Membrane proteins are central to biological signaling pathways, regulating transfer of information and energy across cell membranes. They compose roughly 30% of the human proteome, but due to their key roles in signaling control, represent as much as 50% of therapeutic drug targets. Functionality of membrane proteins depends on interaction with the lipid environment, which makes their study challenging. Removed from their native lipid bilayer, membrane proteins become denatured and aggregated or insoluble. They can be solubilized using detergents, but native structure is disrupted and functionality often lost. Membrane bilayer constructs such as liposomes or bicelles can maintain membrane protein functionality but remain difficult to prepare consistently and to manipulate in an aqueous environment. In addition to these hurdles, the best method of membrane protein preparation for in vitro assays is typically dependent on the target protein and must be worked out for each.
Nanodisc technology, developed by Professor Stephen Sligar at the University of Illinois at Urbana-Champaign, addresses many of these challenges by enabling preparation of membrane proteins in a native-like lipid bilayer that is water soluble.1 Amphipathic helical segments of Apolipoprotein A-1, termed membrane scaffold proteins, surround a phospholipid bilayer containing the target protein, resulting in a particle containing active protein that can be manipulated in an aqueous environment. Proteins in Nanodiscs are monodisperse in solution and offer improved particle uniformity compared to microsomes or proteoliposomes. They are also quite stable, and the system works for a variety of membrane proteins. Nanodiscs have been used to study cytochrome P450s, bacteriorhodopsin, coagulation factors, Tar receptor, SecYEG, and cholera toxin, among others.
Uniform size, robust stability, and the ability to be manipulated in a detergent-free aqueous environment opens study of membrane proteins in Nanodiscs to many of the biochemical and analytical techniques developed for soluble proteins. As a result, there has been significant progress in understanding of structure and function for several types of membrane proteins. The cytochrome P450 CYP17A1 directs cholesterol-derived substrates either toward production of corticoids or toward androgen formation in human steroidogenesis. The use of CYP17A1 in Nanodiscs in conjunction with resonance Raman spectroscopy demonstrated differential hydrogen bonding for hydroxylated progesterone vs hydroxylated pregnenolone at the active site, such that substrate structure dictates conversion toward the respective endpoint.2 In another example, high-resolution NMR of the mTOR activator Rheb tethered to a Nanodisc showed that interaction of the GTPase domain occurs in 2 distinct orientations that depends on the bound nucleotide.3 Rheb is a member of the Ras superfamily of GTPases, which function as molecular switches in multiple signaling networks.
Nanodisc technology is also being used to study interacting partners of membrane embedded molecules. For integral membrane proteins, the lack of detergent allows detection of weakly interacting partners and prevents false positives created by binding to hydrophobic domains exposed by lipid stripping. In a proof of principle study, binding partners for the bacterial SecYEG protein-conducting channel, the maltose transporter MalFGK2, and the membrane integrase YidC were retrieved from a complex cell lysate and identified via SILAC (define) -based mass spectrometry.4 Conversely, rather than use a homogeneous Nanodisc population to fish binding partners out of a heterogenous pool, a whole membrane proteome can be incorporated into Nanodiscs to identify membrane protein targets of an immobilized ligand bait.5 Other types of membrane associated molecules have also been used in Nanodiscs to identify interacting molecules. The glycosphingolipid ganglioside GM1 in intestinal epithelia is a target for bacterial toxins that, upon binding to the gycosyl moiety of GM1, are internalized to the host cell. GM1-embedded Nanodiscs have been used in a co-immunoprecipitation experiment with culture media from E. coli for subsequent identification by peptide mass fingerprinting of specifically bound heat labile enterotoxin.6
One exciting application of the Nanodisc system is delivery of therapeutics. Nanodiscs containing the minor pulmonary surfactant phospholipid palmitoyloleoylphosphatidylglycerol have been used to inhibit respiratory syncytial virus (RSV) in mouse lung, with concurrent suppression of histopathological changes as well as of inflammatory cytokines.7 In another study, it has been shown that Nanodiscs can deliver porphyrin-conjugated lipid in an inactive form that becomes photoactivatable upon disruption of the disc.8 Such a system may lend itself to imaging applications as well as photoactivatable drug delivery to specific sites. In addition, the sub-40 nm Nanodisc size results in better penetration of dense collagen matrices than vesicles used for drug delivery, which may produce more effective therapeutic delivery into fibrous tumors.
Nanodisc technology represents a breakthrough in our ability to study and understand the functionality of membrane proteins, providing improved resolution of key molecular events fundamental to a mechanistic understanding of critical signaling and metabolic functions, with implications for more effective intervention when those events are disrupted in disease states.