Reviving Natural Rubber Synthesis via Native/Large Nanodiscs.

Natural rubber (NR) is utilized in more than 40,000 products, and the demand for NR is projected to reach $68.5 billion by 2026. The primary commercial source of NR is the latex of Hevea brasiliensis. NR is produced by the sequential cis-condensation of isopentenyl diphosphate (IPP) through a complex known as the rubber transferase (RTase) complex. This complex is associated with rubber particles, specialized organelles for NR synthesis. Despite numerous attempts to isolate, characterize, and study the RTase complex, definitive results have not yet been achieved. This review proposes an innovative approach to overcome this longstanding challenge. The suggested method involves isolating the RTase complex without using detergents, instead utilizing the native membrane lipids, referred to as "natural nanodiscs", and subsequently reconstituting the complex on liposomes. Additionally, we recommend the adaptation of large nanodiscs for the incorporation and reconstitution of the RTase complex, whether it is in vitro transcribed or present within the natural nanodiscs. These techniques show promise as a viable solution to the current obstacles. Based on our experimental experience and insights from published literature, we believe these refined methodologies can significantly enhance our understanding of the RTase complex and its role in in vitro NR synthesis.

Structures and Dynamics of Native-State Transmembrane Protein Targets and Bound Lipids.

Membrane proteins work within asymmetric bilayers of lipid molecules that are critical for their biological structures, dynamics and interactions. These properties are lost when detergents dislodge lipids, ligands and subunits, but are maintained in native nanodiscs formed using styrene maleic acid (SMA) and diisobutylene maleic acid (DIBMA) copolymers. These amphipathic polymers allow extraction of multicomponent complexes of post-translationally modified membrane-bound proteins directly from organ homogenates or membranes from diverse types of cells and organelles. Here, we review the structures and mechanisms of transmembrane targets and their interactions with lipids including phosphoinositides (PIs), as resolved using nanodisc systems and methods including cryo-electron microscopy (cryo-EM) and X-ray diffraction (XRD). We focus on therapeutic targets including several G protein-coupled receptors (GPCRs), as well as ion channels and transporters that are driving the development of next-generation native nanodiscs. The design of new synthetic polymers and complementary biophysical tools bodes well for the future of drug discovery and structural biology of native membrane:protein assemblies (memteins).

Multisite interactions of prions with membranes and native nanodiscs.

Although prions are known as protein-only infectious particles, they exhibit lipid specificities, cofactor dependencies and membrane-dependent activities. Such membrane interactions play key roles in how prions are processed, presented and regulated, and hence have significant functional consequences. The expansive literature related to prion protein interactions with lipids and native nanodiscs is discussed, and provides a unique opportunity to re-evaluate the molecular composition and mechanisms of its infectious and cellular states. A family of crystal and solution structures of prions are analyzed here for the first time using the membrane optimal docking area (MODA) program, revealling the presence of structured binding elements that could mediate specific lipid recognition. A set of motifs centerred around W99, L125, Y169 and Y226 are consistently predicted as being membrane interactive and form an exposed surface which includes α helical, ß strand and loop elements involving the prion protein (PrP) structural domain, while the scrapie form is radically different and doubles the size of the membrane interactive site into an extensible surface. These motifs are highly conserved throughout mammalian evolution, suggesting that prions have long been intrinsically attached to membranes at central and N- and C-terminal points, providing several opportunities for stable and specific bilayer interactions as well as multiple complexed orientations. Resistance or susceptibility to prion disease correlates with increased or decreased membrane binding propensity by mutant forms, respectively, indicating a protective role by lipids. The various prion states found in vivo are increasingly resolvable using native nanodiscs formed by styrene maleic acid (SMA) and stilbene maleic acid (STMA) copolymers rather than classical detergents, allowing the endogenous states to be tackled. These copolymers spontaneously fragment intact membranes into water-soluble discs holding a section of native bilayer, and can accommodate prion multimers and mini-fibrils. Such nanodiscs have also proven useful for understanding how ß amyloid and α synuclein proteins contribute to Alzheimer's and Parkinson's diseases, providing further biomedical applications. Structural and functional insights of such proteins in styrene maleic acid lipid particles (SMALPs) can be resolved at high resolution by methods including cryo-electron microscopy (cEM), motivating continued progress in polymer design to resolve biological and pathological mechanisms.

Polymer-Encased Nanodiscs and Polymer Nanodiscs: New Platforms for Membrane Protein Research and Applications.

Membrane proteins (MPs) are essential to many organisms' major functions. They are notorious for being difficult to isolate and study, and mimicking native conditions for studies in vitro has proved to be a challenge. Lipid nanodiscs are among the most promising platforms for MP reconstitution, but they contain a relatively labile lipid bilayer and their use requires previous protein solubilization in detergent. These limitations have led to the testing of copolymers in new types of nanodisc platforms. Polymer-encased nanodiscs and polymer nanodiscs support functional MPs and address some of the limitations present in other MP reconstitution platforms. In this review, we provide a summary of recent developments in the use of polymers in nanodiscs.

Native nanodiscs formed by styrene maleic acid copolymer derivatives help recover infectious prion multimers bound to brain-derived lipids.

Prions are lipidated proteins that interact with endogenous lipids and metal ions. They also assemble into multimers and propagate into the infectious scrapie form known as PrPSc The high-resolution structure of the infectious PrPSc state remains unknown, and its analysis largely relies on detergent-based preparations devoid of endogenous ligands. Here we designed polymers that allow isolation of endogenous membrane:protein assemblies in native nanodiscs without exposure to conventional detergents that destabilize protein structures and induce fibrillization. A set of styrene-maleic acid (SMA) polymers including a methylamine derivative facilitated gentle release of the infectious complexes for resolution of multimers, and a thiol-containing version promoted crystallization. Polymer extraction from brain homogenates from Syrian hamsters infected with Hyper prions and WT mice infected with Rocky Mountain Laboratories prions yielded infectious prion nanoparticles including oligomers and microfilaments bound to lipid vesicles. Lipid analysis revealed the brain phospholipids that associate with prion protofilaments, as well as those that are specifically enriched in prion assemblies captured by the methylamine-modified copolymer. A comparison of the infectivity of PrPSc attached to SMA lipid particles in mice and hamsters indicated that these amphipathic polymers offer a valuable tool for high-yield production of intact, detergent-free prions that retain in vivo activity. This native prion isolation method provides an avenue for producing relevant prion:lipid targets and potentially other proteins that form multimeric assemblies and fibrils on membranes.

The effect of hydrophobic alkyl sidechains on size and solution behaviors of nanodiscs formed by alternating styrene maleamic copolymer.

The development of amphipathic polymers, including various formulations of styrene-maleic acid (SMA) copolymers, has allowed the purification of increasing sizes and complexities of biological membrane protein assemblies in native nanodiscs. However, the factors determining the sizes and shapes of the resulting bio-nano particles remain unclear. Here, we show how grafting on short alkyl amine sidechains onto the polar residues leads to a broad set of nanoparticle sizes with improved solution behavior. The solubilization of lipid vesicles occurs over a wide range of pH levels and calcium concentrations, providing utility across the physiologically relevant range of solution conditions. Furthermore, the active SMA derivatives can contain strictly alternating monomers, which have inherently lower sequence polydispersity. Pronounced differences in the shapes of native nanoparticles were formed from Escherichia coli bacterial outer membrane containing PagP protein using methyl, ethyl and propylamine derivatives of styrene-maleic anhydride. In particular, the methylamine-substituted polymer forms smaller, monodispersed nanodiscs, while the longer alkyl derivatives form worm-like nanostructures. Thus the introduction of hydrophobicity onto the polar sidechains of amphipathic polymers has profound effects on morphology of native nanodisc, with shorter methyl moieties offering more uniformity and utility for structural biology studies.

Factors influencing the solubilization of membrane proteins from Escherichia coli membranes by styrene-maleic acid copolymers.

Styrene-maleic acid (SMA) copolymers are a promising alternative to detergents for the solubilization of membrane proteins. Here we employ Escherichia coli membranes containing KcsA as a model protein to investigate the influence of different environmental conditions on SMA solubilization efficiency. We show that SMA concentration, temperature, incubation time, ionic strength, presence of divalent cations and pH all influence the amount of protein that is extracted by SMA. The observed effects are consistent with observations from lipid-only model membrane systems, with the exception of the effect of pH. Increasing pH from 7 to 9 was found to result in an increase of the solubilization yield of E. coli membranes, whereas in lipid-only model systems it decreased over the same pH range, based on optical density (OD) measurements. Similar opposite pH-dependent effects were observed in OD experiments comparing solubilization of native yeast membranes and yeast lipid-only membranes. We propose a model in which pH-dependent electrostatic interactions affect binding of the polymers to extramembraneous parts of membrane proteins, which in turn affects the availability of polymer for membrane solubilization. This model is supported by the observations that a similar pH-dependence as for SMA is observed for the anionic detergent SDS, but not for the nonionic detergent DDM and that the pH-dependence can be largely overcome by increasing the SMA concentration. The results are useful as guidelines to derive optimal conditions for solubilization of biological membranes by SMA.

Structures and Interactions of Transmembrane Targets in Native Nanodiscs.

Transmembrane proteins function within a continuous layer of biologically relevant lipid molecules that stabilizes their structures and modulates their activities. Structures and interactions of biological membrane-protein complexes or "memteins" can now be elucidated using native nanodiscs made by poly(styrene co-maleic anhydride) derivatives. These linear polymers contain a series of hydrophobic and polar subunits that gently fragment membranes into water-soluble discs with diameters of 5-50 nm known as styrene maleic acid lipid particles (SMALPs). High-resolution structures of memteins that include endogenous lipid ligands and posttranslational modifications can be resolved without resorting to synthetic detergents or artificial lipids. The resulting ex situ structures better recapitulate the in vivo situation and can be visualized by methods including cryo-electron microscopy (cryoEM), electron paramagnetic resonance (EPR), mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, small angle x-ray scattering (SAXS), and x-ray diffraction (XRD). Recent progress including 3D structures of biological bilayers illustrates how polymers and native nanodiscs expose previously inaccessible membrane assemblies at atomic resolution and suggest ways in which the SMALP system could be exploited for drug discovery.

Memtein: The fundamental unit of membrane-protein structure and function.

The concept of a memtein as the minimal unit of membrane function is proposed here, and refers to the complex of a membrane protein together with a continuous layer of biological lipid molecules. The elucidation of the atomic resolution structures and specific interactions within memteins remains technically challenging. Nonetheless, we argue that these entities are critical endpoints for the postgenomic era, being essential units of cellular function that mediate signal transduction and trafficking. Their biological mechanisms and molecular compositions can be resolved using native nanodiscs formed by poly(styrene-co-maleic acid) (SMA) copolymers. These amphipathic polymers rapidly and spontaneously fragment membranes into water-soluble discs holding a section of bilayer. This allows structures of complexes found in vivo to be prepared without resorting to synthetic detergents or artificial lipids. The ex situ structures of memteins can be resolved by methods including cryo-electron microscopy (cEM), X-ray crystallography (XRC), NMR spectroscopy and mass spectrometry (MS). Progress in the field demonstrates that memteins are better representations of how biology actually works in membranes than naked proteins devoid of lipid, spurring on further advances in polymer chemistry to resolve their details.