In proteins where conformational changes are functionally important, the number of accessible states and their dynamics are often difficult to establish. Here we describe a novel ¹⁹F-NMR spectroscopy approach to probe dynamics of large membrane proteins. We labeled a glutamate transporter homolog with a ¹⁹F probe via cysteine chemistry and with a Ni²⁺ ion via chelation by a di-histidine motif. We used distance-dependent enhancement of the longitudinal relaxation of ¹⁹F nuclei by the paramagnetic metal to assign the observed resonances. We identified one inward- and two outward-facing states of the transporter, in which the substrate-binding site is near the extracellular and intracellular solutions, respectively. We then resolved the structure of the unanticipated second outward-facing state by cryo-EM. Finally, we showed that the rates of the conformational exchange are accessible from measurements of the metal-enhanced longitudinal relaxation of ¹⁹F nuclei.

在构象变化在功能上很重要的蛋白质中，可达到的状态的数量及其动态通常很难确定。在这里，我们描述了一种新颖的¹⁹ F-NMR 光谱方法来探测大膜蛋白的动力学。我们通过半胱氨酸化学用¹⁹ F 探针标记谷氨酸转运蛋白同系物，并通过二组氨酸基序螯合用 Ni ²⁺离子标记。我们利用顺磁性金属对¹⁹ F 核纵向弛豫的距离依赖性增强来分配观察到的共振。我们确定了转运蛋白的一种向内和两种向外状态，其中底物结合位点分别靠近细胞外和细胞内溶液。然后，我们通过冷冻电镜解析了意想不到的第二向外态的结构。最后，我们表明构象交换速率可以通过测量¹⁹ F 核的金属增强纵向弛豫来获得。