Characterization of the dynamic conformational changes in membrane protein signaling complexes by nuclear magnetic resonance (NMR) spectroscopy remains challenging. Here we report the site-specific incorporation of 4-trimethylsilyl phenylalanine (TMSiPhe) into proteins, through genetic code expansion. Crystallographic analysis revealed structural changes that reshaped the TMSiPhe-specific amino-acyl tRNA synthetase active site to selectively accommodate the trimethylsilyl (TMSi) group. The unique up-field ¹H-NMR chemical shift and the highly efficient incorporation of TMSiPhe enabled the characterization of multiple conformational states of a phospho-β2 adrenergic receptor/β-arrestin-1(β-arr1) membrane protein signaling complex, using only 5 μM protein and 20 min of spectrum accumulation time. We further showed that extracellular ligands induced conformational changes located in the polar core or ERK interaction site of β-arr1 via direct receptor transmembrane core interactions. These observations provided direct delineation and key mechanism insights that multiple receptor ligands were able to induce distinct functionally relevant conformational changes of arrestin.

通过核磁共振（NMR）光谱表征膜蛋白信号复合物的动态构象变化仍然具有挑战性。在这里，我们报告了通过遗传密码扩展将 4-三甲基硅烷基苯丙氨酸 (TMSiPhe) 定点掺入蛋白质中。晶体分析揭示了结构变化，重塑了 TMSiPhe 特异性氨酰 tRNA 合成酶活性位点，以选择性地容纳三甲基甲硅烷基 (TMSi) 基团。独特的上场¹ H-NMR 化学位移和 TMSiPhe 的高效掺入，仅使用5 μM 蛋白质和 20 分钟光谱积累时间。我们进一步表明，细胞外配体通过直接受体跨膜核心相互作用诱导位于β-arr1的极性核心或ERK相互作用位点的构象变化。这些观察结果提供了直接描述和关键机制见解，即多个受体配体能够诱导视紫红质抑制蛋白的不同功能相关构象变化。