Zhang Yan, Gan Yuefang, Zhao Weijing, Zhang Xuning, Zhao Yongxiang, Xie Huayong, Yang Jun
National Center for Magnetic Resonance in Wuhan, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Wuhan National Laboratory for Optoelectronics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, Wuhan 430071, P. R. China.
University of Chinese Academy of Sciences, Beijing 100049, P. R. China.
JACS Au. 2023 Nov 21;3(12):3412-3423. doi: 10.1021/jacsau.3c00564. eCollection 2023 Dec 25.
The structural characterization of membrane proteins within the cellular membrane environment is critical for understanding the molecular mechanism in their native functional context. However, conducting residue site-specific structural analysis of membrane proteins in native membranes by solid-state NMR faces challenges due to poor spectral sensitivity and serious interference from background protein signals. In this study, we present a new protocol that combines various strategies for cellular membrane sample preparations, enabling us to reveal the secondary structure of the mechanosensitive channel of large conductance from (MscL) in inner membranes. Our findings demonstrate the feasibility of achieving complete resonance assignments and the potential for determining the 3D structures of membrane proteins within cellular membranes. We find that the use of the BL21(DE3) strain in this protocol is crucial for effectively suppressing background protein labeling without compromising the sensitivity of the target protein. Furthermore, our data reveal that the structures of different proteins exhibit varying degrees of sensitivity to the membrane environment. These results underscore the significance of studying membrane proteins within their native cellular membranes when performing structural characterizations. Overall, this study opens up a new avenue for achieving the atomic-resolution structural characterization of membrane proteins within their native cellular membranes, providing valuable insights into the nativeness of membrane proteins.
在细胞膜环境中对膜蛋白进行结构表征对于理解其天然功能背景下的分子机制至关重要。然而，由于光谱灵敏度差以及背景蛋白信号的严重干扰，通过固态核磁共振对天然膜中的膜蛋白进行残基位点特异性结构分析面临挑战。在本研究中，我们提出了一种新方案，该方案结合了多种细胞膜样品制备策略，使我们能够揭示内膜中大电导机械敏感通道（MscL）的二级结构。我们的研究结果证明了实现完全共振归属的可行性以及确定细胞膜中膜蛋白三维结构的潜力。我们发现，在该方案中使用BL21(DE3)菌株对于有效抑制背景蛋白标记而不影响目标蛋白的灵敏度至关重要。此外，我们的数据表明不同蛋白质的结构对膜环境表现出不同程度的敏感性。这些结果强调了在进行结构表征时在其天然细胞膜内研究膜蛋白的重要性。总体而言，本研究为在其天然细胞膜内实现膜蛋白的原子分辨率结构表征开辟了一条新途径，为膜蛋白的天然性提供了有价值的见解。
