Self‐Assembly of Protein‐Containing Lipid‐Bilayer Nanodiscs from Small‐Molecule Amphiphiles

Abstract

native environment while hydrophobic surfaces need to be shielded from the aqueous solution by a membrane mimic. Traditionally, detergents have been applied to extract and purify membrane proteins, although they mimic the native environment only poorly and often lead to denaturation and loss of function. [1] Lipid-bilayer nanodiscs are excellent tools for studying membrane proteins under native-like yet well-controlled in vitro conditions. Such nanodiscs encapsulate membrane proteins in a nanosized membrane patch that, in spite of its small size, provides a nativelike lipid environment. [2,3] All nanodiscs have in common that their lipid-bilayer core is surrounded by a belt composed of amphiphilic molecules that serve to shield the lipid acyl chains at the rim of the patch from contact with water. Yet, different types of nanodiscs drastically differ from one another in terms of their ability to selfassemble and their dynamics once formed. On the one hand, nanodiscs encapsulated by membrane scaffold proteins (MSPs) are kinetically trapped, static structures that require solubilization by conventional detergents before the latter are removed to drive nanodisc assembly. [4] On the other hand, both bicelles ("bilayered micelles") made from certain lipid mixtures [5] as well When membrane proteins are removed from their natural environment, the quality of the membrane-solubilizing agent used is critical for preserving their native structures and functions. Nanodiscs that retain a lipid-bilayer core around membrane proteins have attracted great attention because they offer a much more native-like environment than detergent micelles. Here, two smallmolecule amphiphiles with diglucose headgroups and either a hydrocarbon or a fluorocarbon hydrophobic chain are shown to directly assemble lipids and membrane proteins to form native nanodiscs rather than mixed micelles. Selfassembly of nanodiscs of increasing complexity from both defined, artificial vesicles as well as complex, cellular membranes is demonstrated. A detailed investigation of bilayer integrity and membrane-protein activity in these nanodiscs reveals gentle effects on the encapsulated bilayer core. The fluorinated amphiphile appears particularly promising because its lipophobicity results in gentle, non-perturbing interactions with the nanoscale lipid bilayer. A sequential model of nanodisc self-assembly is proposed that proceeds through perforation of the original membrane followed by saturation and complete solubilization of the bilayer. On this basis, pseudophase diagrams are established for mixtures of lipids and nanodisc-forming diglucoside amphiphiles, and the latter are used for the extraction of a broad range of membrane proteins from cellular membranes.

Cryo-Electron Microscopy Snapshots of Eukaryotic Membrane Proteins in Native Lipid-Bilayer Nanodiscs

“…The same limitation also applies to in-cell cryo-electron tomography, where biomolecules within or proximal to membranes can have similar physical properties. However, advances in detergent-free nanodisc preparation, in conjunction with complementary advances in cryo-FIB SEM technologies (e.g., plasma milling), direct electron detectors, , energy filters, , and AI-based image analysis might alleviate these challenges and eventually distinguish similarly shaped particles to achieve higher resolution. Artificial intelligence tools for structure prediction, such as AlphaFold, in particular for membrane–protein structures, can be used as a hypothesis to annotate observed structural signatures in the future, introducing AI-based structural “purification” within cryo-EM images and tomograms.…”

Tailoring Butyl Methacrylate/Methacrylic Acid Copolymers for the Solubilization of Membrane Proteins: The Influence of Composition and Molecular Weight

“…[41] Finally, new amphiphilic small molecules (nonpolymeric but of higher MW) for membrane protein solubilization have been introduced recently, further blurring the boundary between the originally used detergents and the newly developed (mostly low-MW) polymers. [42,43] Despite the considerable progress made, the new copolymers still share some of the drawbacks of SMA mentioned above, e.g., they incorporate the potentially problematic styrenic or maleic acid moieties or show broad MW distribution. The importance of the latter parameter is being increasingly recognized because the MW (or chain length) of the copolymer is known to impact on the material's membrane solubilization efficiency and stability of the formed nanodiscs.…”

“…[ 41 ] Finally, new amphiphilic small molecules (nonpolymeric but of higher MW) for membrane protein solubilization have been introduced recently, further blurring the boundary between the originally used detergents and the newly developed (mostly low‐MW) polymers. [ 42,43 ]…”

Applications of Synthetic Polymer Discoidal Lipid Nanoparticles to Biomedical Research

“…Even small-molecule amphiphiles have been found to directly assemble lipids and membrane proteins to form native nanodisks. 66) These results could prove inspirational for novel polymer design. Although such recent pioneering works on scaffolding molecules are generally for structural studies of membrane proteins, these nanodisks would change the drug loading capability or the in vivo metabolic pathway as delivery vehicles.…”