Membrane protein isolation and structure determination in cell-derived membrane vesicles

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 120; no. 18; p. e2302325120
• Main Authors: Tao, Xiao, Zhao, Chen, MacKinnon, Roderick
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 02.05.2023
• Subjects: Binding Sites; Biological Sciences; Calcium ions; Calcium-binding protein; Cell Membrane - metabolism; Cell membranes; Cholesterol; Detergents; Integral membrane proteins; Ion channels; Ion Channels - metabolism; Lipids; Membrane Proteins - metabolism; Membrane vesicles; Membranes; Plasma membranes; Polymers; Protein structure; Proteins; Structural analysis; Vesicles; membrane protein; cell membrane vesicle; ion channel; cryo-EM; proteoliposome
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.2302325120

Integral membrane protein structure determination traditionally requires extraction from cell membranes using detergents or polymers. Here, we describe the isolation and structure determination of proteins in membrane vesicles derived directly from cells. Structures of the ion channel Slo1 from total cell membranes and from cell plasma membranes were determined at 3.8 Å and 2.7 Å resolution, respectively. The plasma membrane environment stabilizes Slo1, revealing an alteration of global helical packing, polar lipid, and cholesterol interactions that stabilize previously unresolved regions of the channel and an additional ion binding site in the Ca 2+ regulatory domain. The two methods presented enable structural analysis of both internal and plasma membrane proteins without disrupting weakly interacting proteins, lipids, and cofactors that are essential to biological function.