SUMMARY Membrane proteins play crucial roles in living organisms. Protein-lipid interactions are crucial for the structural and functional integrity of membrane proteins. High-resolution of membrane protein structures are in high-demand for both understanding their biology and structure-based drug discovery. It is often need to extract membrane proteins from cell membranes for biochemical and biophysical study. Currently detergents are predominantly used for membrane protein extraction. Detergents have significant drawbacks in that they destroy lipid bilayer and remove important lipid molecules from membrane proteins. We are developing a detergent-free native cell membrane nanoparticles system for membrane protein research. The native cell membrane nanoparticles system is composed of three components: 1) Membrane active polymers. 2) Protocols for preparation of native cell membrane nanoparticles. 3) Single-particles cryo-EM analysis of the native cell membrane nanoparticles. We propose to develop a system that can be generally applicable to both prokaryotic and eukaryotic membrane proteins. Specific Aim 1 focuses on construction of membrane active polymer library and develop protocols for preparation of native cell membrane nanoparticles using our selected model membrane proteins. We have developed four prototype membrane active polymers, with the similar strategies we plan to expand the membrane active polymers using both commercially available styrene maleic anhydride copolymers and polymers synthesized in laboratories. Specific Ami 2 focuses on developing protocols for high-resolution structure determination of selected prokaryotic model membrane proteins. Specific Aim 3 focuses on developing protocols for high-resolution structures of selected eukaryotic model membrane proteins. The proposed research is significant because it provides a revolutionary detergent-free approach for study membrane proteins in their local lipid environment. As we have demonstrated in our preliminary data, we found the structural information of protein-lipid interaction for membrane proteins is crucial for understanding the structure and function of membrane proteins. This system might transform the membrane protein research field and have a great impact for membrane protein structure based drug discovery and development.

We have developed a prototype detergent-free native cell membrane nanoparticles system for study membrane proteins in their local native lipid environment. The goal of this proposal is to develop a working native cell membrane nanoparticles system for high-resolution structure determination of membrane proteins in general.