Abstract
Previously we introduced peptidiscs as an alternative to detergents to stabilize membrane proteins in solution (Carlson et al., 2018). Here, we present 'on-gradient' reconstitution, a new gentle approach for the reconstitution of labile membrane-protein complexes, and used it to reconstitute Rhodobacter sphaeroides reaction center complexes, demonstrating that peptidiscs can adapt to transmembrane domains of very different sizes and shapes. Using the conventional 'on-bead' approach, we reconstituted Escherichia coli proteins MsbA and MscS and find that peptidiscs stabilize them in their native conformation and allow for high-resolution structure determination by cryo-electron microscopy. The structures reveal that peptidisc peptides can arrange around transmembrane proteins differently, thus revealing the structural basis for why peptidiscs can stabilize such a large variety of membrane proteins. Together, our results establish the gentle and easy-to-use peptidiscs as a potentially universal alternative to detergents as a means to stabilize membrane proteins in solution for structural and functional studies.
Highlights
Integral membrane proteins make up a third of the human proteome and are the target of many therapeutic drugs, but until recently, determining their structure has been very challenging
Substantial efforts have been devoted to finding alternative ways to stabilize membrane proteins in solution, especially for structural studies, which resulted in the introduction of several novel membrane mimetics, such as amphipols (Diab et al, 2007; Tribet et al, 1998), membrane-scaffold protein (MSP)-based lipid nanodiscs (Civjan et al, 2003; Denisov and Sligar, 2017), saposin A-based Salipro particles (Frauenfeld et al, 2016), and styrene maleic acid co-polymer lipid particles (SMALPs) (Bada Juarez et al, 2019; Morrison et al, 2016), all reviewed in Autzen et al (2019)
Our results demonstrate that peptidiscs can be used to stabilize membrane proteins and membrane-protein complexes with very different structures, that they preserve the native conformation of membrane proteins as well as their interaction with associated lipids, and that they allow for high-resolution structure determination by single-particle cryo-electron microscopy (cryo-EM)
Summary
Integral membrane proteins make up a third of the human proteome and are the target of many therapeutic drugs, but until recently, determining their structure has been very challenging. Detergents are the most commonly used way to stabilize membrane proteins in solution outside a lipid bilayer, but detergents are cause for concern Because of their amphipathic nature, small size and dynamic micellar state, detergents cover the hydrophobic belt of a membrane protein, but may destabilize their fold by disrupting hydrophobic interactions. We show that peptidisc-stabilized membrane proteins (in this case reconstituted with the conventional ‘on-beads’ method) can be used for high-resolution structure determination by single-particle cryo-EM. Our results demonstrate that peptidiscs can be used to stabilize membrane proteins and membrane-protein complexes with very different structures, that they preserve the native conformation of membrane proteins as well as their interaction with associated lipids, and that they allow for high-resolution structure determination by single-particle cryo-EM
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