Production of a Human Histamine Receptor for NMR Spectroscopy in Aqueous Solutions.
Production of a Human Histamine Receptor for NMR Spectroscopy in Aqueous Solutions.
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DOI：
10.3390/biom11050632
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发表时间：
2021-04-24
期刊：
影响因子：
5.5
通讯作者：
Eddy MT
中科院分区：
文献类型：
--
作者：
Mulry E;Ray AP;Eddy MT
G蛋白偶联受体（GPCR）结合广泛的细胞外分子并传递启动生理反应的细胞内信号。GPCR的信号转导功能与其结构可塑性内在相关，这可以通过光谱技术在实验上观察到。核磁共振（NMR）光谱特别是研究GPCR的动态行为的特别有利的方法。NMR研究的成功关键取决于含有稳定同位素标记探针的功能性GPCR的产生，这对于大多数人类GPCR来说仍然是一项具有挑战性的奋进。我们报告了在甲醇营养型酵母巴斯德毕赤酵母中生产人组胺H1受体（H1 R）的NMR实验方案。多个表达参数的系统评价导致在确定的培养基中表达的H1 R的产量比最初的努力增加了10倍。表达的受体可以被纯化至同质，并被发现响应于已知的H1 R配体的添加。稳定同位素标记的H1 R的二维横向弛豫优化光谱（TROSY）NMR光谱显示出与正确折叠的蛋白质一致的良好分散和分辨的信号，19 F-NMR数据记录了蛋白质对结合配体功效差异的响应。
G protein-coupled receptors (GPCRs) bind a broad array of extracellular molecules and transmit intracellular signals that initiate physiological responses. The signal transduction functions of GPCRs are inherently related to their structural plasticity, which can be experimentally observed by spectroscopic techniques. Nuclear magnetic resonance (NMR) spectroscopy in particular is an especially advantageous method to study the dynamic behavior of GPCRs. The success of NMR studies critically relies on the production of functional GPCRs containing stable-isotope labeled probes, which remains a challenging endeavor for most human GPCRs. We report a protocol for the production of the human histamine H1 receptor (H1R) in the methylotrophic yeast Pichia pastoris for NMR experiments. Systematic evaluation of multiple expression parameters resulted in a ten-fold increase in the yield of expressed H1R over initial efforts in defined media. The expressed receptor could be purified to homogeneity and was found to respond to the addition of known H1R ligands. Two-dimensional transverse relaxation-optimized spectroscopy (TROSY) NMR spectra of stable-isotope labeled H1R show well-dispersed and resolved signals consistent with a properly folded protein, and 19F-NMR data register a response of the protein to differences in efficacies of bound ligands.
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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
影响因子：
64.5
作者：
Huang, Shuya Kate;Pandey, Aditya;Duy Phuoc Tran;Villanueva, Nicolas L.;Kitao, Akio;Sunahara, Roger K.;Sljoka, Adnan;Prosser, R. Scott
通讯作者：
Prosser, R. Scott
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：
2.7
作者：
Liu, Dongsheng;Wuthrich, Kurt
通讯作者：
Wuthrich, Kurt