Cheng Chu-Chun, Tsai Ruei-Fong, Lin Che-Kai, Tan Kui-Thong, Kalendra Vidmantas, Simenas Mantas, Lin Chun-Wei, Chiang Yun-Wei
Department of Chemistry, National Tsing Hua University, Hsinchu 300-044, Taiwan.
Faculty of Physics, Vilnius University, Sauletekio 3, LT-10257 Vilnius, Lithuania.
JACS Au. 2024 Sep 24;4(10):3766-3770. doi: 10.1021/jacsau.4c00702. eCollection 2024 Oct 28.
Membrane proteins are integral to numerous cellular processes, yet their conformational dynamics in native environments remains difficult to study. This study introduces a nanodelivery method using nanodiscs to transport spin-labeled membrane proteins into the membranes of living cells, enabling direct in-cell double electron-electron resonance (DEER) spectroscopy measurements. We investigated the membrane protein BsYetJ, incorporating spin labels at key positions to monitor conformational changes. Our findings demonstrate successful delivery and high-quality DEER data for BsYetJ in both Gram-negative and Gram-positive membranes. The delivered BsYetJ retains its ability to transport calcium ions. DEER analysis reveals distinct conformational states of BsYetJ in different membrane environments, highlighting the influence of lipid composition on the protein structure. This nanodelivery method overcomes traditional limitations, enabling the study of membrane proteins in more physiologically relevant conditions.
膜蛋白对于众多细胞过程至关重要，然而它们在天然环境中的构象动力学仍难以研究。本研究引入了一种使用纳米圆盘的纳米递送方法，将自旋标记的膜蛋白转运到活细胞的膜中，从而能够进行直接的细胞内双电子-电子共振（DEER）光谱测量。我们研究了膜蛋白BsYetJ，在关键位置掺入自旋标记以监测构象变化。我们的研究结果表明，BsYetJ在革兰氏阴性和革兰氏阳性膜中均成功递送并获得了高质量的DEER数据。递送的BsYetJ保留了其运输钙离子的能力。DEER分析揭示了BsYetJ在不同膜环境中的不同构象状态，突出了脂质组成对蛋白质结构的影响。这种纳米递送方法克服了传统的局限性，能够在更生理相关的条件下研究膜蛋白。
