Yang Limin, Catalano Claudio, Xu Yunyao, Qiu Weihua, Zhang Dongyu, McDermott Ann, Guo Youzhong, Blount Paul
Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9040, United States.
Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA 23298-0540, United States.
BBA Adv. 2021;1. doi: 10.1016/j.bbadva.2021.100011. Epub 2021 Apr 5.
Proteoliposomes mimic the cell membrane environment allowing for structural and functional membrane protein analyses as well as antigen presenting and drug delivery devices. To make proteoliposomes, purified functional membrane proteins are required. Detergents have traditionally been used for the first step in this process However, they can irreversibly denature or render membrane proteins unstable, and the necessary removal of detergents after reconstitution can decrease proteoliposome yields. The recently developed native cell membrane nanoparticles (NCMN) system has provided a variety of detergent-free alternatives for membrane protein preparation for structural biology research. Here we attempt to employ the MCMN system for the functional reconstitution of channels into proteoliposomes. NCMN polymers NCMNP1-1 and NCMNP7-1, members of a NCMN polymer library that have been successful in extraction and affinity purification of a number of intrinsic membrane proteins, were selected for the purification and subsequent reconstitution of three bacterial channels: KcsA and the mechanosensitive channels of large and small conductance (MscL and MscS). We found that channels in NCMN particles, which appeared to be remarkably stable when stored at 4 °C, can be reconstituted into bilayers by simply incubating with lipids. We show that the resulting proteoliposomes can be patched for electrophysiological studies or used for the generation of liposome-based nanodevices. In sum, the findings demonstrate that the NCMN system is a simple and robust membrane protein extraction and reconstitution approach for making high-quality functional proteoliposomes that could significantly impact membrane protein research and the development of nanodevices.
蛋白脂质体模拟细胞膜环境，可用于膜蛋白的结构和功能分析，以及作为抗原呈递和药物递送装置。要制备蛋白脂质体，需要纯化的功能性膜蛋白。传统上，去污剂用于这一过程的第一步。然而，它们可能会使膜蛋白不可逆地变性或变得不稳定，并且在重组后去除去污剂的必要步骤会降低蛋白脂质体的产量。最近开发的天然细胞膜纳米颗粒（NCMN）系统为结构生物学研究中的膜蛋白制备提供了多种无去污剂的替代方法。在这里，我们尝试使用NCMN系统将通道功能性重组到蛋白脂质体中。选择NCMN聚合物库中的成员NCMNP1-1和NCMNP7-1，它们已成功用于多种内在膜蛋白的提取和亲和纯化，用于纯化三种细菌通道并随后进行重组：KcsA以及大电导和小电导机械敏感通道（MscL和MscS）。我们发现，NCMN颗粒中的通道在4℃储存时似乎非常稳定，只需与脂质孵育即可重组到双层膜中。我们表明，所得的蛋白脂质体可用于膜片钳电生理研究或用于生成基于脂质体的纳米装置。总之，这些发现表明，NCMN系统是一种简单而强大的膜蛋白提取和重组方法，可用于制备高质量的功能性蛋白脂质体，这可能会对膜蛋白研究和纳米装置的开发产生重大影响。
