Nanodiscs provide an excellent system for the structure-function investigation of membrane proteins. Its direct advantage lies in presenting a water soluble form of an otherwise hydrophobic molecule, making it amenable to a plethora of solution techniques. Nuclear Magnetic Resonance is one such high resolution approach that looks at the structure and dynamics of a protein with atomic level precision. Recently, there has been a breakthrough in making nanodiscs more susceptible for structure determination by solution NMR, yet it still remains to become the preferred choice for a membrane mimetic. In this practical review, we provide a general discourse on nanodisc and its application to solution NMR. We also offer potential solutions to remediate the technical challenges associated with nanodisc preparation and the choice of proper experimental set-ups. Along with discussing several structural applications, we demonstrate an alternative use of nanodiscs for functional studies, where we investigated the phosphorylation of a cell surface receptor, Integrin. This is the first successful manifestation of observing activated receptor phosphorylation in nanodiscs through NMR. We additionally present an on-column method for nanodisc preparation with multiple strategies and discuss the potential use of alternative nanoscale phospholipid bilayer systems like SMA lipid discs and Saposin-A lipoprotein discs.
Integrin; Nanodisc; SMALP; Saposin-A; Styrene Maleic Acid; beta barrel; membrane proteins; nanoscale phospholipid bilayers; solution NMR; transmembrane.
Nanodiscs models were constructed using the nanodisc builder from CHARMM GUI . (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 grey, oxygen is red, phosphorus is orange, and nitrogen is blue. The lipids are rendered partially transparent in order to distinguish bacteriorhodopsin from the rest of the 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 different lengths. Nanodiscs with smaller diameters are preferable for NMR studies. The largest “MACRODISC” was obtained from a singular 14 amino acid peptide producing a disc with a diameter of 30nm while the smallest disc, with a diameter of 6.4 nm, was obtained from the deletion of helices 4 through 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 . The chemical structure of each lipid is presented underneath (Top Panel). The complete sequence of Apolipoprotein A1 is shown demarking each individual helices. The MSP1 construct, represented by the green lines, was created by deleting the N-terminal region highlighted in the red box (Bottom Panel).
General workflow for nanodisc preparation. (A) Comparison of the regular method to our on-column method. The on-column method is split into two approaches, depending on the immobilization of either MSP or the target protein on 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 leads to the encapsulation of the target protein into nanodiscs while still bound to the resin. This nanodisc encapsulated protein is further eluted from the resin (Bottom Panel).
(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 target proteins (Left Panel). Pictorial representation of larger “MACRODISCS” are shown where the outer annulus is formed by a 14 amino acid amphipathic peptide. These large 30 nm discs have been used to orient molecules for residual dipolar coupling (RDC) 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). The scheme for incorporation of proline residues to further push nanodiscs 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 showing that the D5P nanodiscs (green) are approximately the same size as D5 discs (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 discs, with a diameter of 3.2 nm, are formed where 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 copolymer forms discs by encapsulating lipid within its central cavity. SMA lipid discs or Lipodisq
® have a diameter of 9nm (Bottom Panel).
(A) Overview of the procedure to obtain Integrin α
IIb [trans-membrane and cytoplasmic domains (TMCD)] incorporated nanodiscs. TEV protease treatment of MBP- Integrin α IIb (TMCD) incorporated nanodiscs, cleaves the MBP–tag at the TEV cleavage site engineered between the tag and Integrin. The α IIb incorporated discs 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 of discs. The exact same procedure can also be extended to obtain β 3 (TMCD) incorporated nanodiscs. (B) 1H- 15N- TROSY HSQC spectra of Integrin α IIb and β 3 (TMCD) acquired on 800 MHz magnet (Agilent, USA). 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 β 3 which is relayed intracellularly through the phosphorylation of its cytoplasmic tail by Src kinase (Top Panel). This event is mimicked ( in vitro) in an NMR tube by mixing 15N labeled β 3 incorporated discs with the kinase domain of Src kinase in the presence of ATP molecules. Phosphorylation is manifested through chemical shift perturbations (CSP) observed through the overlay of the 1H- 15N- TROSY HSQC spectra obtained from the un-phosphorylated (black) and bi-phosphorylated (red) β 3 (TMCD) collected on 600 MHz magnet (Agilent, USA). Also shown is the sequence of the β 3 tail where the phosphorylation occurs at the two tyrosine residues.
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