The Application of Nanodiscs in Membrane Proteins


View:1211 Time:2018-02-01


In the previous chapter, we briefly introduced the basic properties of detergents and their use in membrane proteins. In detergent-containing buffers, most membrane proteins can maintain stability and activity. However, a few detergent-sensitive membrane proteins are not suitable for buffer containing detergents and have features including inability to be purified (eg, the purified membrane proteins with very poor purity and low yields), poor stability (susceptible to degradation or significant precipitation) and no activity (probably the membrane protein is sensitive to detergents or its activity requires the structure of the phospholipid bilayer). For this type of membrane protein, we suggest to use nanodiscs technology. Currently, Nanodiscs technology shows important advantages in membrane protein isolation, purification, structure studies and functional characterization. This article describes the composition and structure of nanodiscs, the comparison of nanodiscs with liposomes, and the application of nanodiscs in membrane proteins. Hope it can help you learn more about membrane proteins through our introduction.

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1. Composition and Structure of Nanodiscs

The main components of Nanodiscs are synthetic phospholipids and amphipathic helical proteins, also known as membrane scaffold proteins (MSPs). Membrane-scaffold proteins are associated with serum apolipoproteins, whereas serum apolipoproteins are the major component of high-density lipoproteins. The two membrane scaffold proteins are ribbon-shaped and surround the phospholipids, as shown in Fig 1 [1], ie, the discoid phospholipid bilayers Nanodiscs.

Fig 1. Model of Nanodiscs structure viewed (a) perpendicular to the bilayer and (b) in the plane of the bilayer,

based on the molecular belt model of discoidal HDL. Two monomers of the membrane scaffold protein (blue and cyan) form an amphipathic helical belt around a segment of phospholipid bilayer (in white)~10 nm in diameter.

The model is courtesy of S. C. Harvey[1].

2. Model Membrane

We mainly compare two models of liposomes and nanodiscs.

2.1 Liposomes

Liposomes are amphipathic lipid bilayer vesicles. There are many ways to prepare liposomes. The best-known and easiest one is the Bangham method [2]. The target lipid is dissolved in a volatile organic solvent and dried under a stream of inert gas. The obtained thin-layer lipid is vortexed with water-soluble buffer and then finally transformed into a monolayer bubble by various methods such as sonication, homogenization and extrusion through a filter of known size (typically 100-200 nm pore size). However, this also results in some obvious disadvantages of liposomes, including instability, inhomogeneity, and difficulty of reproduction.

Liposomes is easy to aggregate and fuse, and is often unstable under operation of long period of time or under certain physical manipulations, such as stopping the flow or mixing vigorously. The size of liposomes prepared by the extruder is not homogeneous, there may be significant differences in the preparation of different batches [3]. Membrane proteins assembled into liposomes often result in cloudy and viscous samples, which may limit the use of some biochemical analytical techniques.

2.2 Nanodiscs

The preparation of nanodiscs involves preparation of master mix containing phospholipid, membrane scaffold protein and detergent, and the remaining components are assembled as a discoid phospholipid bilayer 8-16 nm in diameter, namely nanodiscs [4], upon removal of the detergent. The diameter of Nanodiscs is determined by the length of the MSP band and when the phospholipid is in the optimum molar ratio to MSP, uniform nanodiscs are formed.

Nanodiscs are more homogeneous (in one preparation) and consistent (in different batches of preparation) than liposomes; nanodiscs have more precise particle size and can be sized by adjusting the length of MSP sequences; nanodiscs is more stable.

3. Application of Nanodiscs in Membrane Proteins

For the application of Nanodiscs, there is an outstanding review [5]. The structural studies can be found in the following table, which can be applied to structural biology and various technologies such as electron microscopy, NMR, EPR and spectroscopy detection.

Table 1. Methods and tools used with MPs incorporated into nanodiscs


SPR: Surface plasmon resonance; LSPR: localized SPR; CPR: NADPH–cytochrome P450 reductase[5].

4. AQPs are Expressed Using In Vitro E.coli Protein Expression and Nanodiscs Technology

Cusabio incorporates both in vitro E.coli protein expression and nanodiscs technology, adding pre-assembled nanodiscs to in vitro E.coli protein expression systems. The membrane proteins are assembled into nanodiscs while they are translated, forming membrane protein-nanodiscs complexes (Fig. 2). This simulated artificial lipid environment provides new avenues for analyzing the effects of different lipids on membrane proteins, allowing us to better understand membrane proteins.

Fig. 3. Results of SDS-PAGE after successfully assembly of nanodiscs into Escherichia coli Aquaporin Z (aqpZ). As shown in figure 2, there are two bands, one for MSP and one for the target protein of membrane protein aqpZ.

Fig 2. Schematic illustration of a MSP nanodiscs with a 7-transmembrane protein embedded.

Diameter is about 10.6 nm. Picture from Sligar.

Fig 3.

5. Cusabio’s New Strategy for Membrane Protein Expression

Cusabio provides risk-free services for membrane protein expression (detergent preparation) and nanodics assembly for proteins under 300aa. Welcome to contact us !

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CUSABIO TECHNOLOGY (US)

Address: 8400 Baltimore Avenue, Suite 332. College Park, MD 20740

Tel: 301-363-4651 (Available from 9 a.m. to 5 p.m. EST from Monday to Friday)

Email: info@flarebio.com

Web: www.cusabio.com


Past Veview

The Nature of Detergent and Its Application in Membrane Proteins

References

[1]     Applications of Phospholipid Bilayer Nanodiscs in the Study of Membranes and Membrane Proteins. Abhinav Nath, William M. Atkins, and Stephen G. Sligar. Biochemistry, 2007, 46 (8), 2059-2069

[2]     Large volume liposomes by an ether vaporization method. D Deamer, AD Bangham. Biochimica et Biophysica Acta, 443 (1976) 629-~34

[3]     Applications of Phospholipid Bilayer Nanodiscs in the Study of Membranes and Membrane Proteins. Abhinav Nath, William M. Atkins, and Stephen G. Sligar. Biochemistry, 2007, 46 (8), 2059-2069

[4]     Self-assembly of discoidal phospholipid bilayer nanoparticles with membrane scaffold proteins. Timothy H. Bayburt,Yelena V. Grinkova, and Stephen G. Sligar. Nano Letters, 2002, 2 (8), pp 853–856

[5]     Nanodiscs for structural and functional studies of membrane proteins.Ilia G Denisov   & Stephen G Sligar. Nature Structural & Molecular Biology 23, 481–486 (2016)


 

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