Wyant College of Optical Sciences, The University of Arizona, Tucson, AZ, 85721, USA.
Department of Biomedical Engineering, The University of Arizona, Tucson, AZ, 85721, USA.
Nat Commun. 2024 Aug 28;15(1):7445. doi: 10.1038/s41467-024-51320-x.
G-protein coupled receptors help regulate cellular function and communication, and are targets of small molecule drug discovery efforts. Conventional techniques to probe these interactions require labels and large amounts of receptor to achieve satisfactory sensitivity. Here, we use frequency-locked optical microtoroids for label-free characterization of membrane interactions in vitro at zeptomolar concentrations for the kappa opioid receptor and its native agonist dynorphin A 1-13, as well as big dynorphin (dynorphin A and dynorphin B) using a supported biomimetic membrane. The measured affinity of the agonist dynorphin A 1-13 to the κ-opioid receptor was also measured and found to be 3.1 nM. Radioligand assays revealed a dissociation constant in agreement with this value (1.1 nM). The limit of detection for the κOR/DynA 1-13 was calculated as 180 zM. The binding of Cholera Toxin B-monosialotetrahexosyl ganglioside was also monitored in real-time and an equilibrium dissociation constant of 1.53 nM was found. Our biosensing platform provides a method for highly sensitive real-time characterization of membrane embedded protein binding kinetics that is rapid and label-free, for drug discovery and toxin screening among other applications.
G 蛋白偶联受体有助于调节细胞功能和通讯，是小分子药物发现努力的目标。探测这些相互作用的传统技术需要标记物和大量的受体才能达到令人满意的灵敏度。在这里，我们使用频率锁定光学微环来对体外的膜相互作用进行无标记表征，浓度低至飞摩尔，用于 κ 阿片受体及其天然激动剂 dynorphin A 1-13，以及使用支持的仿生膜的大 dynorphin (dynorphin A 和 dynorphin B)。还测量了激动剂 dynorphin A 1-13 与 κ-阿片受体的亲和力，发现为 3.1 nM。放射配体测定法显示出与该值一致的离解常数（1.1 nM）。κOR/DynA 1-13 的检测限计算为 180 zM。霍乱毒素 B-单唾液酸四己糖神经节苷脂的结合也被实时监测，发现平衡离解常数为 1.53 nM。我们的生物传感平台提供了一种快速、无标记的方法，用于高度敏感的实时表征嵌入膜的蛋白质结合动力学，可用于药物发现和毒素筛选等应用。
