The Department of Chemistry and the Institute of Nanotechnology and Advanced Materials, Faculty of Exact Sciences, Bar-Ilan University, Ramat-Gan 529002, Israel.
J Phys Chem B. 2024 Sep 19;128(37):8908-8914. doi: 10.1021/acs.jpcb.4c03676. Epub 2024 Sep 4.
electron paramagnetic resonance (EPR) spectroscopy experiments provide high-resolution data about conformational changes of proteins within the cell. However, one of the limitations of EPR is the requisite of stable paramagnetic centers in a reducing environment. We recently showed that histidine-rich sites in proteins hold a high affinity to Cu(II) ions complexed with a chelator. Using a chelator prevents the reduction of Cu(II) ions. Moreover, this spin-labeling methodology can be performed within the native cellular environment on any overexpressed protein without protein purification and delivery to the cell. Herein, we use this novel methodology to gain spatial information on the extracellular domain of the human copper transporter, hCtr1. Limited structural information on the transmembrane domain of the human Ctr1 (hCtr1) was obtained using X-ray crystallography and cryo-EM. However, these structures are missing information on the disordered extracellular domains of hCtr1. Extracellular domains are sensing or interacting with the environment outside of the cell and therefore play an essential role in any transmembrane protein. Especially in hCtr1, the extracellular domain functions as a gating mechanism for copper ions. Here, we performed EPR experiments revealing structural information about the extracellular N-terminal domain of the full-length hCtr1 in vitro and in situ in insect cells and cell membrane fragments. The comparison revealed that the extracellular domains of the in situ and native membrane hCtr1 are further apart than the structure of the purified protein. These method-related differences highlight the significance of studying membrane proteins in their native environment.
电子顺磁共振（EPR）光谱实验提供了关于细胞内蛋白质构象变化的高分辨率数据。然而，EPR 的一个限制是需要在还原环境中稳定的顺磁中心。我们最近表明，蛋白质中的组氨酸丰富位点对与螯合剂配位的 Cu(II)离子具有高亲和力。使用螯合剂可以防止 Cu(II)离子的还原。此外，这种自旋标记方法可以在不进行蛋白质纯化和递送到细胞的情况下，在任何过表达的蛋白质的天然细胞环境中进行。在这里，我们使用这种新的方法学来获得人铜转运蛋白 hCtr1 的细胞外结构域的空间信息。使用 X 射线晶体学和 cryo-EM 获得了人 Ctr1（hCtr1）跨膜结构域的有限结构信息。然而，这些结构缺少 hCtr1 的无序细胞外结构域的信息。细胞外结构域是与细胞外环境感应或相互作用的，因此在任何跨膜蛋白中都起着至关重要的作用。特别是在 hCtr1 中，细胞外结构域作为铜离子的门控机制。在这里，我们进行了 EPR 实验，揭示了全长 hCtr1 的细胞外 N 端结构域在体外和昆虫细胞原位以及细胞膜片段中的结构信息。比较表明，原位和天然膜 hCtr1 的细胞外结构域彼此之间的距离比纯化蛋白的结构更远。这些与方法相关的差异突出了在天然环境中研究膜蛋白的重要性。
