maleic-acid and diisobutylene

Styrene-maleic acid copolymers (SMAs), and related amphiphilic copolymers, are promising tools for isolating and studying integral membrane proteins in a native-like state. However, they do not exhibit this ability universally, as several reports have found that SMAs and related amphiphilic copolymers show little to no efficiency when extracting specific membrane proteins. Recently, it was discovered that esterified SMAs could enhance the selective extraction of trimeric Photosystem I from the thylakoid membranes of thermophilic cyanobacteria; however, these polymers are susceptible to saponification that can result from harsh preparation or storage conditions. To address this concern, we herein describe the development of α-olefin-maleic acid copolymers (αMAs) that can extract trimeric PSI from cyanobacterial membranes with the highest extraction efficiencies observed when using any amphiphilic copolymers, including diisobutylene-co-maleic acid (DIBMA) and functionalized SMA samples. Furthermore, we will show that αMAs facilitate the formation of photosystem I-containing nanodiscs that retain an annulus of native lipids and a native-like activity. We also highlight how αMAs provide an agile, tailorable synthetic platform that enables fine-tuning hydrophobicity, controllable molar mass, and consistent monomer incorporation while overcoming shortcomings of prior amphiphilic copolymers.

Topics: Alkenes; Lipid Bilayers; Membrane Proteins; Photosystem I Protein Complex; Polystyrenes; Styrene

The study of membrane proteins remains challenging, especially in a native membrane environment. Recently, major progress has been made using maleic acid copolymers, such as styrene maleic acid, to purify membrane proteins and study them directly with native lipids associated with the membrane. Additional maleic acid copolymers, such as diisobutylene maleic acid (DIBMA) membrane-mimetic systems, are being developed and found to have improved spectroscopic properties and pH stability. We studied DIBMA and its lipid particles in solution to better understand its assembly, without and with the lipids, to provide an insight regarding how to use it in solution for better membrane extraction. Using small-angle neutron and X-ray scattering (SANS/SAXS), we show that DIBMA organizes into structures of different size scales at various concentrations and ionic strengths. The polymer performed reasonably well under most solvent conditions except in very low concentrations and high-salt conditions that could result in limited interaction with lipids. To explore DIBMA lipid particles as a suitable membrane-mimetic system for neutron scattering studies of membrane proteins, we measured and determined the contrast-matching point of DIBMA to be ∼12% (v/v) D

Topics: Alkenes; Biomedical Research; Biomimetics; Dimyristoylphosphatidylcholine; Hydrogen-Ion Concentration; Lipid Bilayers; Maleates; Membrane Proteins; Osmolar Concentration; Polymers

Amphiphilic maleic acid-containing polymers allow for the direct extraction of membrane proteins into stable, homogenous, water-soluble copolymer/lipid nanoparticles without the use of detergents. By adjusting the polymer/lipid ratio, the size of the nanoparticles can be tuned at convenience for the incorporation of protein complexes of different size. However, an increase in the size of the lipid nanoparticles may correlate with increased sample heterogeneity, thus hampering their application to spectroscopic and structural techniques where highly homogeneous samples are desirable. In addition, size homogeneity can be affected by low liposome solubilization efficiency by DIBMA, which carries a negative charge, in the presence of high lipid charge density. In this work, we apply biophysical tools to characterize the size and size heterogeneity of large (above 15 nm) lipid nanoparticles encased by the diisobutylene/maleic acid (DIBMA) copolymer at different DIBMA/lipid ratios and percentages of anionic lipids. Importantly, for nanoparticle preparations in the diameter range of 40 nm or below, the size homogeneity of the DIBMA/lipid nanoparticles (DIBMALPs) remains unchanged. In addition, we show that anionic lipids do not affect the production, size and size homogeneity of DIBMALPs. Furthermore, they do not affect the overall lipid dynamics in the membrane, and preserve the functionality of an enclosed membrane protein. This work strengthens the suitability of DIBMALPs as universal, native-like lipid environments for functional studies of membrane proteins and provide useful insight on the suitability of these systems for those structural techniques requiring highly homogeneous sample preparations.

Topics: Alkenes; Anions; Archaeal Proteins; Electron Spin Resonance Spectroscopy; Halobacteriaceae; Lipid Bilayers; Maleates; Membrane Proteins; Nanoparticles; Particle Size; Recombinant Proteins; Spin Labels

Structure and function analysis of human membrane proteins in lipid bilayer environments is acutely lacking despite the fundame1ntal cellular importance of these proteins and their dominance of drug targets. An underlying reason is that detailed study usually requires a potentially destabilising detergent purification of the proteins from their host membranes prior to subsequent reconstitution in a membrane mimic; a situation that is exacerbated for human membrane proteins due to the inherent difficulties in overexpressing suitable quantities of the proteins. We advance the promising styrene maleic acid polymer (SMA) extraction approach to introduce a detergent-free method of obtaining stable, functional human membrane transporters in bilayer nanodiscs directly from yeast cells. We purify the human serotonin transporter (hSERT) following overexpression in Pichia pastoris using diisobutylene maleic acid (DIBMA) as a superior method to traditional detergents or the more established styrene maleic acid polymer. hSERT plays a pivotal role in neurotransmitter regulation being responsible for the transport of the neurotransmitter 5-hydroxytryptamine (5-HT or serotonin). It is representative of the neurotransmitter sodium symporter (NSS) family, whose importance is underscored by the numerous diseases attributed to their malfunction. We gain insight into hSERT activity through an in vitro transport assay and find that DIBMA extraction improves the thermostability and activity of hSERT over the conventional detergent method.

Topics: Alkenes; Humans; Maleates; Polymers; Protein Stability; Recombinant Proteins; Serotonin; Serotonin Plasma Membrane Transport Proteins; Temperature

Amphiphilic diisobutylene/maleic acid (DIBMA) copolymers extract lipid-encased membrane proteins from lipid bilayers in a detergent-free manner, yielding nanosized, discoidal DIBMA lipid particles (DIBMALPs). Depending on the DIBMA/lipid ratio, the size of DIBMALPs can be broadly varied which makes them suitable for the incorporation of proteins of different sizes. Here, we examine the influence of the DIBMALP sizes and the presence of protein on the dynamics of encased lipids. As shown by a set of biophysical methods, the stability of DIBMALPs remains unaffected at different DIBMA/lipid ratios. Coarse-grained molecular dynamics simulations confirm the formation of viable DIBMALPs with an overall size of up to 35 nm. Electron paramagnetic resonance spectroscopy of nitroxides located at the 5th, 12th or 16th carbon atom positions in phosphatidylcholine-based spin labels reveals that the dynamics of enclosed lipids are not altered by the DIBMALP size. The presence of the membrane protein sensory rhodopsin II from

Topics: Alkenes; Biophysical Phenomena; Dimyristoylphosphatidylcholine; Electron Spin Resonance Spectroscopy; Halobacteriaceae; Halorhodopsins; Lipid Bilayers; Maleates; Microscopy, Atomic Force; Microscopy, Electron, Transmission; Molecular Dynamics Simulation; Nanoparticles; Particle Size; Photochemical Processes; Sensory Rhodopsins; Spin Labels

Amphiphilic maleic acid-containing copolymers account for a recent methodical breakthrough in the study of membrane proteins. Their application enables a detergent-free extraction of membrane proteins from lipid bilayers, yielding stable water-soluble, discoidal lipid bilayer particles with incorporated proteins, which are wrapped with copolymers. Although many studies confirm the potential of this approach for membrane protein research, the interactions between the maleic acid-containing copolymers and extracted lipids, as well as possible effects of the copolymers on lipid-embedded proteins deserve further scrutinization. Here, we combine electron paramagnetic resonance spectroscopy and coarse-grain molecular dynamics simulations to compare the distribution and dynamics of lipids in lipid particles of phospholipid bilayers encased either by an aliphatic diisobutylene/maleic acid copolymer (DIBMALPs) or by an aromatic styrene/maleic acid copolymer (SMALPs). Nitroxides located at the 5th, 12th or 16th carbon atom positions in phosphatidylcholine-based spin labels experience restrictions of their reorientational motion depending on the type of encasing copolymer. The dynamics of the lipids was less constrained in DIBMALPs than in SMALPs with the affinity of spin labeled lipids to the polymeric rim being more pronounced in SMALPs.

Topics: Alkenes; Dimyristoylphosphatidylcholine; Electron Spin Resonance Spectroscopy; Lipid Bilayers; Maleates; Membrane Proteins; Molecular Dynamics Simulation; Nanoparticles; Phosphatidylcholines; Phospholipids; Polymers; Polystyrenes; Spin Labels

The use of styrene maleic acid co-polymer (SMA) for membrane protein extraction and purification has grown in recent years. SMA inserts in the membrane and assembles into small discs of bilayer encircled by polymer, termed SMA lipid particles (SMALPs). This allows purification of membrane proteins whilst maintaining their lipid bilayer environment. SMALPs offer several improvements over conventional detergent approaches, however there are limitations, most notably a sensitivity to low pH and divalent cations. Recently it was shown that the aliphatic diisobutylene-maleic acid (DIBMA) copolymer, was also able to directly solubilise membranes forming DIBMALPs (DIBMA lipid particles), and that this polymer overcame some of the limitations of SMA. In this study the ability of DIBMA to solubilise and purify functional membrane proteins has been compared to SMA. It was found that DIBMA is able to solubilise several different membrane proteins from different expression systems, however for some proteins it gives a lower yield and lower degree of purity than SMA. DIBMA extracted G protein-coupled receptors retain ligand- and G protein-binding. DIBMALPS are larger than SMALPs and display a decreased sensitivity to magnesium. However the stability of DIBMALPs appears to be lower than SMALPs. The lower purity and lower stability are likely linked to the larger size of the DIBMALP particle. However, this also offers a potentially less rigid lipid environment which may be more amenable to protein dynamics. Therefore the optimal choice of polymer will depend on which features of a protein are to be investigated.

Topics: Alkenes; Lipid Bilayers; Lipid Droplets; Maleates; Membrane Proteins; Polystyrenes

α-Synuclein (αsyn) is a cytosolic intrinsically disordered protein (IDP) known to fold into an α-helical structure when binding to membrane lipids, decreasing protein aggregation. Model membrane enable elucidation of factors critically affecting protein folding/aggregation, mostly using either small unilamellar vesicles (SUVs) or nanodiscs surrounded by membrane scaffold proteins (MSPs). Yet SUVs are mechanically strained, while MSP nanodiscs are expensive. To test the impact of lipid particle size on α-syn structuring, while overcoming the limitations associated with the lipid particles used so far, we compared the effects of large unilamellar vesicles (LUVs) and lipid-bilayer nanodiscs encapsulated by diisobutylene/maleic acid copolymer (DIBMA) on αsyn secondary-structure formation, using human-, elephant- and whale -αsyn. Our results confirm that negatively charged lipids induce αsyn folding in h-αsyn and e-αsyn but not in w-αsyn. When a mixture of zwitterionic and negatively charged lipids was used, no increase in the secondary structure was detected at 45 °C. Further, our results show that DIBMA/lipid particles (DIBMALPs) are highly suitable nanoscale membrane mimics for studying αsyn secondary-structure formation and aggregation, as folding was essentially independent of the lipid/protein ratio, in contrast with what we observed for LUVs having the same lipid compositions. This study reveals a new and promising application of polymer-encapsulated lipid-bilayer nanodiscs, due to their excellent efficiency in structuring disordered proteins such as αsyn into nontoxic α-helical structures. This will contribute to the unravelling and modelling aspects concerning protein-lipid interactions and α-helix formation by αsyn, paramount to the proposal of new methods to avoid protein aggregation and disease.

Topics: Alkenes; alpha-Synuclein; Humans; Intrinsically Disordered Proteins; Lipid Bilayers; Maleates; Membrane Lipids; Membrane Proteins; Polymers; Protein Aggregates; Protein Conformation, alpha-Helical; Protein Folding; Protein Structure, Secondary; Unilamellar Liposomes

Some amphiphilic copolymers such as diisobutylene/maleic acid (DIBMA) and styrene/maleic acid (SMA) copolymers are able to directly extract cellular membranes into nanosized polymer-bounded lipid-bilayer patches. These polymer-encapsulated nanodiscs offer the possibility to investigate delicate membrane proteins along with their surrounding lipids and, thus, protein/lipid interactions, in a near-native bilayer environment. By dissecting the kinetics of lipid exchange among DIBMA- and SMA-bounded nanodiscs, we have recently shown that the encapsulated lipid bilayer does not represent a static snapshot of the membrane at the time point of solubilisation. Instead, nanoscale lipid-bilayer patches remain in equilibrium with one another through lipid exchange enabled by nanodisc collisions. This finding is important for correctly interpreting any attempts at studying protein/lipid interactions with the aid of polymer-based nanodiscs and will be relevant to characterising the rapidly growing repertoire of new amphiphilic copolymers for membrane extraction. A highly sensitive and efficient technique for measuring the kinetics of lipid transfer among various kinds of nanosized membrane mimics consists in time-resolved Förster resonance energy transfer (TR-FRET) monitored in a stopped-flow apparatus. Here, we provide detailed instructions on how to measure the kinetics and unravel the underlying mechanisms of lipid exchange among lipid-bilayer nanodiscs under various solution conditions.

Topics: Alkenes; Fluorescence Resonance Energy Transfer; Kinetics; Lipid Bilayers; Maleates; Membrane Proteins; Nanostructures; Polymers; Solubility

Co-translational folding studies of membrane proteins lag behind cytosolic protein investigations largely due to the technical difficulty in maintaining membrane lipid environments for correct protein folding. Stalled ribosome-bound nascent chain complexes (RNCs) can give snapshots of a nascent protein chain as it emerges from the ribosome during biosynthesis. Here, we demonstrate how SecM-facilitated nascent chain stalling and native nanodisc technologies can be exploited to capture

Topics: Alkenes; Amino Acid Sequence; Maleates; Membrane Proteins; Micelles; Nanoparticles; Protein Biosynthesis; Protein Stability; Protein Structure, Secondary; Proteins; Ribosomes; SEC Translocation Channels

Most membrane-solubilising amphiphilic copolymers such as diisobutylene/maleic acid (DIBMA) and styrene/maleic acid (SMA) carry high negative charge densities. Their polyanionic character results in strong Coulombic repulsion, both between polymer chains and lipid membranes during the solubilisation process as well as among polymer-encapsulated nanodiscs after solubilisation. Coulombic repulsion is attenuated by charge screening and, more efficiently, by counterion association, which is particularly strong for multivalent cations binding to polyanionic copolymers. Here, we investigated the effects of the two common alkaline earth metal ions Mg

Topics: Alkenes; Calcium; Calorimetry, Differential Scanning; Kinetics; Magnesium; Maleates; Microscopy, Electron, Transmission; Nanostructures; Polymers; Solubility

Rhomboid proteases form a paradigm for intramembrane proteolysis and have been implicated in several human diseases. However, their study is hampered by difficulties in solubilization and purification. We here report on the use of polymers composed of maleic acid and either diisobutylene or styrene for solubilization of rhomboid proteases in lipid nanodiscs, which proceeds with up to 48% efficiency. We show that the activity of rhomboids in lipid nanodiscs is closer to that in the native membrane than rhomboids in detergent. Moreover, a rhomboid that was proteolytically unstable in detergent turned out to be stable in lipid nanodiscs, underlining the benefit of using these polymer-stabilized nanodiscs. The systems are also compatible with the use of activity-based probes and can be used for small molecule inhibitor screening, allowing several downstream applications.

Topics: Alkenes; Humans; Lipids; Maleates; Models, Molecular; Molecular Structure; Nanoparticles; Particle Size; Polymers; Serine Proteases; Small Molecule Libraries

Topics: Alkenes; Lipid Bilayers; Maleates

Once removed from their natural environment, membrane proteins depend on membrane-mimetic systems to retain their native structures and functions. To this end, lipid-bilayer nanodiscs that are bounded by scaffold proteins or amphiphilic polymers such as styrene/maleic acid (SMA) copolymers have been introduced as alternatives to detergent micelles and liposomes for in vitro membrane-protein research. Herein, we show that an alternating diisobutylene/maleic acid (DIBMA) copolymer shows equal performance to SMA in solubilizing phospholipids, stabilizes an integral membrane enzyme in functional bilayer nanodiscs, and extracts proteins of various sizes directly from cellular membranes. Unlike aromatic SMA, aliphatic DIBMA has only a mild effect on lipid acyl-chain order, does not interfere with optical spectroscopy in the far-UV range, and does not precipitate in the presence of low millimolar concentrations of divalent cations.

Topics: Alkenes; Detergents; Escherichia coli; Escherichia coli Proteins; Lipid Bilayers; Liposomes; Maleates; Membrane Proteins; Micelles; Nanostructures; Phospholipids; Polymers; Solubility