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The nanodisc technology is increasingly used for structural studies on membrane proteins and drug delivery. The development of synthetic polymer nanodiscs and the recent discovery of non-ionic inulin-based polymers have significantly broadened the scope of nanodiscs. While the lipid exchange and size flexibility properties of the self-assembled polymer-based nanodiscs are valuable for various applications, the non-ionic polymer nanodiscs are remarkably unique in that they enable the reconstitution of any protein, protein-protein complexes, or drugs irrespective of their charge. However, the non-ionic nature of the belt could influence the stability and size homogeneity of inulin-based polymer nanodiscs. In this study, we investigate the size stability and homogeneity of nanodiscs formed by non-ionic lipid-solubilizing polymers using different biophysical methods. Polymer nanodiscs containing zwitterionic DMPC and different ratios of DMPC:DMPG lipids were made using anionic SMA-EA or non-ionic pentyl-inulin polymers. Non-ionic polymer nanodiscs made using zwitterionic DMPC lipids produced a very broad elution profile on SEC due to their instability in the column, thus affecting sample monodispersity which was confirmed by DLS experiments that showed multiple peaks. However, the inclusion of anionic DMPG lipids improved the stability as observed from SEC and DLS profiles, which was further confirmed by TEM images. Whereas, anionic SMA-EA-based DMPC-nanodiscs showed excellent stability and size homogeneity when solubilizing zwitterionic lipids. The stability of DMPC:DMPG non-ionic polymer nanodiscs is attributed to the inter-nanodisc repulsion by the anionic-DMPG that prevents the uncontrolled collision and fusion of nanodiscs. Thus, the reported results demonstrate the use of electrostatic interactions to tune the solubility, stability, and size homogeneity of non-ionic polymer nanodiscs which are important features for enabling functional and atomic-resolution structural studies of membrane proteins, other lipid-binding molecules, and water-soluble biomolecules including cytosolic proteins, nucleic acids and metabolites.

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Divalent cations, especially Ca2+ and Mg2+, play a vital role in the function of biomolecules and making them important to be constituents in samples for in vitro biophysical and biochemical characterizations. Although lipid nanodiscs are becoming valuable tools for structural biology studies on membrane proteins and for drug delivery, most types of nanodiscs used in these studies are unstable in the presence of divalent metal ions. To avoid the interaction of divalent metal ions with the belt of the nanodiscs, synthetic polymers have been designed and demonstrated to form stable lipid nanodiscs under such unstable conditions. Such polymer-based nanodiscs have been shown to provide an ideal platform for structural studies using both solid-state and solution NMR spectroscopies because of the near-native cell-membrane environment they provide and the unique magnetic-alignment behavior of large-size nanodiscs. In this study, we report an investigation probing the effects of Ca2+ and Mg2+ ions on the formation of polymer-based lipid nanodiscs and the magnetic-alignment properties using a synthetic polymer, styrene maleimide quaternary ammonium (SMA-QA), and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipids. Phosphorus-31 NMR experiments were used to evaluate the stability of the magnetic-alignment behavior of the nanodiscs for varying concentrations of Ca2+ or Mg2+ at different temperatures. It is remarkable that the interaction of divalent cations with lipid headgroups promotes the stacking up of nanodiscs that results in the enhanced magnetic alignment of nanodiscs. Interestingly, the reported results show that both the temperature and the concentration of divalent metal ions can be optimized to achieve the optimal alignment of nanodiscs in the presence of an applied magnetic field. We expect the reported results to be useful in the design of nanodisc-based nanoparticles for various applications in addition to atomic-resolution structural and dynamics studies using NMR and other biophysical techniques.

Membrane-anchored flavin mononucleotide binding domain (FBD) of the CYP450 reductase and native lipids were directly extracted from E. coli using charge-free pentyl-inulin polymer. FBD in native-nanodiscs was conformationally homogenous and enabled high-resolution NMR probing. (31)P NMR revealed the polymer’s no preference for any specific E. coli lipids and identified the lipid-types in native-nanodiscs.

Structural studies of membrane proteins in native-like environments require the development of diverse membrane mimetics. Currently there is a need for nanodiscs formed with non-ionic belt molecules to avoid non-physiological electrostatic interactions between the membrane system and protein of interest. Here, we describe the formation of lipid nanodiscs from the phospholipid DMPC and a class of non-ionic glycoside natural products called saponins. The morphology, surface characteristics, and magnetic alignment properties of the saponin nanodiscs were characterized by light scattering and solid-state NMR experiments. We determined that preparing nanodiscs with high saponin:lipid ratios reduced their size, diminished their ability to spontaneously align in a magnetic field, and favored insertion of individual saponin molecules in the lipid bilayer surface. Further, purification of saponin nanodiscs allowed flipping of the orientation of aligned nanodiscs by 90°. Finally, we found that aligned saponin nanodiscs provide a sufficient alignment medium to allow the measurement of residual dipolar couplings (RDCs) in aqueous cytochrome c.

Recent developments in lipid nanodiscs technology have successfully overcome the major challenges in the structural and functional studies of membrane proteins and drug delivery. Among the different types of nanodiscs, the use of synthetic amphiphilic polymers created new directions including the applications of solution and solid-state NMR spectroscopy. The ability to magnetically-align large size (>20 nm diameter) polymer nanodiscs and flip them using paramagnetic lanthanide ions have enabled high-resolution studies on membrane proteins using solid-state NMR techniques. The use of polymer-based macro-nanodiscs (>20 nm diameter) as an alignment medium to measure residual dipolar couplings (RDCs) and residual quadrupole couplings by NMR experiments has also been demonstrated. In this study, we demonstrate the use of magnetically aligned and 90°-flipped polymer nanodiscs as alignment media for structural studies on proteins by solution NMR spectroscopy. These macro-nanodiscs, composed of negatively-charged SMA-EA polymers and DMPC lipids, were used to measure residual (1)H-(15)N dipolar couplings (RDCs) from the water-soluble ~21-kDa uniformly-(15)N-labeled flavin mononucleotide binding domain (FBD) of cytochrome-P450-reductase. The experimentally measured (1)H-(15)N RDC values are compared with the values calculated from crystal structures of cytochrome-P450 reductase that lacks the transmembrane domain. The N-H RDCs measured using aligned and 90°-flipped nanodiscs show a modulation by the function (3cos(2)θ−1), where θ is the angle between the N-H bond vector and the applied magnetic field direction. This successful demonstration of the use of two orthogonally oriented alignment media should enable structural studies on a variety of systems including small molecules, DNA and RNA.

The ability of amphipathic polymers to self-assemble with lipids and form nanodiscs has been a boon for the field of functional reconstitution of membrane proteins. In a field dominated by detergent micelles, a unique feature of polymer nanodiscs is their much-desired ability to align in the presence of an external magnetic field. Magnetic alignment facilitates the application of solid-state nuclear magnetic resonance (NMR) spectroscopy and aids in the measurement of residual dipolar couplings via well-established solution NMR spectroscopy. In this study, we comprehensively investigate the magnetic alignment properties of styrene maleimide quaternary ammonium (SMA-QA) polymer-based nanodiscs by using (31)P and (14)N solid-state NMR experiments under static conditions. The results reported herein demonstrate the spontaneous magnetic alignment of large-sized (≥20 nm diameter) SMA-QA nanodiscs (also called as macro—nanodiscs) with the lipid bilayer normal perpendicular to the magnetic field direction. Consequently, the orientation of macro—nanodiscs is further shown to flip the alignment axis parallel to the magnetic field direction upon the addition of a paramagnetic lanthanide salt. These results demonstrate the use of SMA-QA polymer nanodiscs for solid-state NMR applications including structural studies on membrane proteins.

Despite their denaturing properties, detergents are used to purify and study membrane proteins. Herein, we demonstrated a polymer-based detergent-free extraction of the membrane protein cytochrome-b5 along with E. coli lipids. Nuclear magnetic resonance experiments revealed the suitability of using nanodiscs for high-resolution studies and revealed the types of native lipids associated with the protein.

Paramagnetic relaxation enhancement (PRE) is commonly used to speed up spin lattice relaxation time (T(1)) for rapid data acquisition in NMR structural studies. Consequently, there is significant interest in novel paramagnetic labels for enhanced NMR studies on biomolecules. Herein, we report the synthesis and characterization of a modified poly(styrene-co-maleic acid) polymer which forms nanodiscs while showing the ability to chelate metal ions. Cu(2+)-chelated nanodiscs are demonstrated to reduce the T(1) of protons for both polymer and lipid-nanodisc components. The chelated nanodiscs also decrease the proton T(1) values for a water-soluble DNA G-quadruplex. These results suggest that polymer nanodiscs functionalized with paramagnetic tags can be used to speed-up data acquisition from lipid bilayer samples and also to provide structural information from water-soluble biomolecules.

Experimentally measured residual dipolar couplings (RDCs) are highly valuable for atomic-resolution structural and dynamic studies of molecular systems ranging from small molecules to large proteins by solution NMR spectroscopy. Here we demonstrate the first use of magnetic-alignment behavior of lyotropic liquid-crystalline polymer macro-nanodiscs (>20 nm in diameter) as a novel alignment medium for the measurement of RDCs using high-resolution NMR. The easy preparation of macro-nanodiscs, their high stability against pH changes and the presence of divalent metal ions, and their high homogeneity make them an efficient tool to investigate a wide range of molecular systems including natural products, proteins, and RNA.

Despite lipid-nanodiscs are increasingly used in the structural studies of membrane proteins, drug delivery and other applications, the interaction between the nanodisc-belt and the protein to be reconstituted is a major limitation. To overcome this limitation and to further broaden the scope of nanodiscs, a family of non-ionic amphiphilic polymers synthesized by hydrophobic functionalization of fructo-oligosaccharides/inulin is reported. We show the stability of lipid-nanodiscs formed by these polymers against pH and divalent metal ions, and their magnetic-alignment properties. The reported results also demonstrate that the non-ionic polymers extract membrane proteins with unprecedented efficiency.

Natural-abundance (17)O NMR experiments are used to investigate the hydrated water in magnetically alligned synthetic polymer based lipid-nanodiscs. Residual quadrupole couplings (RQCs) measured from the observed five (17)O (central and satellite) transitions, and molecular dynamics simulations, are used to probe the ordering of water moecules across the lipid bilayer.

Lipid bilayer nanodiscs serve as excellent membrane mimetics for structural and functional studies of mem-brane proteins. Peptide-based lipid nanodiscs are unique in rendering the flexibility in size and exchange of membrane components. While the ability to form flexible size lipid-nanodiscs has been well utilized in the structural studies of membrane-bound protein-protein complexes and to demonstrate the formation of lipid domains, mechanistic insights into these processes have been unknown. Here, for the first time, the dynamic nature of peptide-based lipid-nanodiscs is investigated by high speed atomic force microscopy (HS-AFM), which is capable of acquiring several images per second. 31P NMR, SEC and DLS were also used to monitor the association of lipid nanodiscs. The real time monitoring of nanodiscs in unprecedented time and space resolu-tions provides evidence for nanodisc association and the time scale of their fusion. This study provides the first experimental real-time direct evidence for the n...

Although membrane environment is known to boost drug metabolism by mammalian cytochrome P450s, the factors that stabilize the structural folding and enhance protein function are unclear. In this study, we use peptide-based lipid nanodiscs to "trap" the lipid boundaries of microsomal cytochrome P450 2B4. We report the first evidence that CYP2B4 is able to induce the formation of raft domains in a biomimetic compound of the endoplasmic reticulum. NMR experiments were used to identify and quantitatively determine the lipids present in nanodiscs. A combination of biophysical experiments and molecular dynamics simulations revealed a sphingomyelin binding region in CYP2B4. The protein-induced lipid raft formation increased the thermal stability of P450 and dramatically altered ligand binding kinetics of the hydrophilic ligand BHT. These results unveil membrane/protein dynamics that contribute to the delicate mechanism of redox catalysis in lipid membrane.

Polymer lipid nanodiscs have provided an invaluable system for structural and functional studies of membrane proteins in their near-native environment. In spite of the recent advances in the development and usage of polymer lipid nanodisc systems, lack of control over size and poor tolerance to pH and divalent metal ions are major limitations for further applications. Here we report a facile modification of a low molecular weight styrene maleic acid copolymer to form monodispersed lipid bilayer nanodiscs that show ultra-stability towards a pH range of 2.5 to 10 and divalent metal ion concentration. The macro-nanodiscs (>20 nm diameter) show magnetic-alignment properties that can be exploited for high-resolution structural studies of membrane proteins and amyloid proteins using solid-state NMR techniques. As demonstrated in this study, the new polymer, SMA-QA, nanodisc is a robust membrane mimetic tool that offers significant advantages over currently reported nanodisc systems.

Lipids are critical for the function of membrane proteins. NADPH-cytochrome-P450-reductase, the sole electron transferase for microsomal oxygenases, possesses a conformational dynamics entwined with its topology. Here, we use peptide-nanodiscs to unveil cytochrome-P450-reductase's lipid boundaries, demonstrating a protein-driven enrichment of ethanolamine lipids (by 25%) which ameliorates by 3-fold CPR's electron-transfer ability.

There is a growing interest in the use of lipid bilayer nanodiscs for various biochemical and biomedical applications. Among the different types of nanodiscs, the unique features of synthetic polymer-based nanodiscs have attracted additional interest. A styrene-maleic acid (SMA) copolymer demonstrated to form lipid nanodiscs has been used for structural biology related studies on membrane proteins. However, the application of SMA polymer based lipid nanodiscs is limited because of the strong absorption of the aromatic group interfering with various experimental measurements. Thus, there is considerable interest in the development of other molecular frameworks for the formation of polymer-based lipid nanodiscs. In this study, we report the first synthesis and characterization of a library of polymethacrylate random copolymers as alternatives to SMA polymer. In addition, we experimentally demonstrate the ability of these polymers to form lipid bilayer nanodiscs through the fragmentation of lipid vesicles by means of light scattering, electron microscopy, differential scanning calorimetry, and solution and solid-state NMR experiments. We further demonstrate a unique application of the newly developed polymer for kinetics and structural characterization of the aggregation of human islet amyloid polypeptide (also known as amylin) within the lipid bilayer of the polymer nanodiscs using thioflavin-T-based fluorescence and circular dichroism experiments. Our results demonstrate that the reported new styrene-free polymers can be used in high-throughput biophysical experiments. Therefore, we expect that the new polymer nanodiscs will be valuable in the structural studies of amyloid proteins and membrane proteins by various biophysical techniques.

Polymer-based nanodiscs are valuable tools in biomedical research that can offer a detergent-free solubilization of membrane proteins maintaining their native lipid environment. Here, we introduce a novel ~1.6 kDa SMA-based polymer with styrene:maleic acid moieties that can form nanodiscs containing a planar lipid bilayer which are useful to reconstitute membrane proteins for structural and functional studies. The physicochemical properties and the mechanism of formation of polymer-based nanodiscs are characterized by light scattering, NMR, FT-IR, and TEM imaging experiments. A remarkable feature is that nanodiscs of different sizes, from nanometer to sub micrometer diameter, can be produced only by varying the lipid-to-polymer ratio. As demonstrated, the small size nanodiscs (up to ~30 nm diameter) can be used for solution NMR studies whereas the magnetic-alignment of macro-nanodiscs (diameter of >~40 nm) can be exploited for solid-state NMR studies on membrane proteins.

Microsomal cytochrome b5 (cytb5) is a membrane-bound protein capable of donating the second electron to cytochrome P450s (CYP P450s) in the CYP P450s monooxygenase reactions. Recent studies have demonstrated the importance of the transmembrane domain of cytb5 in the interaction with cytP450 by stabilizing its monomeric structure. While recent NMR studies have provided high-resolution insights into the structural interactions between the soluble domains of ~16-kDa cytb5 and ~57-kDa cytP450 in a membrane environment, there is need for studies to probe the residues in the transmembrane region as well as to obtain intermolecular distance constraints to better understand the very large size cytb5-cytP450 complex structure in a near native membrane environment. In this study, we report the expression, purification, functional reconstitution of19F-labeled full-length rabbit cytb5 in peptide based nanodiscs for structural studies using NMR spectroscopy. Size exclusion chromatography, dynamic light scattering, transmission electron microscopy, and NMR experiments show a stable reconstitution of cytb5 in 4F peptide-based lipid-nanodiscs. Our results demonstrate that the use of peptide-nanodiscs containing cytb519F-labeled with 5-fluorotryptophan (5FW) enables, for the first time, the detection of residues from the transmembrane domain in different lipid compositions by solution19F NMR experiments.19F NMR results revealing the interaction of the transmembrane domain of cytb5 with the full-length rabbit Cytochrome P450 2B4 (CYP2B4) are also presented. We expect the results presented in this study to be useful to devise approaches to probe the structure, dynamics and functional roles of transmembrane domains of a membrane protein, and also to measure intermolecular19F-19F distance constraints to determine the structural interactions between the transmembrane domains.

The advent of ultrahigh magnetic field and fast magic-angle-spinning (MAS) probe technology has led to dramatically enhanced spectral resolution and sensitivity in solid-state NMR spectroscopy. In particular, proton-based multidimensional solid-state NMR techniques have become feasible to investigate the structure and dynamics at atomic resolution, due to the increased chemical shift span and spectral resolution. Herein, the benefits of faster MAS and higher magnetic field are demonstrated on a self-assembled diphenylalanine (Phe-Phe) nanomaterial. Proton-detected 2D 1H/1H single-quantum/single-quantum (SQ/SQ) correlation, double-quantum/single-quantum (DQ/SQ) correlation, and 1H chemical shift anisotropy/chemical shift (CSA/CS) correlation spectra obtained at two different spinning speeds (60 and 100 kHz) and two different magnetic fields (600 and 900 MHz) are reported. The dramatic enhancement of proton spectral resolution achieved with the use of a 900 MHz magnetic field and 100 kHz MAS is remarkable and enabled the measurement of proton CSA tensors, which will be useful to better understand the self-assembled structures of Phe-Phe nanotubes. We also show through numerical simulations that the unaveraged proton-proton dipolar couplings can result in broadening of CSA lines, leading to inaccurate determination of CSA tensors of protons. Thus, our results clearly show the insufficiency of a 600 MHz magnetic field to resolve 1H spectra lines and the inability of a moderate spinning speed of 60 kHz to completely suppress 1H–1H dipolar couplings, which further justify the pursuit of ultrahigh magnetic field beyond 1 GHz and ultrafast MAS beyond 100 kHz.

The ability of amphipathic polymers to self-assemble with lipids and form nanodiscs has been a boon for the field of functional reconstitution of membrane proteins. In a field dominated by detergent micelles, a unique feature of polymer nanodiscs is their much-desired ability to align in the presence of an external magnetic field. Magnetic alignment facilitates the application of solid-state NMR spectroscopy and aids in the measurement of residual dipolar couplings (RDCs) via well-established solution NMR spectroscopy. In this study, we comprehensively investigate the magnetic-alignment properties of SMA-QA polymer based nanodiscs by using 31P and 14N solid-state NMR experiments under static conditions. The results reported herein demonstrate the spontaneous magnetic-alignment of large-size (≥ 20 nm diameter) SMA-QA nanodiscs (also called as macro-nanodiscs) with the lipid-bilayer-normal perpendicular to the magnetic field direction. Consequently, the orientation of macro-nanodiscs are further shown to flip their alignment axis parallel to the magnetic field direction upon the addition of a paramagnetic lanthanide salt. These results demonstrate the use of SMA-QA polymer nanodiscs for solid-state NMR applications including structural studies on membrane proteins.

Residual dipolar couplings (RDCs) have been shown to be valuable for the structural studies of systems ranging from small molecules to large proteins. Here we demonstrate the lyotropic liquid crystal behavior of polymer macro-nanodiscs (> 20 nm in diameter) and enable the measurement of RDCs using high resolution NMR.

Investigating the interplay in a minimal redox complex of cytochrome-P450 and its reductase is crucial for understanding cytochrome-P450's enzymatic activity. Probing the hotspots of dynamic structural interactions using NMR revealed the engagement of loop residues from P450-reductase to be responsible for the enhanced affinity of CYP450 towards its obligate redox partner.

There is considerable interest in the development of membrane mimetics to study the structure, dynamics and function of membrane proteins. Polymer nanodiscs have been useful as a membrane mimetic by not only providing a native-like membrane environment, but also have the ability to extract the desired membrane protein directly from the cell membrane. In spite of such great potential, polymer nanodiscs have their disadvantages including lack of size control and instability at low pH and with divalent metals. In this review, we discuss how these limitations have been overcome by simple modifications of synthetic polymers commonly used to form nanodiscs. Recently, size control has been achieved using an ethanolamine functionalization of a low molecular weight polymer. This size control enabled the use of polymer-based lipid-nanodiscs in solution NMR and macro-nanodiscs in solid-state NMR applications. The introduction of quaternary ammonium functional groups has been shown to improve the stability in the presence of low pH and divalent metal ions, forming highly monodispersed nanodiscs. The polymer charge has been shown to play a significant role on the reconstitution of membrane proteins due to the high charge density on the nanodisc's belt. These recent developments have expanded the applications of polymer nanodiscs to study the membrane proteins using wide variety of techniques including NMR, Cryo-EM and other biophysical techniques.

Although there is a growing interest in using polymer lipid-nanodiscs, the polymer charge poses limitations for studies on membrane proteins. Here, we demonstrate the functional reconstitution of a large soluble-domain containing positively-charged ∼57 kDa cytochrome-P450 and negatively-charged ∼16 kDa cytochrome-b5 in lipid-nanodiscs, and the role of the polymer charge for high-resolution studies on membrane proteins.

Polymer lipid nanodiscs have enabled some exciting structural biology and nanobiotechnology applications. The use of a small molecular weight polymer (SMA-EA) has been demonstrated to dramatically increase the size of nanodiscs (up to ∼60 nm diameter). Here, we report the first demonstration of the formation of macro-nanodiscs for a variety of lipids, and solid-state NMR experiments utilizing their magnetic-alignment properties.

Heme's spin-multiplicity is key in determining the enzymatic function of cytochrome P450 (cytP450). The origin of the low-spin state in ferric P450 is still under debate. Here, we report the first experimental demonstration of P450's membrane interaction altering its spin equilibrium which is accompanied by a stronger affinity for cytochrome b5. These results highlight the importance of lipid membrane for the function of P450.

Polymer nanodisks have shown great potential as membrane mimetics that enable the study of functional membrane protein structural biology and also have a wider application in other fields such as drug delivery. To achieve these research goals, the ability to have a cheap, simple, fully customizable platform for future nanodisks technology applications is paramount. Here, a facile functionalization of polyacrylic acid (PAA) with varying hydrophobic groups that form nanodisks at different sizes is successfully demonstrated. The study shows that the choice of hydrophobic group can have a noticeable effect on the polymer solubilization properties and polymer-induced perturbation to the encased lipid bilayer. Due to this robust, tunable chemical synthesis method, PAA is an exciting platform for the future optimization of the hydrophobic, hydrophilic, or direct purposed functionalizations for polymer nanodisks.

Retraction of ‘Polymer nanodiscs and macro-nanodiscs of a varying lipid composition’ by Venkata Sudheer Kumar Ramadugu et al., Chem. Commun., 2017, 53, 10824–10826.