The lipid-dependent structure and function of LacY can be recapitulated and analyzed in phospholipid-containing detergent micelles

Abstract

Membrane proteins play key roles in cellular functions, their activity mainly depending on their topological arrangement in membranes. Structural studies of membrane proteins have long adopted a protein-centric view regarding the determinants of membrane protein topology and function. Several studies have shown that the orientation of transmembrane domains of polytopic membrane proteins with respect to the plane of the lipid bilayer can be largely determined by membrane lipid composition. However, the mechanism by which membrane proteins exhibit structural and functional duality in the same membrane or different membranes is still unknown. Here we show that lipid-dependent structural and functional assessment of a membrane protein can be conducted in detergent micelles, opening the possibility for the determination of lipid-dependent high-resolution crystal structures. We found that the lactose permease purified from Escherichia coli cells exhibiting varied phospholipid compositions exhibits the same topology and similar function as in its membrane of origin. Furthermore, we found several conditions, including protein mutations and micelle lipid composition, that lead to increased protein stability, correlating with a higher yield of two-dimensional crystal formation. Altogether, our results demonstrate how the membrane lipid environment influences membrane protein topology and arrangement, both in native membranes and in mixed detergent micelles.

Monoclonal antibody 4B1 influences the pKa of Glu325 in lactose permease (LacY) from Escherichia coli: evidence from SEIRAS

“…Half of the loaded vesicles were incubated with 4B1 antibody (10 µg affinity‐purified 4B1 AB for 100 µg of total membrane protein) for 30 min at room temperature. Aliquots (2 μL) were diluted 200‐fold into 100 m m KPi (pH 7.5) in the absence (efflux) or presence (exchange) of 10 m m unlabeled lactose, and reactions were terminated by dilution and rapid filtration at a given times [19]. For counterflow measurements, WT RSO vesicles were loaded overnight with ‘cold’ lactose (10 m m final) and incubated afterward with 4B1 antibody for 30 min at room temperature.…”

“…Cys‐scanning mutagenesis of 31 single Cys mutants in helices I, V, VII, VIII X, and XI revealed that 4B1 binding induced a small structural change [18]. The monoclonal antibody 4B1 was also used in studies focusing on the role of lipids in structure and function as it recognizes in a conformation‐specific manner native LacY via the epitope in domain P7 [19].…”

Membrane Protein Stabilization Strategies for Structural and Functional Studies

“…In fact, detergent micelles do not fully resemble the native environment as they remove protein–lipid interactions that could be key for protein function and/or structure [ 82 , 83 ]. Additionally, the removal of specific lipids during purification—a process called delipidation—can severely impair protein stability and function [ 83 , 84 , 85 ]. The structural biology of membrane proteins provides insight into the roles of specific membrane lipids within the protein–lipid environment.…”

“…The lipidic environment modulates membrane protein functionality [ 12 , 82 , 83 ] by molecular interactions between the protein and lipids of the phospholipid bilayer. To answer key questions regarding the requirement of specific phospholipids for optimal activity or stabilization of particular conformations, nanodisc technology provides a controllable membrane environment by regulating the lipid composition with precision [ 96 ].…”

“…This process often destabilizes membrane proteins as they are removed from the native membrane and inserted into a non-native membrane environment. Additionally, functionally critical protein–protein and protein–lipid interactions may be lost during protein purification [ 83 , 84 , 85 ]. Finally, the asymmetry in lipid composition between the inner and the outer leaflets observed in cell membranes is difficult to reproduce in nanodiscs [ 102 ].…”