Nanodiscs

Lipid Nanodiscs

Lipid nanodisc (LND) is an alternative name for the original nanodisc, employed to distinguish them from newer variants described below,⁷ which uses ApoA1-based MSP as the stabilizing agent.

LNDs are made by a self-assembly process from a starting point of detergent-solubilized MSP, membrane protein and lipids of choice (Figure 2), the specific ratio of which can dictate nanodisc size, yield and monodispersity.⁴ Detergent is then removed by the addition of hydrophobic beads. For this approach to be successful, the protein must tolerate and be amenable to detergent solubilization and be stable enough for the somewhat lengthy purification process.

Figure 2: Workflow for lipid nanodisc generation. Membrane proteins are first purified using detergents before combining with MSP and lipids. Detergents are removed with biobeads.

LNDs were the first to be applied to studying membrane proteins⁸ and have a wide variety of applications, including in biophysical and biochemical protein research, high-throughput drug discovery, and as imaging agents.¹ They exhibit many of the advantages of all classes of nanodisc, such as a stable environment for membrane proteins, and access to the protein from both sides of the membrane for the full elucidation of signaling pathways.⁷

Despite their uses, LND applications are more limited than their synthetic counterparts.⁷ Detergents are required during LND generation to extract the membrane protein, meaning that the lipid bilayer composition is selected rather than native.⁷ This selection has the advantage of great control over lipid composition, but may not reflect the true lipid-protein interactions in the endogenous cell membrane. The proteinaceous MSP of LNDs can also interfere with some applications, such as background spectroscopic absorbance.⁹

Synthetic Nanodiscs

Synthetic nanodiscs, sometimes called polymer-encased nanodiscs or native nanodiscs,^7,10 replace the MSP of LNDs with an artificial copolymer, removing the MSP as a source of interference and greatly simplifying the purification process. Styrene maleic acid (SMA) is an amphipathic copolymer that was the first to be used in nanodisc generation,^9,11 but SMA modifications and other copolymers such as diisobutylene/maleic acid (DIBMA) have since been introduced as alternative polymers. These address issues of SMA lipid particles (SMALPs) including nanodisc size, instability at acidic pH and precipitation in the presence of divalent cations.⁷ As with LNDs, synthetic nanodisc size can be varied by altering the polymer to lipid ratio.^12,13

Synthetic nanodiscs share many advantages for researching membrane proteins with LNDs, such as significantly improved stability over other purification methods and access to both sides of the transmembrane protein to detect functional or structural changes following binding of ligands or protein modulators.⁷ One distinct advantage of synthetic nanodiscs is the simplification of nanodisc production (Figure 3). SMA and other copolymers can directly extract proteins from the cell membrane, maintaining the native lipid-protein interface, removing the need for detergents and quickening the process.^4,9 This helps to preserve function and structural conformation of the membrane protein during nanodisc preparation, making them suitable for a whole host of applications.

Figure 3: Workflow for synthetic nanodisc generation. Synthetic nanodiscs containing full-length transmembrane proteins in a phospholipid bilayer are generated directly from native cell membranes. Target nanodiscs can then be affinity-purified.

Research Applications

Nanodiscs are an ideal platform for studying the structure and function of membrane proteins with a wide range of biochemical and biophysical techniques.⁴ Different sizes of proteins or protein complexes can be accommodated by changing the size of the nanodisc with altered lipid to polymer ratios. This control over their composition and size results in a high degree of homogeneity, which is particularly useful for structural studies.

Structural Studies

Structural studies of membrane proteins benefit from several aspects of nanodiscs. A structural investigation ideally reveals not only the physical conformation of the target protein, but also its topology within a lipid bilayer and how it interacts with the lipids around it.⁴ Nanodiscs are well-suited to this, containing proteins within their native membrane environment. High concentrations of protein are often required for structural techniques such as nuclear magnetic resonance (NMR), but this is challenging when working with membrane proteins due to their propensity to aggregate. The protection of hydrophobic domains afforded by the lipid bilayer of nanodiscs prevents this, meaning high concentrations can be achieved without aggregation or high viscosity.

As a result of these traits, nanodiscs are applicable to structural techniques such as:

• Cryo-electron microscopy (cryo-EM)
• NMR
• X-ray crystallography

Functional Studies

Nanodiscs can be used to study the function of membrane proteins thanks to their low viscosity and turbidity in aqueous solution, the access they provide to multiple domains of the protein, and the ability to immobilize the protein without interfering with the natural conformation of the protein.⁴ In single molecule studies, the protein can be immobilized to a solid surface via tags on the scaffold or lipid components, leaving the protein to interact unimpeded with ligands or binding partners.¹⁴ Nanodiscs can also act as sensors by tagging them with a split fluorophore.¹⁵

Nanodiscs are therefore suitable for a number of functional approaches, including:

• Atomic force microscopy
• Linear dichroism optical spectroscopy
• Resonance Raman spectroscopy
• Surface plasmon resonance (SPR)
• Förster resonance energy transfer (FRET)

Clinical and Therapeutic Applications

Drug Screening and Drug Discovery

As with functional studies of membrane proteins, nanodiscs are useful in drug screening approaches due to their maintenance of protein activity, accessibility to ligands (such as small molecules), and the ease of immobilizing them to solid surfaces.¹⁶ Nanodiscs can be used to generate membrane protein libraries from tissue fractions while preserving their activity, which can then be paired with high-throughput screening.¹⁷

Antibody Development

Nanodiscs are also useful when developing and characterizing antibodies that bind to membrane proteins.^18,19 Antibody binding kinetics to membrane proteins can be examined by coating protein-containing nanodiscs on to a plate, and then using SPR to measure on- and off-rates of antibody binding.²⁰ Pure membrane protein may not be feasible to obtain, purify, or keep in a stable and active conformation, all of which are required for standard antibody generation and can be solved by using nanodiscs.

Raising antibodies against membrane proteins benefits from using nanodiscs to prevent aggregation, which can block antigenic sites and lower an immune response. Membrane proteins within nanodiscs can also be used in phage- and yeast-display, a preferable alternative to using detergents.^21,22

Drug Delivery

Novel methods of drug delivery are highly sought after to improve the pharmacokinetics of drugs (what happens to the drug once in the body), target drugs to specific areas, and control drug release. Based on HDLs, which are a natural transportation solution in the blood stream for lipids, proteins and RNA, nanodiscs are an ideal carrier to deliver drugs to specific sites in the body, particularly following surface modification of nanodiscs.^23,24 Nanodiscs can improve the bioavailability of drugs, and load compounds that would otherwise be poorly water soluble. They have also been shown to accumulate more in tumors than other nano-particle delivery systems, in part due to their small size, stability, and long circulation half-life.²⁴

Nanodisc delivery systems have thus far shown promise in a wide-range of therapeutic applications, including treatments for cancer, infectious diseases and neurodegenerative conditions, as well as vaccine development, diagnostic imaging, and specific targeting of drugs to the brain.^23–27