What are nanodiscs?

What are nanodiscs?

The term nanodiscs describe a small (7-50 nm in diameter) disc-shapes structure that finds use proteomics and biomedicine. It consists of two main components:
  1. Phosphopeptides, either of artificial origin or from the cell membrane
  2. stabilizer that holds the phosphopeptides together. This is either a MSP protein or a synthetic polymer.
Nanodisc ImageA schematic depiction of a nanodiscs that stabilizes a hypothetical membrane protein.
Green: The stabilizer - A MSP protein in this case
Grey: Phosphopeptides
Orange: Stabilized membrane protein

The purpose of nanodiscs

Their aim is to mimic the native phospholipid bilayer of cells for target molecules (often membrane proteins). Membrane proteins are the key for communication between cells. They mediate fundamental biological processes such as signal transduction, transport processes across membranes, sensing of chemical signals, and coordination of cell–cell interactions.
Numerous diseases in humans are linked to membrane proteins, making them important targets for drug development. So it seems surprising, that membrane proteins are encoded up ∼23% of genes but represent <1 % of known protein structures (1).
stabilisation of membrane proteins with nanodisc
Fig. 1: Membrane proteins have hydrophobic and hydrophilic parts. Nanodiscs make them soluble in aqueous solutions.
The problem is, that membrane proteins, unlike soluble proteins, are difficult to analyze in their native environment, due to their insertion in the lipidic membrane. Surfaces of membrane proteins in the hydrophobic core of the lipid bilayer are also hydrophobic, whereas surface areas in contact with the aqueous membrane environment are as hydrophilic as the surfaces of ordinary soluble proteins (2). The presence of extensive hydrophobic and hydrophilic surface on the same molecule is characteristic for membrane proteins. As a result, membrane proteins are not soluble in standard aqueous buffers without a solubilising agent. For example nanodiscs are required to solubilize them to mimic the amphipathic environment of a lipid bilayer whilst maintaining the structure of the membrane protein in a physiologically relevant state.

Two types of nanodisc

nanodisc cartoonFigure 2: Two types of nanodiscs exist: MSP nanodiscs (green stabilizer) and Synthetic nanodiscs (blue stabilizer)
As mentioned before nanodiscs can be differentiated between their phospholipid composition and most importantly, their type of stabilizer. This stabilizer is the reason why nanodisc in total are split into two main categories: MSP nanodiscs and Synthetic nanodiscs.
The respective names originate from the type of stabilizer that are used to keep the nanodiscs together and form them in the first place. It also decides how the lipid composition of the nanodisc is made up of. MSP nanodiscs always contain an artificial lipid composition. Meaning you have full control of it. In contrast: Synthetic nanodiscs use the native cell phospholipids to create the nanodisc out of. A direct comparison of the individual advantages of both nanodiscs can be found here.
Table 1: Small comparison between MSP and Synthetic nanodiscs
  Synthetic NanodiscMembrane scaffold protein (MSP) Nanodisc
Belt properties / Stabilizer synthetic polymers (e.g. DIBMA, SMA) MSP proteins
Lipids Native cell membrane lipids Artificial phospholipid enviroments (e.g. phospholipids)
Examples Diisobutylene-maleic acid (DIBMA), styrene maleic acid (SMA), amphipols Pre-Assembled nanodiscs
But first, let us introduce both kind of nanodiscs on their own, starting with the MSP nanodiscs.

MSP nanodiscs

MSP nanodiscs
Figure 3: Schematic depition of a MSP nanodisc. Similar to figure 1.
MSP nanodiscs are held together by membrane scaffold proteins (MSPs). MSPs can be truncated truncated forms of apolipoprotein (apo) A-I which wrap around a patch of a lipid bilayer to form a disc-like particle or nanodisc (5). MSPs provide a hydrophobic surface facing the lipids, and a hydrophilic surface at the outside. This setup makes nanodiscs highly soluble in aqueous solutions. Once assembled into nanodiscs, membrane proteins can be kept in solution in the absence of detergents (5).

Size: The size of a MSP nanodisc can range between 7 - 17 nm. It is determined by the used membrane scaffolding protein. Table 2 depicts the membrane scaffold proteins that Cube Biotech offers and which nanodisc sizes it leads to. MSP nanodiscs of the same MSP protein are uniformous in size and only differ +/- 1 nm in diameter. This suits them perfectly for Cryo-EM studies.


Table 2: Overview of MSP proteins and the resulting nanodisc sizes.
MSP typediameter (nm)Ref
MSP1D1ΔH5 8.2 (+/- 0.6) (5)
MSP1D1 9.5 (+/- 1.1) (5)
MSP1E3D1 13 (+/-1) (15)
MSP2N2 17 (+/-1) (21)
Other advantages of MSP nanodiscs
MSP nanodiscs have a number of advantages compared to other systems for membrane protein solubilization and reconstitution, in particular for ligand binding studies, analysis of conformational dynamics, and protein interaction studies (6). Nanodiscs can be used to reconstitute membrane proteins such as GPCRs or transporters in an artificial environment resembling the native membrane.
These nanodisc-stabilized proteins can be directly purified by standard chromatographic procedures. The resulting purified membrane protein-nanodisc complex can be used in applications that require access to both the physiologically intracellular and extracellular surfaces of the protein and thus allows unrestricted access of antagonists, agonists, G proteins and other interaction partners (7).

How to generate MSP nanodisc + protein - complexes

nanodisc assembly
Fig. 4: Schematic image of three ways to reconstitute proteins into nanodiscs. A: Assembled nanodiscs are added to a cell-free reaction. The nascent protein can insert spontaneously. B: Already solubilized membrane protein (orange) is mixed with phospholipids (light gray), detergent (dark gray), and MSP protein (green). Upon detergent removal, the protein-nanodisc complex forms. C: Detergent and MSP are added to membranes expressing the protein of interest. A complex of membrane phospholipids, proteins, and MSP forms.
A: Combining nanodiscs and cell-free expression systems
Starting from an expression plasmid, membrane proteins can be produced in cell-free systems. Pre-assembled nanodiscs are supplied in the mixture that integrate the nascent membrane protein (8). Addition of detergents is not required, which minimizes possible artifacts. Optionally, modifications such as biotinylation or isotope labelling can be included.
B: Two-step reconstitution of detergent-solubilized proteins
Starting from a purified membrane protein in suitable detergent, membrane scaffold proteins and phospholipids are added. Nanodiscs containing the membrane protein form spontaneously, and can be purified by affinity or size exclusion chromatography (6, 7).
C: Direct solubilization from membranes
Starting from membranes expressing the protein of interest, detergent and membrane scaffold protein are added. Membrane phospholipids, membrane protein and MSP assemble to form the nanodisc complex (5). Here, a mixture of nanodisc complexes representing the membrane protein population is obtained, which may be used for proteomics studies. If required, individual membrane protein-nanodisc complexes can be purified by affinity chromatography. Compared to method B, exposure time to detergents is significantly shorter (hours vs. days).

Choice of phospholipids - the key to proper protein activity

As already mentioned the phophopeptide composition of a MSP nanodisc is artificial. Meaning the used phosphopeptides that should make up the artificial membrane environment for the membrane protein of interest must be decided in before. But there are numerous phosphopeptides to choose from out there, so which to choose from? 

Refer to this list of our most commonly used phosphopeptides for MSP nanodiscs, when faced with this question.


Dimyristoyl-glycero-phosphocholine (DMPC) DMPC phosphopeptide structure

Palmitoyl-oleoyl-phosphatidylcholine (POPC) POPC phosphopeptide structure

Phosphatidylglycerol (DMPG)DMPG phosphopeptide structure
This selection, but also many other phospholipids have been successfully used alone or in combination (8,25). The choice of lipids has been shown to be crucial for protein activity (8), for example in cases where lipids promote protein oligomerization (25). Cell-free expression using assembled nanodiscs is a fast and easy way to screen a variety of lipids and lipid mixtures for their effect on the protein. When proteins are solubilized directly from the membrane fraction, endogenous phospholipids are carried along and incorporated into the nanodisc complex, which may enhance protein activity.

Examples for MSP nanodisc applications in science

Nanodiscs provide the perfect environment to stabilize membrane proteins to study binding of ligands, agonists or antagonists by methods such as NMR and SPR (9,10). Nanodiscs were shown to increase resolution of membrane-spanning protein regions in Cryo-EM (22,26). Membrane scaffold proteins can be tagged with histidines to facilitate purification, detection, and immobilization of the protein-nanodisc complex. Other nanodisc applications include resonance Raman (11), MALDI (13), non-covalent mass spectrometry (25), protein activation studies (14), time-resolved fluorescence spectroscopy (15) , and protein crystallization (24). Antigens reconstituted into nanodiscs have been used to raise immunogenic response in mice, showing their potential to be used as vaccines (16). In addition, the entire membrane proteome of E.coli was reconstituted into nanodiscs, thereby creating a solubilized membrane protein library (15). Proteins reconstituted in nanodicscs can be transferred to bicelles to improve NMR resolution (23). Even soluble, lipid-interacting proteins were analyzed with the help of nanodiscs (20). Table 2 lists examples for nanodisc applications.
 
The following consits of publication that include MSP proteins:
 
MSP Nanodiscs were first described by Sligar and coworkers (3,4).

Synthetic nanodiscs

Nanodisc ImageFig, 5: Schematic depiction of a synthetic nanodiscs that stabilizes a hypothetical membrane protein. The figure legend mentions SMA as the used stabilizing polymer, but e.g. DIBMA could fill this role as well.
Synthetic nanodiscs are the second big option in the field of nanodisc. They differ in certain key aspects to their MSP counterparts, but also share certain similarities.

Creation of Synthetic nanodiscs
In contrast to the three creation ways of MSP nanodiscs (figure 4), synthetic nanodiscs can only be created directly from intact cells. The used synthetic polymer has a dual function during this process. First it dissolves the cell membrane, similar to a detergent. Then it form a nanodisc structure around membrane proteins using the native cell phospholipids. A good analog to this process is a cookie cutter that stamp the cookies out of the dough.

Size
Synthetic nanodiscs are variable in their size. The main factor that decides their diameter is the size of the membrane protein complex that they surround & stabilize. Therefore a definitive size cannot be given for a synthetic nanodiscs. But they all range in the size range that can also be found in MSP nanodiscs (table 2). This applies to all established polymers so far. If uniformous nanodisc size is desired for a synthetic nanodiscs complex, a size-exclusion chromatography (SEC) has to be performed after the stabilized membrane protein of interest has been purified through e. g. affinity chromatography with the Rho1D4-tag.
Which polymer? SMA or DIBMA?
SMA and DIBMA comparisonFigure 6: DIBMA and SMA seem to be interchangeable on the first view, but there are some differences.
The question regarding which synthetic polymer is suited better for your specific membrane protein is a tricky one to answer. It mostly comes down to a screening process between the two polymers DIBMA and SMA . A key difference between the two however is their absorbance in a photometer. SMA has a aborbance spectrum that covers 280 nm wavelength and therefore overlaps with the absorbance of proteins.
This interferes with quantification of protein attempts of proteins using a photometer. This problem however does not apply to DIBMA. Its own absorbance spectrum lies far off 280 nm. Because of that protein quantification with DIBMA based synthetic nanodiscs are easiest to perform.
More in depth information regarding SMA and DIBMA individually can be found here:

MSP or Synthetic nanodiscs?

So after all of this the question remains what type of nanodisc best suits your project. Both MSP and synthetic nanodiscs are meant for the solubilization & stabilization of membrane proteins by mimicking a cell-membrane environment. However, as mentioned before, there are some key differences between the two. Table 3 lists all differences and their respective advantages & disadvantages.


Table 3: Direct comparison between MSP and Synthetic nanodiscs.
MSP nanodiscsSynthetic nanodiscs
Size

Depending on used MSP protein. Uniformous (+/-1 nm) for each MSP protein)

Advantage: Uniformous sizes makes MSP nanodiscs perfect tools for applications like Cryo-EM.
Size

Variable, due to different lengths of the polymer chains.

Advantage: The variability of the diameter skips screening steps that are necessary when working with MSP nanodiscs.
Lipid composition

Artificial. Provided by the scientist.

Advantage: The scientist has complete control over the phospholipid composition.
Lipid composition

Made up from native cell membrane lipids.

Advantage: The membrane protein is stabilized in a part of its native environment.
UV absorption

Overlaps with membrane protein due to the presence of the MSP proteins.

Note: Due to the MSP proteins, the nanodisc itself has a UV signal at wavelength at 280 nm and interferes with protein quantification attempts via absorbance.
UV absorption

SMA behaves like MSP nanodiscs, but DIBMA based nanodiscs do not absorb at wavelength 280 nm.

Advantage: With DIBMA based nanodiscs the protein quantity can easily be determined by measuring the absorbance of the solution at wavelength 280 nm.
Creation

Able to be created in 3 different ways (see figure 4).

Advantage: The different situations from which MSP nanodiscs can stabilize membrane proteins make them to go-to option often.
Creation

Only directly from the cell.

Note: Since the synthetic polymers use native cell membrane material to create the nanodiscs, only membrane proteins from living cell material can be stabilized.
Involvement of detergents

Involved in the beginning before the MSP protein form the nanodisc around the protein of interest.

Note: A detergent has to be chosen that does not impact the folded protein's structure. This can result in some extra work.
Involvement of detergents

No detergent necessary.

Advantage: The polymers both act as solbulizers and stabilizers simultaneously. Therefore no additional detergents are needed.




Literature references
  1. Douglas, Shawn M., James J. Chou, and William M. Shih. "DNA-nanotube-induced alignment of membrane proteins for NMR structure determination." Proceedings of the National Academy of Sciences 104.16 (2007): 6644-6648.
  2. Yeates, T. O., et al. "Structure of the reaction center from Rhodobacter sphaeroides R-26: membrane-protein interactions." Proceedings of the National Academy of Sciences 84.18 (1987): 6438-6442.
  3. Bayburt, T.H. et al. Reconstitution and imaging of a membrane protein in a nanometer-size phospholipid bilayer. J. Struct. Biol. (1998), 123(1):37-44
  4. Civjan, N.R. et al. Direct solubilization of heterologously expressed membrane proteins by incorporation into nanoscale lipid bilayers. BioTechniques (2003) 35:556-563
  5. Hagn, F. et al. Optimized phospholipid bilayer nanodiscs facilitate high-resolution structure determination of membrane proteins. J.Am.Chem. Soc. (2013), 135:1919-1925
  6. Serebryany et al. Artificial membrane-like environments for in vitro studies of purified G-protein coupled receptors. Biochim. Biophys. Acta (2012), 181:225-233
  7. Leitz, J. et al. Functional reconstitution of beta2-adrenergic receptors utilizing self-assembling nanodisc technology. BioTechniques (2006), 40:601-612
  8. Proverbio D., et al. Functional properties of cell-free expressed human endothelin A and endothelin B receptors in artifical membrane environments. Biochim.Biophys. Acta (2013), 1828(9):2182-92
  9. Glueck, J.M. et al. Integral membrane proteins in nanodiscs can be studied by solution NMR spectroscopy. J.Am.Chem.Soc. (2009), 131(34):12060-1
  10. Glueck, J.M. et al. Nanodiscs allow the use of integral membrane proteins as analytes in surface plasmon resonance studies. Anal. Biochem. (2011), 408(1):46-52
  11. Mak, P.J. et al. Defining CYP3A4 structural responses to substrate binding. Raman spectroscopic studies of a nanodisc-incorporated mammalian cytochrome P450. J.Am.Chem.Soc. (2011) 133(5):1357-66
  12. Frauenfeld, J. et al. Cryo-EM structure of the ribosome-SecYE complex in the membrane environment. Nature Struct. Mol. Biol. (2011), 5:614-21
  13. Marty M.T., et al. Ultra-thin layer MALDI mass spectrometry of membrane proteins in nanodiscs. Anal. Bioanal. Chem. (2012) 402(2):721-9
  14. Wang, Z. et al. Tyrosine phosphorylation of Mig6 reduces its inhibition of the epidermal growth factor receptor. ACS Chem. Biol. (2013) 8(11):2372-6.
  15. Pandit A., et al. Assembly of the major light-harvesting complex II in lipid nanodiscs. Biophys. J. (2011) 101:2507-2515
  16. Bhattacharya, P. et al. Nanodisc-incorporated hemagglutinin provides protective immunity against influenza virus infection. J. Virology (2010) 361-371
  17. Marty M.T. et al., Nanodisc-solubilized membrane protein library reflects the membrane proteome. Anal. Bioanal. Chem. (2013) 405(12):4009-16
  18. Moers et al., Modified lipid and protein dynamics in nanodiscs. Biochim. Biophys. Acta (2013), 1828(4):1222-9.
  19. Nasr. et al., Radioligand binding to nanodisc-reconstituted membrane transporters assessed by the scintillation proximity assay. Biochemistry (2014), 14;53(1):4-6.
  20. Kobashigawa. et al., Phosphoinositide-incorporated lipid-protein nanodiscs: A tool for studying protein-lipid interactions. Anal. Biochem. 410 (2011), 77-83
  21. Grinkova, Y.V., et. al., Engineering extended membrane scaffold proteins for self-assembly of soluble nanoscale lipid bilayers. Protein Engineering, Design & Selection (2010) 23(11):843-848
  22. Gatsogiannis, C, et. al., Membrane insertion of a Tc toxin in near-atomic detail. Nat. Struct. Mol Biol. (2016) Oct;23(10):884-890.
  23. Laguerre, A. et al. From nanodiscs to isotropic bicelles: A procedure for solution nuclear magnetic resonance studies of detergent-sensitive integral membrane proteins. Structure (2016) 24, 1-12.
  24. Nikolaev, M. et al. Integral membrane proteins can be crystallized directly from nanodiscs. Cryst. Growth Des. (2017) 17(3), 945–948
  25. Henrich, E., et al. Analyzing native membrane protein assembly in nanodiscs by combined non-covalent mass spectrometry and synthetic biology. eLife (2017) 6:e20954.
  26. Gao, Y. et al. TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action. Nature (2016) 534(7607):347-351. doi:10.1038/nature17964
MSP Nanodiscs are protected by US Patents 7,691,414; 7,662,410; 7,622,437; 7,592,008; 7,575,763; 7,083,958; 7,048,949
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