Extrinsic Tryptophans as NMR Probes of Allosteric Coupling in Membrane Proteins: Application to the A(2A) Adenosine Receptor.
Extrinsic Tryptophans as NMR Probes of Allosteric Coupling in Membrane Proteins: Application to the A(2A) Adenosine Receptor.
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外在色氨酸作为膜蛋白变构耦合的NMR探针：应用于A（2a）腺苷受体。
DOI：
10.1021/jacs.8b03805
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发表时间：
2018-07-05
影响因子：
15
通讯作者：
Wüthrich K
中科院分区：
文献类型：
--
作者：
Eddy MT;Gao ZG;Mannes P;Patel N;Jacobson KA;Katritch V;Stevens RC;Wüthrich K
色氨酸吲哚15N-1H信号在蛋白质的核磁共振（NMR）光谱中分离得很好。因此，吲哚15N-1H信号的分配使人们能够获得超大分子系统中复杂蛋白质的位点特异性信息，即使在主干15N-1H共振的广泛分配具有挑战性时也是如此。在这里，我们利用独特的吲哚15N-1H化学位移，在膜蛋白中明智地选择位置引入外源性色氨酸报告残基，以增加核磁共振对结构和功能的覆盖。我们用人类A2A腺苷受体（A2AAR）的三种变体证明了这种方法，A2AAR是一种a类G蛋白偶联受体，每种变体分别在螺旋V， VI或VII中，在受体细胞内表面附近含有单个外源性色氨酸。我们发现在这些A2AAR变体中保留了天然的A2AAR全局蛋白折叠和配体结合活性。来自外源性色氨酸报告残基的吲哚15N-1H信号对与受体正构腔结合的药物的不同疗效表现出不同的响应，并且在螺旋VI的胞内末端引入的色氨酸的吲哚15N-1H化学位移对与GαS胞内伴侣蛋白羧基端多肽相互作用引起的构象变化敏感。将外源性色氨酸引入复杂超分子体系的蛋白质中，为溶液中的核磁共振研究开辟了新的途径。
Tryptophan indole 15N–1H signals are well separated in nuclear magnetic resonance (NMR) spectra of proteins. Assignment of the indole 15N–1H signals therefore enables one to obtain site-specific information on complex proteins in supramacromolecular systems, even when extensive assignment of backbone 15N–1H resonances is challenging. Here we exploit the unique indole 15N–1H 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 A2A adenosine receptor (A2AAR), 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 A2AAR global protein fold and ligand binding activity are preserved in these A2AAR variants. The indole 15N–1H signals from the extrinsic tryptophan reporter residues show different responses to variable efficacy of drugs bound to the receptor orthosteric cavity, and the indole 15N–1H 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 GαS intracellular partner protein. Introducing extrinsic tryptophans into proteins in complex supramolecular systems thus opens new avenues for NMR investigations in solution.
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影响因子：
64.5
作者：
Manglik A;Kim TH;Masureel M;Altenbach C;Yang Z;Hilger D;Lerch MT;Kobilka TS;Thian FS;Hubbell WL;Prosser RS;Kobilka BK
通讯作者：
Kobilka BK
影响因子：
7.3
作者：
Munk C;Isberg V;Mordalski S;Harpsøe K;Rataj K;Hauser AS;Kolb P;Bojarski AJ;Vriend G;Gloriam DE
通讯作者：
Gloriam DE
DOI：
10.1126/science.1215802
发表时间：
2012-03-02
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Liu JJ;Horst R;Katritch V;Stevens RC;Wüthrich K
通讯作者：
Wüthrich K
影响因子：
6.4
作者：
Asada H;Uemura T;Yurugi-Kobayashi T;Shiroishi M;Shimamura T;Tsujimoto H;Ito K;Sugawara T;Nakane T;Nomura N;Murata T;Haga T;Iwata S;Kobayashi T
通讯作者：
Kobayashi T
影响因子：
64.8
作者：
Hino, Tomoya;Arakawa, Takatoshi;Iwanari, Hiroko;Yurugi-Kobayashi, Takami;Ikeda-Suno, Chiyo;Nakada-Nakura, Yoshiko;Kusano-Arai, Osamu;Weyand, Simone;Shimamura, Tatsuro;Nomura, Norimichi;Cameron, Alexander D.;Kobayashi, Takuya;Hamakubo, Takao;Iwata, So;Murata, Takeshi
通讯作者：
Murata, Takeshi