究开辟了新途径。
