The following material related to Nanodisc Technology is adapted from on-line content of the research group of Professor Stephen Sligar of the University of Illinois at Urbana-Champaign, with the kind permission of Professor Sligar.
The concept of membrane scaffold proteins was originally derived from the naturally occurring apoliporotein A-I component of high-density lipoprotein. Phospholipid bilayer Nanodiscs have been used to incorporate a wide variety of anchored and transmembrane proteins. Nanodiscs can provide improved solubility, homogeneity, and more native conditions over traditional microsomal preparations.
Figure 1.Membrane Scaffold
Membrane proteins have historically been difficult to investigate mechanistically, because many biophysical and chemical methods useful for soluble enzymes work poorly with insoluble protein aggregates. The challenge is to have a membrane protein of interest in a solubilized state for applications such as the following:
Historically, researchers have used detergents to solubilize membrane proteins, to give mixed detergent-protein lipid micelles. However, detergents pose risks to membrane protein stability. Excess micellar phases can also interfere with many assay techniques and often have non-ideal optical properties, like absorbance and light scattering. There can also be undesired partitioning of substrates and products.
Detergents also present technical obstacles during membrane protein manipulation, because detergents often co-concentrate with the protein target and can denature or inactivate those proteins. Furthermore, many membrane protein systems require specific types of phospholipids to maintain active function. Liposome preparations have been used to incorporate membrane proteins. This approach has been useful when compartmentalization of each side of the bilayer is needed, like for assays of ion channels. Liposomes are, however, large, unstable, and difficult to prepare with precisely controlled size and stoichiometry.
Nanodisc technology offers a means to overcome some of these challenges.1-4 To prepare Nanodiscs (soluble nanoscale membrane assemblies), the membrane protein target is solubilized transiently with a detergent in the presence of phospholipids and an encircling amphipathic helical protein belt. This protein belt is referred to as a membrane scaffold protein (MSP). Upon removal of the detergent, typically via adsorption to hydrophobic beads, the target membrane protein simultaneously assembles with phospholipids into a discoidal bilayer. The MSP length controls the eventual Nanodisc size. The membrane protein finds itself in a native membrane environment, where the encircling MSP belt renders the entire assembly soluble.
Nanodisc technology has proven applicable to a wide variety of membrane proteins and protein classes, such as the following:
Our MSP products enable the incorporation of a broad range of target protein sizes. The phospholipid content will vary depending on size of the target protein incorporated in the Nanodisc. The thickness of a Nanodisc can vary depending on the type of phospholipid incorporated (typically 4.6-5.6 nm).
We offer an enhanced variety of MSP products, including MSP constructs with biotin or FLAG tags.
Figure 2.Membrane Scaffold Protein Constructs
Fifteen Membrane Scaffold Protein Constructs are available for applications that require tagging, chemical modifications, and accommodations for various-sized membrane proteins.
Figure 3.Nanodisc Containing 7-TM Protein
Our 15 MSP constructs allow the incorporation of a broad range of target protein sizes, generating Nanodiscs of varying diameters.
The generation of Nanodiscs involves self-assembly of their constituent parts from a detergent micellar state. The constituent parts are as follows:
It is necessary to have enough of the correct detergent to generate this micellar starting state.1 It is likewise extremely critical for successful Nanodisc construction to have the correct stoichiometry of phospholipids (PL), scaffold protein (MSP) and target protein. Different degrees of deviation from the ideal lipid:MSP:target stoichiometry can lead to various problems with Nanodisc formation:
Many problems in the generation of homogeneous Nanodisc structures tend to result from incorrect lipid:MSP ratios. Another notable cause of problems is the use of other amphipathic helical MSP sequences which have not been optimized to produce homogeneous and monodisperse entities.2
When including a detergent-solubilized target protein in the self-assembly mixture, a general problem is that it’s not known at the outset how many PL that the target protein will displace in the assembly. In other words, the net number of PL in the resultant target protein incorporated into the Nanodiscs is not known. Although reasonable estimates are possible for how many lipids are displaced from the bare discs for some systems, like 7-transmembrane (7-TM) proteins, anchored P450s and some other systems, the best general approach is to try 3-5 different PL:MSP:target protein ratios, based on a thorough review of the literature.
One fairly common question relates to protocols that incorporate more than one protein into a Nanodisc. Several references discuss this question with respect to the assembly of bacteriorhodopsin for some phospholipids.3-6 The procedure is facilitated by the use of target proteins which have a purification tag. In general, to incorporate monomeric proteins into Nanodiscs, one strategy is to self-assemble with an excess of PL and MSP over the target. This generates bare discs, and other discs with the target. The latter can then be separated, most ideally by making use of the tag on the target protein.
Another general question of whether Nanodisc technology works for all proteins. It is certainly possible to form Nanodiscs with a great variety of membrane proteins and membrane protein assemblies. However, Nanodisc assembly will not work if the target protein is already in an aggregated soluble state. Such aggregated soluble proteins may probably be inactive, but are otherwise stable in aqueous solution by default. Interestingly, it is possible sometimes to have direct insertion into pre-formed Nanodiscs (e.g. comparably simple anchored proteins like cytochrome P450 reductase and cytochrome b5). However, in general, it is necessary to assemble integral membrane proteins from the detergent-solubilized state.
Lyophilized MSP powder may be reconstituted with sterile water to a concentration of 5 mg/mL. The solution will contain the MSP in 20 mM Tris (pH 7.4), 0.1 M NaCl, and 0.5 mM EDTA. This buffer formulation is a good general “standard buffer” for MSP use. If desired, sodium azide may be added to a final concentration of 0.01% as an antimicrobial. Reconstituted MSP can be kept at 4 °C for up to several days. For long term storage of the lyophilized protein of solutions, we recommend temperatures below -20 °C.
The MSP concentration is determined spectrophotometrically and is reported on the Certificate of Analysis (CofA) for the specific lot of the given product number. Each MSP has its own molar extinction coefficient, reported on its specific Product Information Sheet. Each MSP solution has its own information on its solution formulation components. MSP solution products can be kept at 4 °C for up to several days. For long-term storage of the MSP solution products, storage in aliquots at or below -20 °C is recommended.
Stock PL solutions are prepared in chloroform at 50-100 mM, and are stored long-term at -20 ºC in glass vials (e.g. 4 mL) with PTFE-lined screw caps. The PL concentration of the stock solution is determined by phosphate analysis. A phosphate analysis is available in the separate Supplemental Protocol titled “Procedure for Determination of Total Phosphorus”.
Hamilton syringes are recommended to measure volumes of organic solutions. Plastic (e.g. polypropylene, or PP) tips are at risk of organic solvents partly dissolving the tips, and the dissolved plastic can, in turn, contaminate the lipid solutions. Thus it is not advisable to use plastic pipette tips to dispense PL solutions in organic solvents.
We offer Amberlite® XAD-2 under several product numbers:
The beads should be washed with methanol in advance and thoroughly rinsed with water.
The self-assembly process is started upon removal of the detergent (cholate). Two general processes can be utilized to remove the cholate:
Nanodisc samples are fractionated on a column of Superdex® 200, or a pre-packed column such as a Superdex® 200 10/300 GL column, with the column equilibrated in MSP standard buffer (20 mM Tris (pH 7.4), 0.1 M NaCl, and 0.5 mM EDTA, with 0.01% sodium azide if desired), with a flow rate of 0.5 mL/min.
The upper limit for injection volume is 0.5 mL. Samples are filtered before injection and fractions collected at every minute. A sample chromatogram is shown below:
Figure 4.Sample Chromatogram
Thyroglobulin, from bovine thyroid (17 nM)
Ferritin, from horse spleen (12.2 nM)
Catalase, from bovine liver (10.4 nM)
Albumin, from bovine serum (7.1 nM)
The following is an example of a summary general protocol to incorporate a membrane protein into POPC Nanodiscs:
The general methods behind membrane protein incorporation into Nanodiscs can be summarized as follows:
The important parameters are:
The lipid:MSP ratio depends on the particular lipid and MSP.1,2
The temperature should be close to the lipid melting temperature. For example, with POPC, it is advisable to conduct the assembly on ice. The lipid and detergent concentrations in the reconstitution mixture should be high enough for self-assembly to work properly. The exact value depends on the temperature, the cholate:lipid ratio, and on the presence and concentration of the second detergent. When the cholate:lipid ratio is 2:1, a suggested range of lipid concentration in the final mixture is 7 mM - 18 mM. If it is necessary to perform assembly at lower lipid concentrations, additional cholate should be introduced into the reconstitution mixture, so that the final cholate concentration will be > 14 mM.
It should be noted that the choice and concentration of the secondary detergent are entirely dependent on the membrane protein, and must be determined empirically for every new target. In particular, since it is usually not known in advance how many lipids a particular protein will displace in the fully assembled Nanodisc, titration at various lipid:MSP:target ratios is usually required. In the presence of secondary detergent, larger amounts of beads are often needed. For example, when detergents with low CMC, such as Emulgen 913 or Triton X-100, are used, a suggested range of beads is 0.8-1 g of wet beads per mL of reconstitution mixture.
One general scenario is to use excess MSP relative to target protein, and subsequently to separate Nanodiscs with incorporated membrane protein from "empty" Nanodiscs. One effective combination pairs size-exclusion chromatography (SEC) and affinity chromatography, when the membrane protein has a tag.
It must also be noted that the membrane protein must be soluble in a detergent that can be removed by dialysis and/or hydrophobic bead treatment. For instance, soluble aggregated proteins will not self-assemble into Nanodiscs Because the entire procedure can be done quickly, the target protein does not need to be stable in the detergent for very long time periods (e.g. times for standard protein purification procedures), but the target protein must be initially solubilized so that the process of self-assembly can follow. It is also potentially important to consider the speed of detergent removal in the assembly process, particularly for oligomeric proteins or situations where one wishes to incorporate a complex of membrane proteins. Thus the time of detergent removal, as modulated by quantity and hydrophobic bead treatment protocol, may need to be varied.
Another factor to note is that relatively high concentrations of glycerol interfere with the self-assembly. The final glycerol concentration in the Nanodisc reconstitution mixture should be below 3% It is acceptable, however, to add glycerol to the final Nanodisc sample.
Reagents and Equipment:
10% Ascorbic acid solution:
2.5% Ammonium molybdate(VI) tetrahydrate solution:
Prepare sample tubes:
Digestion of organic sample to inorganic phosphate:
Spectrophotometric analysis of samples:
Alternatively, a 96-well plate and a microplate reader-capable spectrophotometer can be used.
The samples should be treated as chemical waste and collected in an appropriately labeled waste container.
We supply the complete platform of reagents required for Nanodisc assembly and analysis.
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