Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, Florida 33620, United States.
Nuclear Magnetic Resonance Center, University of Guelph, Guelph, Ontario NIG2W1, Canada.
Bioconjug Chem. 2021 Jan 20;32(1):99-105. doi: 10.1021/acs.bioconjchem.0c00670. Epub 2020 Dec 30.
Conformational dynamics and transitions of biologically active molecules are pivotal for understanding the physiological responses they elicit. In the case of receptor activation, there are major implications elucidating disease mechanisms and drug discovery innovation. Yet, incorporation of these factors into drug screening systems remains challenging in part due to the lack of suitable approaches to include them. Here, we present a novel strategy to probe the GPCR domain rotation by utilizing the fluorine signal variability of a trifluorinated keto-enol (TFKE) chemical equilibrium. The method takes advantage of the high sensitivity of the TFKE tautomerism toward microenvironmental changes resulting from receptor conformational transitions upon ligand binding. We validated the method using the adenosine AR receptor as a model system in which the TFKE was attached to two sites exhibiting opposing motions upon ligand binding, namely, V229C on transmembrane domain VI (TM6) and A289C on TM7. Our results demonstrated that the TFKE switch was an excellent reporter for the domain rotation and could be used to study the conformational transition and dynamics of relative domain motions. Although further studies are needed in order to establish a quantitative relationship between the rotational angle and the population distribution of different components in a particular system, the research presented here provides a foundation for its application in studying receptor domain rotation and dynamics, which could be useful in drug screening efforts.
生物活性分子的构象动力学和转变对于理解它们引起的生理反应至关重要。在受体激活的情况下，阐明疾病机制和药物发现创新具有重要意义。然而，由于缺乏合适的方法将这些因素纳入药物筛选系统，因此在这方面仍然具有挑战性。在这里，我们提出了一种新的策略，通过利用三氟代酮-烯醇（TFKE）化学平衡的氟信号变化来探测 GPCR 结构域的旋转。该方法利用了 TFKE 互变异构体对配体结合引起的受体构象转变过程中微环境变化的高灵敏度。我们使用腺苷 A1 受体作为模型系统验证了该方法，其中 TFKE 连接到两个在配体结合时表现出相反运动的位点，即跨膜域 VI（TM6）上的 V229C 和 TM7 上的 A289C。我们的结果表明，TFKE 开关是结构域旋转的极好报告器，可用于研究构象转变和相对结构域运动的动力学。尽管需要进一步的研究以建立特定系统中旋转角度与不同成分的分布之间的定量关系，但这里提出的研究为其在研究受体结构域旋转和动力学中的应用提供了基础，这在药物筛选工作中可能是有用的。
