Nanodiscs and solution NMR: preparation, application and challenges

1 Introduction

Membrane proteins act like a conduit, communicating the internal of a cell with its outer environment. They make up for roughly 30% of the eukaryotic genome [1] and 50% of all drug targets [2]. Membrane proteins have long been the focal point of both academic and pharmaceutical research. Not surprisingly, their dysregulation, misfolding, and/or mutations have been associated with numerous diseases [3], [4]. Despite their importance and relative abundance, very few have been structurally and functionally characterized, which reflects through the poor representation (less than 3%) of their available structures in the protein database. Membrane proteins are notorious for the difficulties associated with their overexpression, purification, low yield and stability. The inability to obtain protein samples which are stable, pure and in large quantities for X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo electron microscopy (cryo-EM) greatly hampers the structural elucidation of these elusive proteins. Initial success in isolating membrane proteins was offered through the use of detergents, which helped in the biochemical investigation of several proteins. Detergents, however, do not provide an ideal solvent condition mimicking a “native like” environment for membrane proteins [5] and may not be optimal for studying signaling across the membrane as they may lead to the unfolding of the soluble interacting partners. Thus, there stands a need for a proper membrane mimetic that stably houses membrane proteins and makes them amenable to in vitro investigations.

Solution NMR has been successfully used to study membrane proteins solubilized in different membrane mimicking systems, including organic solvent mixtures, amphipols, micelles, and bicelles [6], [7]. These media, though useful, present themselves with a number of caveats, including surface curvature artifacts, limited diversity of detergent or lipid molecules, heterogeneity of the sample preparation, and a debilitating inability to study any interaction with their soluble binding partners. These problems were remedied by the introduction of nanodiscs, a class of soluble membrane mimetic which provided a stable lipid bilayer system close to a native membrane environment. The success of nanodisc has been vividly exemplified through the biophysical characterization of several receptors, enzymes, channels, and transporters [8], [9]. In this review, we discuss the application of nanodiscs to solution NMR. We also provide interesting strategies for incorporating membrane proteins into nanodisc along with discussing other potential alternative nanoscale phospholipid bilayer systems. Finally, we present a previously unpublished original study of looking at the activation of a cell surface receptor, integrin, encapsulated within nanodisc. All together, we hope to make a compelling argument towards the use of nanodisc in NMR and provide a lingering excitement that encourages researchers to appreciate and indulge in the use of this attractive system.

2 What are nanodiscs?

Nanodiscs are discoidal nanoscale lipoprotein complexes that are composed of a phospholipid bilayer held together by two anti-parallel strips of amphipathic helical membrane scaffold protein (MSP) (Figure 1A). Nanodiscs were originally developed by Sligar and colleagues in the late nineties [12], [13], [14], [15]. MSP is derived from apolipoprotein A-1 (apoA-1), which normally functions to transport cholesterol from the tissue cells to the liver for excretion. The concept of a disc-shaped structure came from the very nature of apolipoprotein A-1 which forms nascent discoidal particles by utilizing phospholipids from the serum. These phospholipid particles later on ingests cholesterol from the tissue cells with the help of transporter ABCA1, growing from a discoidal to a spherical form and eventually ending up in the liver [16]. Full length apoA-1 is a helical protein with an N-terminal four helix bundle and two C-terminal helices (PDB: 2A01). An N-terminal deletion construct apoA-1 (Δ1–43) yielded a structure that was arranged in a circular belt of amphipathic helices (PDB: 1AV1) [17], which led to the advent of the present nanodisc technology. This deletion construct consisting of 10 α-helical repeats was referred to as MSP1, which (Figure 1C: bottom panel) formed a belt encompassing lipid molecules. Another larger construct MSP2 was designed by joining two MSP1 molecules through a stable linker. MSP1, MSP2 [13], and other variants are broadly referred to as MSP. The variants are denoted by a letter and a number that follows the parent MSP1 or MSP2 from which they are derived through either deletions or insertions of helices. The presence of a protein belt constrains the dimensions of the bilayer, trapping lipid molecules within its central core. This way, the system ensures a monodisperse size distribution. MSP variants differ in the size of their total length resulting in discs of different diameters. MSP with varying length in permutation with different types of lipid molecules, including DMPC (dimyristoyl-phosphocholine), dimyristoyl-phosphorylglycerol, POPC (palmitoyl-oleoyl-phosphocholine), DPPC (dipalmitoyl-phosphocholine), and Escherichia coli lipid extracts, etc. [18], offer a wide variety of customizable discs catering to the stability requirements of different membrane proteins. Lipid molecules differing in their acyl chain lengths provide a bilayer with altered thickness (Figure 1C: top panel) which may be required for membrane proteins that either differ in their expanse of the bilayer region or undergo relative movements in the transmembrane region as a result of their activation. Nanodiscs provide a competitive edge over other systems due to their soluble nature, ease of concentration, monodispersity, temperature stability, and compatibility with cell free expression system offering themselves as a more viable alternative for investigation of membrane proteins.

Figure 1:

Nanodisc models were constructed using the nanodisc builder from CHARMM GUI [10]. (A) Visual representation of a nanodisc that is either empty or contains bacteriorhodopsin (PDB: 1R2N). The outer belt protein MSP is shown in green while the lipid molecules are colored by atom type, where carbon is gray, oxygen is red, phosphorus is orange, and nitrogen is blue. The lipids are rendered partially transparent in order to distinguish bacteriorhodopsin from nanodisc. (B) Schematic representation of the overall length and nanodisc diameters for several MSP1D1 variants. The arrow indicates the scheme for the addition or deletion of helices from the core MSP1D1 leading to MSP with varying lengths. Nanodiscs with smaller diameters are preferred for NMR studies. The largest “MACRODISC” was obtained from a singular 14 amino acid peptide producing a disc with a diameter of 30 nm while the smallest disc, with a diameter of 6.4 nm, was obtained by deleting helices 4–6 from the MSP1D1 construct. (C) Depiction of the approximate hydrocarbon thickness of nanodisc bilayers composed of commonly used phosphatidylcholines. As shown, DMPC is the smallest, DPPC and POPC have similar thickness, and DSPC is the largest [11]. The chemical structure of each lipid is presented underneath (top panel). The complete sequence of apolipoprotein A-1 is shown emphasizing each individual helix. MSP1 construct, represented by the green lines, was created by deleting the N-terminal region highlighted in the red box (bottom panel).

3 Preparation of nanodiscs

For NMR experiments, optimal sample preparation is paramount to getting good data. The following simple, but key, concepts should be taken into consideration before preparing nanodisc samples.

3.2 Incorporation into nanodisc

3.2.1 Preparation of reagents

The primary reagents consists of two Tris-NaCl buffers with 20 mm Tris-HCl and 100 mm NaCl at pH 7.5. The second buffer, in addition, contains 60 mm sodium cholate detergent. NaCl concentration in the above buffers can be significantly reduced for membrane proteins that are sensitive to salt. The lipid stocks used for nanodisc preparations comes either in a soluble form, dissolved in chloroform or as solid powders (Avanti Polar Lipids, AL, USA). Chloroform is removed from the solubilized lipid stock using a stream of nitrogen gas and later left in a vacuum desiccator to eliminate any leftover residual amounts. Alternatively, one could use the powdered stock that can be directly weighted in appropriate quantities for disc preparation. Though we have not directly compared the soluble and powder form, we routinely use the powdered form which has worked well without any drawbacks. Protein incorporation into disc can be carried out at different temperatures which may improve their stability or reduce protein degradation. Lipid molecules that differ in their phase transition temperatures viz., POPC at ~4°C, DMPC at ~25°C, and DPPC at ~37°C [20] are chosen based on the desirable operating temperature required for disc preparation. The most popular utilization has been with using DMPC at room temperature. Alternatively, one could also prepare or use lipid extracts from multiple sources to ensure proper stability of specific membrane proteins.

3.2.2 Preparation of nanodiscs

Lipid molecules are reconstituted in the cholate containing buffer. The amount of lipids required depends on the type of MSP molecule that is utilized. Generally, the reconstituted lipid molecules are added in molar excess as follows: 100–120 molar excess for MSP1E3D1, 80 molar excess for MSP1D1, 50 molar excess for ΔH5 and 20 molar excess for the smaller constructs, D7 or ΔH4H5. A typical reaction mixture consists of, MSP and lipid molecules for preparing empty discs; MSP, lipids, and target membrane protein for protein incorporated discs. After the addition of MSP and lipids in the desired molar ratios, the volume is made up by the aforementioned two buffers such that the final concentration of cholate in the reaction volume is between 12 and 40 mm. This reaction mixture is kept on a rocker for an hour. Meanwhile, pretreatment of Bio-beads SM-2 (Bio-Rad, CA, USA), which functions to pull out the detergent and initiate nanodisc formation, are carried out. The beads are washed with methanol (1×), water (3×), and buffer one (1×) from the above two buffers. Bio-beads are added to the reaction mixture in a ratio of 0.5–1.5 g per milliliter of volume and left rocking at room temperature, such that the beads remain gently suspended in the solution, for 3–4 h or overnight for samples with high amount of total detergents. Later, the sample is separated from beads, concentrated, and run on an SEC column to obtain purified nanodiscs. For incorporating protein into nanodisc, the reaction mixture typically contains MSP at a concentration that is twice in molar excess of the target protein. A protein embeds itself into disc by occupying the volume of the lipid molecules that it displaces. Hence, the addition of lipids can be done at a reduced molar ratio than what is required for making empty disc. Empirical optimization to ensure maximum incorporation of the target protein into nanodisc is achieved by monitoring peaks in the void volume arising from the aggregated or unincorporated membrane protein; and at the position of free MSP, implicating inefficient disc formation. Both can be overcome by varying the concentration of lipids, MSP, or target protein.

3.2.3 On-column nanodisc formation

As with any project, a typical NMR sample is prepared in several iterations to obtain the best NMR spectrum. Preparing nanodisc samples for NMR applications are time intensive and require multiple purifications to separate empty discs from the incorporated ones. We developed a quick, alternative, on-column method where the protein incorporated disc was formed on the resin either through the bound MSP (approach-1) or the bound target protein (approach-2) [21]. The advantages of this method were several-fold, including reduced preparation time, concomitant separation of empty and incorporated discs and better yields, which collectively improved the quality of our NMR spectrum. For on-column preparation, the required reagents remain the same as in the original protocol while we explore the differential permutation of affinity tags to achieve our goals. Briefly, for approach-1, the harvested cells containing over-expressed His-tagged MSP is bound to Ni-NTA resin in a gravity column and washed as per the regular protocol. Prior to elution, the resin is mixed with lipids and target protein in the same column for an hour before the addition of Bio-beads. After passage of the stipulated time, discs are formed on the resin attached through MSP (shown as inset in Figure 2A for approach-1). Once the reaction is over, elution is carried out in the same elution buffer previously used for the purification of MSP. The eluate contains a mixture of empty and protein incorporated discs further separated through chromatography. For approach-2, we took the advantage of the MBP (maltose binding protein) tag by engineering the transmembrane and cytoplasmic tail regions of integrin-α_IIb fused to MBP (in pMAL-C2 vector) with a tobacco etch virus (TEV) cut site present between the tag and protein. MBP helps solubilize the otherwise insoluble transmembrane region of integrin and the TEV cut site helps in the removal of MBP tag after their incorporation into discs using TEV protease (Figure 4A: left panel). Alternatively, other tags could be used in combination with a TEV cut site. The MBP-fused construct was expressed in E. coli BL-21 (DE3) similar to MSP. Harvested cells were lysed and mixed with amylose resin (NEB, USA), which is later washed with Tris-NaCl buffer containing 20 mm Tris-HCl and 100 mm NaCl at pH 7.5. Amylose resin retains the MBP fused protein through its affinity for MBP. Previously purified MSP and lipids are added to the resin, followed by the addition of bio-beads. Discs are formed around the MBP fused protein attached to the amylose resin (shown as inset in Figure 2A for approach-2). After the formation of discs, the resin is rinsed with Tris-NaCl buffer, which washes out the empty discs leaving behind the protein incorporated ones. Elution is carried out in Tris-NaCl buffer with 20 mm maltose and the eluate is further subjected to SEC for buffer exchange and removal of unincorporated target protein. For NMR sample, elution was carried out in the presence of TEV to help remove the MBP tag. Approach-2 is faster and more efficient of the two approaches. A pictorial representation summarizing the different approaches is shown in Figure 2A. There are two caveats to the on-column method. (i) Estimation of the protein concentrations: we usually express MSP/target protein in large volumes of LB and aliquot 1 l harvests in falcon tubes. From one such tube, we estimate the protein concentration and use that to decide the MSP to target protein or vice versa ratios. We admit that this may not be accurate, but the convenience of sampling through several proteins grossly outweighs the effects of inaccuracy arising from minor discrepancies in protein estimations. This problem is usually alleviated by the addition of MSP slightly in excess to its required concentration. (ii) Retrieval of chromatography resin after mixing with Bio-beads: a simple solution to this problem is to use magnetic beads in lieu of agarose beads, which can be easily recovered from the solution mixture using any of the commercial magnetic apparatus. Alternatively, a net filter can be utilized with a pore size large enough for the resin but small enough for the beads, as both differ in size by several orders of magnitude.

Figure 2:

General workflow for nanodisc preparation. (A) Comparison of the regular method to the on-column method. The on-column method is split into two approaches, depending on the immobilization of either MSP or the target protein onto the resin. (B) Schematic representation of a target protein with its corresponding soluble extra cellular/MBP tag domain, a transmembrane hydrophobic domain, and soluble cytoplasmic domain. Also shown is a nanodisc containing the encapsulated target protein with MSP and lipid molecules (top panel). A more detailed workflow for approach-2 from the on-column method is illustrated. The target protein is bound to the resin either through its affinity for the MBP tag or through the crosslinked antibody specific to the extracellular domain (ED). The addition of MSP, lipids, and bio-beads lead to the encapsulation of the target protein into nanodisc while still bound to the resin. This nanodisc encapsulated protein is finally eluted from the resin (bottom panel).

An extension of approach-2, though not useful for NMR applications lies in the use of antibodies to pull larger protein receptors/complexes from any cell lysate. This strategy may find wide applicability among general and pharmaceutical researchers. Antibody-based pull down is a routinely used well-established technique. We propose to utilize antibodies in conjunction with our on-column method by immobilizing antibodies through covalent crosslinking to the resin bead using Crosslink IP/Crosslink Magnetic IP Kit (Pierce, MA, USA). The crosslinked antibodies (specific to the protein or a small tag) can then be used to fish out native receptors expressed in a mammalian cell line. This can be further washed and mixed with MSP and lipid molecules. Nanodiscs with the incorporated pulled receptor can be obtained either with the appropriate elution buffer or through TEV protease cutting at a TEV site engineered between the tag and the protein. A schematic representation, comparing the antibody vs. the MBP tag using approach-2, is depicted in Figure 2B.

3.4.1 Small discs

The original MSP construct, MSP1, that contained 10 amphipathic helical repeats was modified at its first helix through partial deletion and further addition of a Histidine-tag to obtain the MSP1D1 construct [26], which formed nanodisc with a diameter of ~10 nm and MW of ~150 kDa. MSP1D1 became the base construct from which most of the variant discs were generated (Figure 1B). For NMR applications, a larger disc size is unfavorable as its transverse relaxation rate is faster, leading to peak broadening and lower resolution. This problem was ameliorated by creating truncated constructs of MSP, which yielded smaller discs. Sligar’s group had previously developed MSP1E3D1 [26] which are larger discs of ~13 nm diameter, obtained through additional insertion of helices 4–6 (Figure 1B). This proved that repetition of helices in the middle region of the protein does not affect its property to form discs, which also indirectly meant that the middle region is amenable to alterations without impacting the formation of disc. Wagner’s group adopted this idea and successively removed the same three helices 4–6 obtaining smaller nanodiscs. The smallest obtained disc ΔH4–H6 was of ~6.4 nm, but was unstable and formed larger assemblies of ~11 nm over time. The next smaller disc, which was stable enough to retain its small size, was from the MSP construct ΔH4–H5 with a diameter of ~7.3 nm and MW of ~70 kDa (Figure 3A) [27].

Figure 3:

(A) Small nanodiscs that have been employed in solution NMR studies. ΔH5, ΔH4H5, and D7 provide discs with smaller diameters that can be used for structural investigation of encapsulated proteins (left panel). Pictorial representation of larger “MACRODISC” is shown where the outer annulus is formed by a 14 amino acid amphipathic peptide. This large 30 nm discs has been used to orient molecules for residual dipolar coupling experiments (right panel). (B) Schematic representation of MSP1D1/D10 with the positioning of prolines and the number of amino acids present in each helix. Pictorial representation of a nanodisc showing the presence of prolines at helix turns (top panel). A scheme for incorporating proline residues further forcing nanodisc to a smaller size is shown. An altered construct D5P, is created from D5 through the strategic placement of additional prolines, increasing the number of individual helices and potentially offering additional turns at proline residues. Each individual helix in the D5P construct is of identical length and is maintained at a size of 11 amino acids, representing the smallest building block in the MSP1D1 construct (bottom left panel). The elution profile from size exclusion chromatography shows that the D5P nanodisc (green) is approximately the same size as D5 disc (black). Furthermore, D5 and D5P nanodiscs are similar in size to the MSP1D1/D10 discs (brown) (bottom right panel). (C) Alternative nanoscale phospholipid bilayer systems: saposin-A lipoprotein disc formed by the addition of detergent molecules is shown. Saposin-A in its detergent free form adopts a closed conformation which becomes extended when bound to detergent molecules (PDB ID: 4DDJ). Saposin-A lipoprotein disc, with a diameter of 3.2 nm, is formed when two saposin-A proteins are brought together by its lipid core (top panel). Styrene maleic acid lipid particle (SMALP) is shown where the synthetic styrene-maleic acid (SMA) copolymer forms a disc by encapsulating lipids within its central cavity. SMA lipid disc or Lipodisq^® have a diameter of 9 nm (bottom panel).

At the same time, our group independently arrived at a similar conclusion through a different scheme of deletion constructs. We successively removed individual helix from the C-terminal end and obtained the smallest disc from the deletion of the last three helices ΔH8–H10, which we refer to as D7 discs, as this deletion construct contained the first seven helices. D7 disc has a diameter of ~7 nm with a molecular weight of ~62 kDa (Figure 3A). In the MSP1D1 construct, all helices are roughly of similar lengths except for helix 3 and 9 which are half the standard length [Figure 3B: top panel (MSP1D1 or D10)]. Both D7 and ΔH4–H6 constructs were devoid of three helices, with our deletion ΔH8–H10 containing one short helix along with two standard helices while Wagner’s deletion removed three standard length helices. This translates into our D7 construct being slightly larger than the ΔH4–H6 construct though both have deletions by three helices. Interestingly, this was quite accurately corroborated in the distribution of disc sizes where our D7 (ΔH8–H10) disc size of ~7 nm falls right in between Wagner’s discs ΔH4–H5 of ~7.3 nm and ΔH4–H6 of ~6.4 nm. Furthermore, we found that any successive reduction beyond seven helices led to the formation of discs with larger diameters. For example, our D5 construct, which contains the first five helices, produced a size similar to that of the full length MSP1D1 (D10) construct with ten helices. This can be conceptualized as four D5 molecules coming together as two dimers of D5 form either half of the circular belt. These rings present an edge-to-edge arrangement mimicking a single ring of 10 helices. The most intriguing finding from our D7 construct was that the terminal helices do not regulate the formation of nanodiscs, which we believe might have been the initial concern that led to the addition or deletion of helices exclusive to the middle region. A point to be noted here is that though Wagner’s ΔH4–H5 gave the smallest disc, the ΔH5 construct with a slightly larger diameter of ~8.4 nm (Figure 3A: left panel) provided the best NMR spectrum and was adopted for structure determination.

Once we reached the smallest possible size through the deletion constructs, we still argued if we could possibly break the size barrier and force the system to even smaller diameters. A closer look at the protein sequence of MSP1D1 showed the strategic presence of proline residues present in between helices (Figure 3B: top panel). We hypothesize whether the inter-helical turns are an outcome of proline residues. Additionally, we observed that most helices had a standard length of ~22 amino acids (aa) with the smallest helix being 11 aa long. The natural presence of smaller helices led us to wonder whether reducing the helix length would facilitate further size reduction. Using all of this information, we attempted to redesign our D5 MSP construct, which previously gave us larger discs. D5 contains 5 helices with one 11 aa and 4 22 aa blocks. Our idea was to break down D5 into blocks of 11 aa each, such that it gives us a total of 9 helices roughly resembling the natural 10 helix arrangement in MSP1D1. We achieved this by adding proline residues breaking the 22 aa bocks down to 11 aa and left the naturally occurring 11 aa block untouched (Figure 3B: bottom left panel). Unfortunately, this approach was unsuccessful as can be seen from the SEC data (Figure 3B: bottom right panel), where the elution volumes of D5 and D5P (proline mutant) are nearly identical, indicating a similar stokes radius and hence similar diameters. In effect, we were unable to decrease the size of nanodisc any further through selective placement of additional prolines. While we tried several permutations of prolines insertion, we only present the case with maximum number of inserted prolines for the sake of simplicity.

4 Application of nanodiscs for structural studies by solution NMR

Though presently there are few structure articles, there still are several examples of successful application of nanodiscs with solution NMR. The initial use of nanodisc was demonstrated for two membrane proteins: (i) CD4 mutant, containing a single transmembrane and cytoplasmic tail, where the aliphatic resonances in ¹H-¹³C heteronuclear single quantum coherence (HSQC) spectra were compared to that in dodecylphosphocholine (DPC) micelles [41]; and (ii) VDAC-1, human anion channel protein, where ¹H-¹⁵N TROSY HSQC spectra were compared between LDAO isotropic bicelles and MSP1D1 nanodiscs [42]. Additionally, the occurrence of chemical shift perturbations due to NADH binding was demonstrated. A similar study was later performed with VDAC-2 [43]. Following this, several other proteins were investigated in nanodiscs. The voltage sensing domain (VSD) of KvAP channel, containing four consecutive transmembrane helices, was incorporated into (MSP1D1) nanodiscs with different types of lipid molecules and was found to maintain a proper conformation only in zwitterionic environments [44]. A 30 nm MACRODISC, discussed earlier, was developed as a medium for RDC measurements [29]. Bacteriorhodopsin, a hepta-helical transmembrane protein, was studied comparatively across different mimetics comprising micelles, amphipols, and (MSP1D1) nanodisc [45]. ¹H-¹⁵N TROSY HSQC spectra of the trans-membrane and cytoplasmic domains of Integrin α_IIb spectra was obtained in smaller D7 nanodisc [21]. YgaP, an E. coli integral membrane protein composed of two transmembrane helices and cytoplasmic Rhodanese domain, was studied in mixed micelles, FC12 micelles, and MSP1D1 nanodisc. This study demonstrated the improvement in quality of ¹H-¹⁵N TROSY HSQC spectrum in the presence of deuterated d54-DMPC lipids in nanodisc. Interestingly, the spectral quality in nanodisc was relatively better in comparison to FC12, which reflects the perturbed structural integrity of Rhodanese domain in micelles [46]. Sequential assignments of OmpA, an E. coli β barrel protein, were achieved in (MSP1D1) nanodisc albeit with the help of assignments from Fos-10 micelles. Additionally, application of micro-coil NMR was demonstrated to find optimized solution conditions for OmpA in nanodisc [47]. ¹H-¹⁵N TROSY HSQC spectra of two β-barrel outer membrane proteins OprG and OprH from P. aeruginosa were compared between DHPC micelles and different truncation mutants of MSP ΔH4, ΔH5, ΔH4H5, and ΔH4–H6 (developed by Wagner’s group [48]). In agreement with Wagner’s findings, it was found that ΔH5 offered the best spectrum [49]. Protein-protein interactions have also been studied through the incorporation of P450-cyt_b in a 22 amino acid long peptide based nanodisc [48]. Another approach, using HADDOCK simulations [50] with distance restraints derived from paramagnetic relaxation enhancement, allowed to distinguish the reorientation of the effector binding site on K-RAS4B GTPase anchored to (MSP1D1) nanodisc that was previously occluded through the anionic membrane [51]. The most rigorous and compelling study came from Wagner’s group, where they solved the structure of a β barrel protein, OmpX, in nanodisc using small disc formed by the ΔH5 deletion construct of MSP [27]. A later study performing RDC measurements of OmpX nanodisc carried out using Pf1 phage alignment medium, has helped better define the orientation of N–H bonds thereby improving the overall structure of OmpX in ΔH5 disc [52].

With the aim to further nanodisc application beyond structure determination using NMR-based approaches, we hereby demonstrate how a nanodisc can be used to study the functional activity of a membrane protein. Membrane protein phosphorylation has been previously reported in nanodisc [53]. In our present study we show, for the first time, the molecular details of phosphorylation through NMR. To demonstrate this, we used the major platelet integrin α_IIbβ₃ as an example. Integrins represent a class of human cell surface receptor heterodimers, formed by a combination of α and β subunits. Upon stimulation with the extracellular domain, integrin undergoes phosphorylation at the β₃ cytoplasmic tail on two tyrosine residues by Src kinase, schematically shown in Figure 4C (top panel). Integrin constructs, used in our study, consisted of an MBP tag (maltose binding protein) fused to the transmembrane and cytoplasmic domain (TMCD) of either α_IIb or β₃ [54]. MBP tag enhances the solubility of the TMCD region, which aids in the purification and incorporation of α_IIb or β₃ into nanodiscs. Once integrin subunits are incorporated into the disc, the tag is excised with TEV protease which acts on the TEV cut site engineered between MBP and the rest of the protein, providing nanodiscs containing just the TMCD region (Figure 4A). We employed our small D7 MSP construct (roughly similar to Wagner’s ΔH4H5 construct) to form nanodiscs. By themselves, α_IIb forms homo-dimers and β₃ forms homo-trimers [21], [55]. Thus, at least two or three transmembrane helixes fit comfortably within the internal lipid area of the discs, which have an estimated inner diameter of ~60 Å. The ¹H-¹⁵N TROSY HSQC spectra of the individual α_IIb or β₃ incorporated discs were collected on 800 MHz magnet (Agilent, CA, USA) and are shown in Figure 4B. The overall size of the assembly, even within the smallest nanodisc, is still large, resulting in broader peaks corresponding to the transmembrane portion of the protein. Additional local motion of cytoplasmic tails, which protrudes out from the disc, were manifested by sharper peaks. We exploited this advantage to study the downstream effect of integrin activation through the phosphorylation of Integrin β₃ on 600 MHz magnet (Agilent, CA, USA). Lack of deuteration, makes it difficult to observe peaks from the transmembrane region, making it easier to selectively focus only on the cytoplasmic tail. We simply mixed the ¹⁵N-labeled β₃ discs with the kinase domain of Src kinase along with ATP molecules in an NMR tube and collected a ¹H-¹⁵N TROSY HSQC spectrum. By overlaying the un-phosphorylated and bi-phosphorylated spectra, one can clearly see shifts arising due to the phosphorylation of β₃ tails in nanodiscs (Figure 4C: right panel). The shifts correspond to the region around the tyrosine residues at the C-terminus of the cytoplasmic tail where phosphorylation occurs as shown from our previous studies using just the short cytoplasmic tail regions [56]. Further assignments will be required to exactly pinpoint the specific residues perturbed in the process. Thus, here we show a simple application, free from the need for deuteration, which can be routinely applied with a medium field NMR magnet to study receptors signaling. It is important to note that our D7 disc is similar in size to Wagner’s ΔH4–H5 disc. As Wagner’s ΔH5 construct, which is of a larger diameter than the ΔH4–H5 construct, gave a better NMR spectra for OmpX, further experimentation would be required to ascertain the ability of our D7 discs to study β barrel proteins, though our larger D5 discs definitely offer an equal advantage as the ΔH5 discs. Considering all the above different examples, we truly believe that nanodiscs are a promising membrane mimetic system that can be utilized successfully for a multitude of NMR applications.

Figure 4:

(A) Overview of the procedure to obtain integrin α_IIb[trans-membrane and cytoplasmic domains (TMCD)] incorporated nanodisc. TEV protease treatment of MBP-integrin α_IIb (TMCD) incorporated nanodisc, cleaves the MBP–tag at the TEV cleavage site engineered between the tag and Integrin. The α_IIb incorporated disc can then be purified from the rest of the cut products using SEC. Inset: negatively stained TEM image of α_IIb containing nanodiscs displaying a homogeneous population. The exact same procedure can also be extended to obtain β₃ (TMCD) incorporated nanodisc. (B) ¹H-¹⁵N- TROSY HSQC spectra of Integrin α_IIb and β₃ (TMCD) acquired on 800 MHz magnet. The sharp peaks correspond to the cytoplasmic tail region while the broader peaks correspond to the transmembrane region. (C) Applicability of nanodisc systems for studying signaling pathways. A schematic showing the activation events of Integrin β₃ relayed intracellularly through the phosphorylation of its cytoplasmic tail by Src kinase (top panel). This event is mimicked (in vitro) within an NMR tube by mixing ¹⁵N labeled β₃ incorporated discs with the kinase domain of Src kinase in the presence of ATP molecules. Phosphorylation is manifested through chemical shift perturbations observed through the overlay of the ¹H-¹⁵N-TROSY HSQC spectra obtained from un-phosphorylated (black) and bi-phosphorylated (red) β₃ (TMCD) collected on 600 MHz magnet. Also shown is the sequence of the β₃ tail highlighting the tyrosine residues involved in phosphorylation.

5 Technical challenges and possible solutions

Despite the advantage of using nanodisc, there are certain challenges that need to be overcome. We categorize them into: a) preparation of protein-incorporated nanodisc, and b) uncertainties with experimental setups. We have previously discussed potential solution to the former in the section “Preparation of nanodiscs”. We now address some strategies to overcome the latter.

Smaller discs are definitely more advantageous than larger ones. One can easily employ the small nanodiscs developed specifically for this purpose either through our (D7) or Wagner’s (ΔH4H5 or ΔH5) constructs. If the plasmid for MSP1D1 is readily available, D7 construct can be easily generated by placing a stop codon at the end of the seventh helix using site-directed mutagenesis. The ~7 nm diameter discs reflects sufficient lateral space to incorporate smaller porins, such as 8-stranded OmpX [27], or helical membrane proteins with at least two or three transmembrane helices, as has been demonstrated above through α_IIb homo-dimers and β₃ homo-trimers (Figure 4B). To make assignments feasible, complete deuteration of the system is required along with the use of deuterated lipids to minimize dipolar-dipolar relaxations. All NMR experiments need to be carried out on high field magnets like 800 MHz or above to take full advantage of the TROSY-based experiments for complete backbone assignments [57]. Furthermore, novel pulse sequences specifically designed for Ile/Leu/Val side-chain assignments in perdeuterated samples with selectively protonated methyl groups can be used [25], [58]. These assigned methyls, in addition to the backbone amides utilized for the structural characterization of β-barrelporins [27], are absolutely necessary for the high-resolution structure determination of α-helical proteins as the number of long distance restraints obtained exclusively from HN-HN would be insufficient. Interestingly, chemical shifts of membrane protein acquired in other membrane mimetic systems, such as micelles, bicelles or amphipols, often overlap (or are similar) with corresponding chemical shifts in nanodisc, especially for the residues located within the hydrophobic core, and could potentially be utilized to expedite the assignment process [27], [43].

Nanodiscs are especially advantageous in their ability to achieve high concentrations in the high μm to low mm ranges. A sample with higher concentration drastically increases the signal to noise ratio and provides better spectrum quality. Nevertheless, proper care should be taken to maintain the homogeneity of these concentrated samples. We have shown that smaller constructs of MSP form larger discs [21]. This suggests that the purity of your MSP preparation is very crucial. Impure preparations with degraded MSP might lead to a heterogeneous population consisting of a mixture of small and large discs. This will effectively reduce the concentration of small discs, compromising sensitivity. An easy way to check this is through negative stained transmission electron microscopy (TEM) images, which clearly displays sample heterogeneity (inset in Figure 4A). Moreover, if your sample contains a β barrel porin, you can check the number of pores present in the population of your disc which will manifest itself as black dots within your disc’s image. This will help ascertain the oligomeric state of the porin within a disc. As an example, demonstrating the visualization of pores in nanodiscs, we previously carried out image analysis of a trimeric β barrel protein TprC (C-terminal domain) in large MSP1E3D1 discs. The image averaged result shows the presence of three pores in the backdrop of a nanodisc [59].

Large macromolecular systems, including nanodiscs, are notorious for peak broadenings in most of the triple-resonance NMR experiments, resulting in a low signal-to-noise ratio. Even though nanodisc with an incorporated membrane protein can be easily concentrated, the problem of sensitivity still remains. This can be partially alleviated by data acquisition at higher temperatures with longer time durations. The downside of higher temperatures lies in the detrimental effect on protein stability. Another approach to increase the sensitivity (and resolution if necessary) without significantly raising acquisition times is through the utilization of nonuniform sampling (NUS) [60], [61]. NUS has recently emerged as a powerful technique to speed up the acquisition of multidimensional NMR experiments. It is based on the rationale where a subset of the linearly sampled data in the Nyquist grid is measured. For this reason, NUS requires processing algorithms different from the normal Fourier transform. A number of sophisticated methods have been developed for reconstructing sparsely sampled NMR data with minimal artifacts. NMR Pipe (http://www.nmrscience.com/nmrpipe.html), a UNIX-based collection of programs and scripts developed by Delaglio [62], provides tools for reconstruction of NUS data, including iterative shrinkage thresholding (IST) decomposition and options for maximum entropy reconstruction (MEM). The NMR Pipe compatible scripts for istHMS, developed by Wagner’s group, could be found at their website: http://gwagner.med.harvard.edu/intranet/istHMS/index.html. The IST software itself may be requested from the same group. MEM processing script generator, developed by Jeff Hoch and coworkers is available as part of RNMRTK (http://rnmrtk.uchc.edu/rnmrtk/RNMRTK.html) and can be found at the website: http://sbtools.uchc.edu/nmr/nmr_toolkit/.

6 Conclusion

In this review, we discuss the advantages and challenges associated with nanodiscs for studying membrane proteins. We provide an extensive view on the preparation and application of nanodiscs. This could serve as a general guide that should aid any future user in successfully carrying out nanodisc-based experiments; either through the selection of specific discs or through the strategies offered for the incorporation of typical membrane proteins. The feasibility of NMR-based experiments has been clearly emphasized with the need for deuteration to achieve desirable results for assignments or structure solution. We also demonstrate a simple application that can be used for studying protein signaling events. Finally, we offer other promising alternatives that may provide better viability with NMR applications and be suitable for any tailor-made requirements needed for specific membrane proteins. Nanodisc-based system clearly has the potential for both structural and functional investigations and is constantly progressing in terms of utilization and development. With the current pace of developments, we are hopeful that nanodiscs will become the preferred choice of membrane mimetic system for investigating membrane proteins.