Structural and Mechanistic Evidence for Calcium Interacting Sites in the HIV Transmembrane Protein gp41 Involved in Membrane Fusion.

The HIV envelope protein gp160 comprises two subunits, gp120 and gp41, responsible for receptor binding and membrane fusion during viral entry, respectively. In the course of the membrane fusion process, gp41 undergoes a conformational change, leading to the formation of a six-helix bundle (SHB), which ultimately drives membrane fusion. The gp41 C-terminal and N-terminal heptad repeats (CHR and NHR) interact with one another to form the SHB, and this step can be targeted by peptide inhibitors, which are used in the clinic to mitigate HIV infection. Here, we discover the calcium interaction motifs (CIMs) in the gp41 CHR and NHR regions via NMR spectroscopy. We find that the assembly of the CHR-NHR SHB is facilitated in Ca2+-containing media and impaired in CIM mutants. Of note, the clinically approved, gp41-derived fusion inhibitor T20, which does not contain the CIM motif, exhibits reduced inhibitory efficiency when challenged with calcium. This finding could have important implications for the development of better fusion inhibitors for HIV.

Non-Ionic Inulin-Based Polymer Nanodiscs Enable Functional Reconstitution of a Redox Complex Composed of Oppositely Charged CYP450 and CPR in a Lipid Bilayer Membrane.

Although polymer-based lipid nanodiscs are increasingly used in the structural studies of membrane proteins, the charge of the belt-forming polymer is a major limitation for functional reconstitution of membrane proteins possessing an opposite net charge to that of the polymer. This limitation also rules out the reconstitution of a protein-protein complex composed of oppositely charged membrane proteins. In this study, we report the first successful functional reconstitution of a membrane-bound redox complex constituting a cationic cytochrome P450 (CYP450) and an anionic cytochrome P450 reductase (CPR) in non-ionic inulin-based lipid nanodiscs. The gel-to-liquid-crystalline phase-transition temperature (Tm) of DMPC:DMPG (7:3 w/w) lipids in polymer nanodiscs was determined by differential scanning calorimetry (DSC) and 31P NMR experiments. The CYP450-CPR redox complex reconstitution in polymer nanodiscs was characterized by size-exclusion chromatography (SEC), and the electron transfer kinetics was carried out using the stopped-flow technique under anaerobic conditions. The Tm of DMPC:DMPG (7:3 w/w) in polymer nanodiscs measured from 31P NMR agrees with that obtained from DSC and was found to be higher than that for liposomes due to the decreased cooperativity of lipids present in the nanodiscs. The stopped-flow measurements revealed the CYP450-CPR redox complex reconstituted in nanodiscs to be functional, and the electron transfer kinetics was found to be temperature-dependent. Based on the successful demonstration of the use of non-ionic inulin-based polymer nanodiscs reported in this study, we expect them to be useful in studying the function and structures of a variety of membrane proteins/complexes irrespective of the charge of the molecular components. Since the polymer nanodiscs were shown to align in an externally applied magnetic field, they can also be used to measure residual dipolar couplings (RDCs) and residual quadrupolar couplings (RQCs) for various molecules ranging from small molecules to soluble proteins and nucleic acids.

Measurement of Residual Dipolar Couplings Using Magnetically Aligned and Flipped Nanodiscs.

Recent developments in lipid nanodisc 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 has 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 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipids, were used to measure residual 1H-15N dipolar couplings (RDCs) from the water-soluble ∼21 kDa uniformly 15N-labeled flavin mononucleotide binding domain (FBD) of cytochrome-P450 reductase. The experimentally measured 1H-15N RDC values are compared with the values calculated from the 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 (3 cos2 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.

Solid-State NMR Study to Probe the Effects of Divalent Metal Ions (Ca2+ and Mg2+) on the Magnetic Alignment of Polymer-Based Lipid Nanodiscs.

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.

Synthesis, Characterization, and Nanodisc Formation of Non-ionic Polymers*.

Although 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.

Magnetic Alignment of Polymer Nanodiscs Probed by Solid-State NMR Spectroscopy.

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 31P and 14N 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.

Hydrophobic Functionalization of Polyacrylic Acid as a Versatile Platform for the Development of Polymer Lipid Nanodisks.

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.

Magnetic Alignment of Polymer Macro-Nanodiscs Enables Residual-Dipolar-Coupling-Based High-Resolution Structural Studies by NMR Spectroscopy.

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.

Metal-Chelated Polymer Nanodiscs for NMR Studies.

Paramagnetic relaxation enhancement (PRE) is commonly used to speed up spin lattice relaxation time (T1 ) 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. Cu2+ -chelated nanodiscs are demonstrated to reduce the T1 of protons for both polymer and lipid-nanodisc components. The chelated nanodiscs also decrease the proton T1 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.

Styrene maleic acid derivates to enhance the applications of bio-inspired polymer based lipid-nanodiscs.

Membrane mimetics are essential to study the structure, dynamics and function of membrane-associated proteins by biophysical and biochemical approaches. Among various membrane mimetics that have been developed and demonstrated for studies on membrane proteins, lipid nanodiscs are the latest developments in the field and are increasingly used for various applications. While lipid-nanodiscs can be formed using an amphipathic membrane scaffold protein (MSP), peptide, or synthetic polymer, the synthetic polymer based nanodiscs exhibit unique advantages because of the ability to functionalize them for various applications. In addition to the use of synthetic polymers to extract membrane proteins directly from the cell membranes, recent advances in the development of polymers used for nanodiscs formation are attracting new attention to the field of nanodiscs technology. Here we review the developments of novel polymer modifications that overcome the current limitations and enhance the applications of polymer based nanodiscs to a wider variety of biophysical techniques used to study membrane proteins. A summary of the functionalization of poly(Styrene-co-Maleic Acid), SMA, polymers developed by our research and their advantages are also covered in this review article.

Formation of pH-Resistant Monodispersed Polymer-Lipid Nanodiscs.

Polymer lipid nanodiscs are an invaluable system for structural and functional studies of membrane proteins in their near-native environment. Despite 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. A facile modification of a low-molecular-weight styrene maleic acid copolymer is demonstrated to form monodispersed lipid bilayer nanodiscs that show ultra-stability towards divalent metal ion concentration over a pH range of 2.5 to 10. 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. The new polymer, SMA-QA, nanodisc is a robust membrane mimetic tool that offers significant advantages over currently reported nanodisc systems.

Cytochrome-P450-Induced Ordering of Microsomal Membranes Modulates Affinity for Drugs.

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.

Spontaneous Lipid Nanodisc Fomation by Amphiphilic Polymethacrylate Copolymers.

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.

pH Tunable and Divalent Metal Ion Tolerant Polymer Lipid Nanodiscs.

The development and applications of detergent-free membrane mimetics have been the focus for the high-resolution structural and functional studies on membrane proteins. The introduction of lipid nanodiscs has attracted new attention toward the structural biology of membrane proteins and also enabled biomedical applications. Lipid nanodiscs provide a native lipid bilayer environment similar to the cell membrane surrounded by a belt made up of proteins or peptides. Recent studies have shown that the hydrolyzed form of styrene maleic anhydride copolymer (SMA) has the ability to form lipid nanodiscs and has several advantages over protein or peptide based nanodiscs. SMA polymer lipid nanodiscs have become very important for structural biology and nanobiotechnological applications. However, applications of the presently available polymer nanodiscs are limited by their instability toward divalent metal ions and acidic conditions. To overcome the limitations of SMA nanodiscs and to broaden the potential applications of polymer nanodiscs, the present study investigates the tunability of SMA polymer nanodiscs by systematically modifying the maleic acid functional group. The two newly developed polymers and subsequent lipid nanodiscs were characterized using solid-state NMR, FT-IR, TEM, and DLS experiments. The pH dependence and metal ion stability of these nanodiscs were studied using static light scattering and FTIR. The reported polymer nanodiscs exhibit unique pH dependent stability based on the modified functional group and show a high tolerance toward divalent metal ions. We also show these tunable nanodiscs can be used to encapsulate and stabilize a polyphenolic natural product curcumin.

Bioinspired, Size-Tunable Self-Assembly of Polymer-Lipid Bilayer Nanodiscs.

Polymer-based nanodiscs are valuable tools in biomedical research that can offer a detergent-free solubilization of membrane proteins maintaining their native lipid environment. Herein, we introduce a novel ca. 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. A remarkable feature is that nanodiscs of different sizes, from nanometer to sub-micrometer diameter, can be produced by varying the lipid-to-polymer ratio. The small-size nanodiscs (up to ca. 30 nm diameter) can be used for solution NMR spectroscopy studies whereas the magnetic-alignment of macro-nanodiscs (diameter of > ca. 40 nm) can be exploited for solid-state NMR studies on membrane proteins.

Factors influencing the detergent-free membrane protein isolation using synthetic nanodisc-forming polymers.

The detergent-free isolation of membrane proteins using synthetic polymers is becoming the desired approach for functional and structural studies of membrane proteins. Since the expression levels for many membrane proteins are low and a high yield of functionalized reconstituted membrane proteins is essential for in vitro studies, it is crucial to optimize the experimental conditions for a given polymer to solubilize target membranes/proteins effectively. The factors that affect membrane solubilization and subsequently the isolation of a target membrane protein include polymer concentration, polymer charge, temperature, pH, and concentration of divalent metal ions. Therefore, it is important to have knowledge about the efficacy of different types of polymers in solubilizing cell membranes. In this study, we evaluate the efficacy of inulin-based non-ionic polymers in solubilizing E. coli membranes enriched with rat flavin mononucleotide binding-domain (FBD) of cytochrome-P450-reductase (CPR) and rabbit cytochrome-b5 (Cyt-b5) under various solubilization conditions. Our results show that a 1:1 (w/w) membrane:polymer ratio, low temperature, high pH and sub-millimolar concentration of metal ions favor the solubilization of E. coli membranes enriched with FBD or Cyt-b5. Conversely, the presence of excess divalent metal ions affected the final protein levels in the polymer-solubilized samples. We believe that the results from this study provide knowledge to assess and plan the use of non-ionic polymers in membrane protein studies.

Enhancing the stability and homogeneity of non-ionic polymer nanodiscs by tuning electrostatic interactions.

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.

Factors influencing the detergent-free membrane protein isolation using synthetic nanodisc-forming polymers.

The detergent-free isolation of membrane proteins using synthetic polymers is becoming the desired approach for functional and structural studies of membrane proteins. Since the expression levels for many membrane proteins are low and a high yield of functionalized reconstituted membrane proteins is essential for in vitro studies, it is crucial to optimize the experimental conditions for a given polymer to effectively solubilize target membranes/proteins. The factors that affect membrane solubilization and subsequently the isolation of a target membrane protein include polymer concentration, polymer charge, temperature, pH, and concentration of divalent metal ions. Therefore, it is important to have knowledge about the efficacy of different types of polymers in solubilizing cell membranes. In this study, we evaluate the efficacy of inulin-based non-ionic polymers in solubilizing E. coli membranes enriched with rat flavin mononucleotide binding-domain (FBD) of cytochrome-P450-reductase (CPR) and rabbit cytochrome-b5 (Cyt-b5) under various solubilization conditions. Our results show that a 1:1 (w/w) membrane:polymer ratio, low temperature, high pH and sub-millimolar concentration of metal ions favor the solubilization of E. coli membrane enriched with FBD or Cyt-b5. Conversely, the presence of excess divalent metal ions affected the final protein levels in the polymer-solubilized samples. We believe that the results from this study provides knowledge to assess and plan the use of non-ionic polymers in membrane protein studies.