Department of Chemistry, University of Florida, Gainesville, FL 32611, USA.
Biomolecules. 2021 Apr 24;11(5):632. doi: 10.3390/biom11050632.
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 H receptor (HR) in the methylotrophic yeast for NMR experiments. Systematic evaluation of multiple expression parameters resulted in a ten-fold increase in the yield of expressed HR over initial efforts in defined media. The expressed receptor could be purified to homogeneity and was found to respond to the addition of known HR ligands. Two-dimensional transverse relaxation-optimized spectroscopy (TROSY) NMR spectra of stable-isotope labeled HR show well-dispersed and resolved signals consistent with a properly folded protein, and F-NMR data register a response of the protein to differences in efficacies of bound ligands.
G 蛋白偶联受体（GPCRs）结合广泛的细胞外分子，并传递起始生理反应的细胞内信号。GPCR 的信号转导功能与其结构的可塑性固有相关，这可以通过光谱技术进行实验观察。特别是核磁共振（NMR）光谱学是研究 GPCR 动态行为的一种特别有利的方法。NMR 研究的成功关键依赖于包含稳定同位素标记探针的功能性 GPCR 的生产，这对大多数人类 GPCR 来说仍然是一项具有挑战性的工作。我们报告了在甲基营养酵母中生产用于 NMR 实验的人组氨酸 H 受体（HR）的方案。对多个表达参数的系统评估导致表达的 HR 产量比最初在确定的培养基中的努力增加了十倍。表达的受体可以纯化为均相，并被发现对已知的 HR 配体的添加有反应。稳定同位素标记的 HR 的二维横向弛豫优化光谱（TROSY）NMR 谱显示出与正确折叠的蛋白质一致的良好分散和分辨信号，F-NMR 数据记录了蛋白质对结合配体效力差异的响应。
