What are nanodiscs?

Nanodiscs were first described by Sligar and coworkers (1,2). Nanodiscs provide a phospholipid bilayer system held together by membrane scaffold proteins (MSPs). MSPs are truncated forms of apolipoprotein (apo) A-I which wrap around a patch of a lipid bilayer to form a disc-like particle or nanodisc (3). 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.

 

The resulting nanobilayer particles are about 7-17 nm in diameter, depending on the mutation variant of MSP used (see Table 1). Most widely employed are MSP1D1 and MSP1D1-deltaH5, but also other deletion mutants of MSP1D1 are suitable for the generation of nanodiscs (3).

 

        

 

Schematic image of a membrane protein reconstituted into a nanodisc containing membrane scaffold protein (MSP), phospholipids, and the protein of interest.

Fig. 1: Schematic image of a membrane protein reconstituted into a nanodisc.
Green: Membrane scaffold protein (MSP), Gray: Phospholipids, Orange: Reconstituted membrane protein.

In addition, larger scaffold protein variants named MSP2N2 and MSP2N3 have been created (19). Membrane scaffold proteins derived from mouse and rat apo A-I protein instead of the human homologue improve antibody specificity when human target protein-nanodisc complexes are used for immunization.

 

Why nanodiscs?

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 (4). 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 (5).

 

How to generate membrane-protein:nanodisc complexes

Reconstitution of proteins into nanodiscs
 
Fig. 2: 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 (6). 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 (4,5).


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 (3). 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).

 

Protein activity: Choice of phospholipids

Most commonly used phospholipids are dimyristoyl-glycero-phosphocholine (DMPC) or palmitoyl-oleoyl-phosphatidylcholine (POPC), but many other phospholipids have been successfully used alone or in combination (6,23). The choice of lipids has been shown to be crucial for protein activity (6), for example in cases where lipids promote protein oligomerization (23). 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, endogeneous phospholipids are carried along and incorporated into the nanodisc complex, which may enhance protein activity.

 

Nanodisc applications

Nanodiscs provide the perfect environment to stabilize membrane proteins to study binding of ligands, agonists or antagonists by methods such as NMR and SPR (7,8). Nanodiscs were shown to increase resolution of membrane-spanning protein regions in cryo-EM (20,24). 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 (9), MALDI (11), non-covalent mass spectrometry (23),  protein activation studies (12), time-resolved fluorescence spectroscopy (13) , and protein crystallization (22). Antigens reconstituted into nanodiscs have been used to raise immunogenic response in mice, showing their potential to be used as vaccines (14). 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 (21). Even soluble, lipid-interacting proteins were analyzed with the help of nanodiscs (18). Table 2 lists examples for nanodisc applications.

 

Getting started with nanodiscs

Cube Biotech offers a variety of lyophilized membrane scaffold proteins and phospholipids along with detailed protocols to assemble your own nanodiscs.

For even higher reproducibility, pre-assembled and quality controlled nanodiscs are available in different diameters (see Table 1), and with different phospholipid compositions.

 

Get started with nanodiscs.

 

 

      

 

MSP type

 

diameter (nm)

Ref.
  MSP1D1dH5 8.2 (+/- 0.6) (3)
  MSP1D1 9.5 (+/- 1.1) (3)
  MSP1E3D1 13 (+/-1) (13)
  MSP2N2 16 (+/-1) (19)
Table 1. Overview of nanodisc sizes.

 

 

Examples for nanodisc applications

           
Application   Protein studied  

Origin

  Reference
Agonist/antagonist binding
and radiolabel exchange in G proteins
 
beta andrenergic receptor 2
 
human
  (5)
Radioligand binding /
scintillation proximity assay
 
LeuT transporter
 
bacteria
  (17)
Surface plasmon resonance (SPR)
 
CD4 mutant
 
human
  (8)
Resonance Raman spectroscopy
 
cytochrome P450
 
mammalian
  (9)
Time-resolved fluorescence spectroscopy
 
light harvesting complex II (LHCII)
 
spinach
  (13)
Matrix assisted laser desorption/ionization
mass spectrometry (MALDI-MS)
  various   various   (11,15)
Non-covalent mass spectrometry (LILBID-ESI-MS)
 
KcsA LspA, EmrE, Hv1, proteorhodopsin, MraY
 
human bacteria
  (23)
Electron microscopy (EM)
 
light harvesting complex II (LHCII)
 
spinach
  (13)
Cryo electron microscopy (EM)
 
cySecYEG complex
TcdA1 toxin complex
TRPV1 tetramer
 
bacteria bacteria human
  (10,20,24)
Mouse vaccination
 
hemagglutinin (HA)
 
influenza
virus
  (14)
Nuclear magnetic resonance (NMR)
 
OmpX
CD4 mutant
 
human bacteria
  (3,7)
Solid state NMR  

green proteorhodopsin

 
marine
bacteria
  (16)
NMR analysis of lipid-interacting,
soluble proteins
 
phospho-inositol
binding proteins
 
human
  (18)
Transfer into bicelles for NMR
 
lipoprotein signal peptidase II (LspA)
   human   (21)
In meso phase crystallization
 
bacteriorhodopsin
(BR)
 
archae-
bacteria
  (22)
Protein phosphorylation / activation studies
 
epidermal growth factor
receptor (EGFR)
 
human
  (12)
Table 2. Examples for applications successfully performed with membrane proteins reconstituted into nanodiscs.

 

 

Literature references

1.
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
2.
Civjan, N.R. et al. Direct solubilization of heterologously expressed membrane proteins by incorporation into nanoscale lipid bilayers. BioTechniques (2003) 35:556-563
3.
Hagn, F. et al. Optimized phospholipid bilayer nanodiscs facilitate high-resolution structure determination of membrane proteins. J.Am.Chem. Soc. (2013), 135:1919-1925
4.
Serebryany et al. Artificial membrane-like environments for in vitro studies of purified G-protein coupled receptors. Biochim. Biophys. Acta (2012), 181:225-233
5.
Leitz, J. et al. Functional reconstitution of beta2-adrenergic receptors utilizing self-assembling nanodisc technology. BioTechniques (2006), 40:601-612
6.
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
7.
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
8.
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
9.
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
10.
Frauenfeld, J. et al. Cryo-EM structure of the ribosome-SecYE complex in the membrane environment. Nature Struct. Mol. Biol. (2011), 5:614-21
11.
Marty M.T., et al. Ultra-thin layer MALDI mass spectrometry of membrane proteins in nanodiscs. Anal. Bioanal. Chem. (2012) 402(2):721-9
12.
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.
13.
Pandit A., et al. Assembly of the major light-harvesting complex II in lipid nanodiscs. Biophys. J. (2011) 101:2507-2515
14.
Bhattacharya, P. et al. Nanodisc-incorporated hemagglutinin provides protective immunity against influenza virus infection. J. Virology (2010) 361-371
15.
Marty M.T. et al., Nanodisc-solubilized membrane protein library reflects the membrane proteome. Anal. Bioanal. Chem. (2013) 405(12):4009-16
16.
Moers et al., Modified lipid and protein dynamics in nanodiscs. Biochim. Biophys. Acta (2013), 1828(4):1222-9.
17.
Nasr. et al., Radioligand binding to nanodisc-reconstituted membrane transporters assessed by the scintillation proximity assay. Biochemistry (2014), 14;53(1):4-6.
18.
Kobashigawa. et al., Phosphoinositide-incorporated lipid-protein nanodiscs: A tool for studying protein-lipid interactions. Anal. Biochem. 410 (2011), 77-83
19.
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
20.
Gatsogiannis, C, et. al., Membrane insertion of a Tc toxin in near-atomic detail. Nat. Struct. Mol Biol. (2016) Oct;23(10):884-890.
21.
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.
22.
Nikolaev, M. et al. Integral membrane proteins can be crystallized directly from nanodiscs. Cryst. Growth Des. (2017) 17(3), 945–948
23.
Henrich, E., et al. Analyzing native membrane protein assembly in nanodiscs by combined non-covalent mass spectrometry and synthetic
biology. eLife (2017) 6:e20954.
24.
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
 

 

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