Two separate mechanisms are involved in membrane permeabilization during lipid oxidation.

Lipid oxidation is a universal degradative process of cell membrane lipids that is induced by oxidative stress and reactive oxygen and nitrogen species (RONS) in multiple pathophysiological situations. It has been shown that certain oxidized lipids alter membrane properties, leading to a loss of membrane function. Alteration of membrane properties is thought to depend on the initial membrane lipid composition, such as the number of acyl chain unsaturations. However, it is unclear how oxidative damage is related to biophysical properties of membranes. We therefore set out to quantify lipid oxidation through various analytical methods and determine key biophysical membrane parameters using model membranes containing lipids with different degrees of lipid unsaturation. As source for RONS, we used cold plasma, which is currently developed as treatment for infections and cancer. Our data revealed complex lipid oxidation that can lead to two main permeabilization mechanisms. The first one appears upon direct contact of membranes with RONS and depends on the formation of truncated oxidized phospholipids. These lipids seem to be partly released from the bilayer, implying that they are likely to interact with other membranes and potentially act as signaling molecules. This mechanism is independent of lipid unsaturation, does not rely on large variations in lipid packing, and is most probably mediated via short-living RONS. The second mechanism takes over after longer incubation periods and probably depends on the continued formation of lipid oxygen adducts such as lipid hydroperoxides or ketones. This mechanism depends on lipid unsaturation and involves large variations in lipid packing. This study indicates that polyunsaturated lipids, which are present in mammalian membranes rather than in bacteria, do not sensitize membranes to instant permeabilization by RONS but could promote long-term damage.

Membrane-Catalyzed Aggregation of Islet Amyloid Polypeptide Is Dominated by Secondary Nucleation.

Type II diabetes is characterized by the loss of pancreatic ß-cells. This loss is thought to be a consequence of membrane disruption, caused by the aggregation of islet amyloid polypeptide (IAPP) into amyloid fibrils. However, the molecular mechanisms of IAPP aggregation in the presence of membranes have remained unclear. Here, we use kinetic analysis to elucidate the aggregation mechanism of IAPP in the presence of mixed zwitterionic and anionic lipid membranes. The results converge to a model in which aggregation on the membrane is strongly dominated by secondary nucleation, that is, the formation of new nuclei on the surface of existing fibrils. The critical nucleus consists of a single IAPP molecule, and anionic lipids catalyze both primary and secondary nucleation, but not elongation. The fact that anionic lipids promote secondary nucleation implies that these events take place at the interface between the membrane and existing fibrils, demonstrating that fibril growth occurs at least to some extent on the membrane surface. These new insights into the mechanism of IAPP aggregation on membranes may help to understand IAPP toxicity and will be important for the development of therapeutics to prevent ß-cell death in type II diabetes.

Synthesis and Evaluation of a Library of Alternating Amphipathic Copolymers to Solubilize and Study Membrane Proteins.

Amphipathic copolymers such as poly(styrene-maleic acid) (SMA) are promising tools for the facile extraction of membrane proteins (MPs) into native nanodiscs. Here, we designed and synthesized a library of well-defined alternating copolymers of SMA analogues in order to elucidate polymer properties that are important for MP solubilization and stability. MP extraction efficiency was determined using KcsA from E. coli membranes, and general solubilization efficiency was investigated via turbidimetry experiments on membranes of E. coli, yeast mitochondria, and synthetic lipids. Remarkably, halogenation of SMA copolymers dramatically improved solubilization efficiency in all systems, while substituents on the copolymer backbone improved resistance to Ca2+. Relevant polymer properties were found to include hydrophobic balance, size and positioning of substituents, rigidity, and electronic effects. The library thus contributes to the rational design of copolymers for the study of MPs.

Iterative RAFT-Mediated Copolymerization of Styrene and Maleic Anhydride toward Sequence- and Length-Controlled Copolymers and Their Applications for Solubilizing Lipid Membranes.

The use of poly(styrene-co-maleic acid) (SMA) for the solubilization of lipid membranes and membrane proteins is becoming more widespread, and with this, the need increases to better understand the chemical properties of the copolymer and how these translate into membrane solubilization properties. SMA comes in many different flavors that include the ratio of styrene to maleic acid, comonomer sequence distribution, average chain length, dispersity, and potential chemical modifications. In this work, the synthesis and membrane active properties are described for 2:1 (periodic) SMA copolymers with Mw varying from ∼1.4 to 6 kDa. The copolymers were obtained via an iterative RAFT-mediated radical polymerization. Characterization of these polymers showed that they represent a well-defined series in terms of chain length and overall composition (FMAnh ∼ 0.33), but that there is heterogeneity in comonomer sequence distribution (FMSS ∼ 0.50) and some dispersity in chain length (1.1 < D < 1.6), particularly for the larger copolymers. Investigation of the interaction of these polymers with phosphatidylcholine lipid self-assemblies showed that all copolymers inserted equally effectively into lipid monolayers, independent of the copolymer length. Nonetheless, smaller polymers were more effective at solubilizing lipid bilayers into nanodiscs, possibly because longer polymers are more prone to become intertwined with each other, thereby hampering their solubilization efficiency. Nanodisc sizes were independent of the copolymer length. However, nanodiscs formed with larger copolymers were found to undergo slower lipid exchange, indicating a higher stability. The results highlight the usefulness of having well-defined copolymers for systematic studies.

Membrane Solubilization by Styrene-Maleic Acid Copolymers: Delineating the Role of Polymer Length.

Styrene-maleic acid (SMA) copolymers have attracted interest in membrane research because they allow the solubilization and purification of membrane-spanning proteins from biological membranes in the form of native-like nanodisks. However, our understanding of the underlying SMA-lipid interactions is hampered by the fact that SMA preparations are very polydisperse. Here, we obtained fractions of the two most commonly used SMA preparations: SMA 2:1 and SMA 3:1 (both with specified Mw ∼10 kD), with different number-average molecular weight (Mn) and styrene content. The fractionation is based on the differential solubility of styrene-maleic anhydride (SMAnh) in hexane and acetone mixtures. SMAnh fractions were hydrolyzed to SMA and added to lipid self-assemblies. It was found that SMA fractions inserted in monolayers and solubilized vesicles to a different extent, with the highest efficiency being observed for low-Mn SMA polymers. Electron microscopy and dynamic light scattering size analyses confirmed the presence of nanodisks independent of the Mn of the SMA polymers forming the belt, and it was shown that the nanodisks all have approximately the same size. However, nanodisks bounded by high-Mn SMA polymers were more stable than those bounded by low-Mn polymers, as indicated by a better retention of the native lipid thermotropic properties and by slower exchange rates of lipids between nanodisks. In conclusion, we here present a simple method to separate SMAnh molecules based on their Mn from commercial SMAnh blends, which allowed us to obtain insights into the importance of SMA length for polymer-lipid interactions.

Activation of the bacterial thermosensor DesK involves a serine zipper dimerization motif that is modulated by bilayer thickness.

DesK is a bacterial thermosensor protein involved in maintaining membrane fluidity in response to changes in environmental temperature. Most likely, the protein is activated by changes in membrane thickness, but the molecular mechanism of sensing and signaling is still poorly understood. Here we aimed to elucidate the mode of action of DesK by studying the so-called "minimal sensor DesK" (MS-DesK), in which sensing and signaling are captured in a single transmembrane segment. This simplified version of the sensor allows investigation of membrane thickness-dependent protein-lipid interactions simply by using synthetic peptides, corresponding to the membrane-spanning parts of functional and nonfunctional mutants of MS-DesK incorporated in lipid bilayers with varying thicknesses. The lipid-dependent behavior of the peptides was investigated by circular dichroism, tryptophan fluorescence, and molecular modeling. These experiments were complemented with in vivo functional studies on MS-DesK mutants. Based on the results, we constructed a model that suggests a new mechanism for sensing in which the protein is present as a dimer and responds to an increase in bilayer thickness by membrane incorporation of a C-terminal hydrophilic motif. This results in exposure of three serines on the same side of the transmembrane helices of MS-DesK, triggering a switching of the dimerization interface to allow the formation of a serine zipper. The final result is activation of the kinase state of MS-DesK.

Solubilization of human cells by the styrene-maleic acid copolymer: Insights from fluorescence microscopy.

Extracting membrane proteins from biological membranes by styrene-maleic acid copolymers (SMAs) in the form of nanodiscs has developed into a powerful tool in membrane research. However, the mode of action of membrane (protein) solubilization in a cellular context is still poorly understood and potential specificity for cellular compartments has not been investigated. Here, we use fluorescence microscopy to visualize the process of SMA solubilization of human cells, exemplified by the immortalized human HeLa cell line. Using fluorescent protein fusion constructs that mark distinct subcellular compartments, we found that SMA solubilizes membranes in a concentration-dependent multi-stage process. While all major intracellular compartments were affected without a strong preference, plasma membrane solubilization was found to be generally slower than the solubilization of organelle membranes. Interestingly, some plasma membrane-localized proteins were more resistant against solubilization than others, which might be explained by their presence in specific membrane domains with differing properties. Our results support the general applicability of SMA for the isolation of membrane proteins from different types of (sub)cellular membranes.

The effectiveness of styrene-maleic acid (SMA) copolymers for solubilisation of integral membrane proteins from SMA-accessible and SMA-resistant membranes.

Solubilisation of biological lipid bilayer membranes for analysis of their protein complement has traditionally been carried out using detergents, but there is increasing interest in the use of amphiphilic copolymers such as styrene maleic acid (SMA) for the solubilisation, purification and characterisation of integral membrane proteins in the form of protein/lipid nanodiscs. Here we survey the effectiveness of various commercially-available formulations of the SMA copolymer in solubilising Rhodobacter sphaeroides reaction centres (RCs) from photosynthetic membranes. We find that formulations of SMA with a 2:1 or 3:1 ratio of styrene to maleic acid are almost as effective as detergent in solubilising RCs, with the best solubilisation by short chain variants (<30kDa weight average molecular weight). The effectiveness of 10kDa 2:1 and 3:1 formulations of SMA to solubilise RCs gradually declined when genetically-encoded coiled-coil bundles were used to artificially tether normally monomeric RCs into dimeric, trimeric and tetrameric multimers. The ability of SMA to solubilise reaction centre-light harvesting 1 (RC-LH1) complexes from densely packed and highly ordered photosynthetic membranes was uniformly low, but could be increased through a variety of treatments to increase the lipid:protein ratio. However, proteins isolated from such membranes comprised clusters of complexes in small membrane patches rather than individual proteins. We conclude that short-chain 2:1 and 3:1 formulations of SMA are the most effective in solubilising integral membrane proteins, but that solubilisation efficiencies are strongly influenced by the size of the target protein and the density of packing of proteins in the membrane.

Solubilization of lipids and lipid phases by the styrene-maleic acid copolymer.

A promising tool in membrane research is the use of the styrene-maleic acid (SMA) copolymer to solubilize membranes in the form of nanodiscs. Since membranes are heterogeneous in composition, it is important to know whether SMA thereby has a preference for solubilization of either specific types of lipids or specific bilayer phases. Here, we investigated this by performing partial solubilization of model membranes and analyzing the lipid composition of the solubilized fraction. We found that SMA displays no significant lipid preference in homogeneous binary lipid mixtures in the fluid phase, even when using lipids that by themselves show very different solubilization kinetics. By contrast, in heterogeneous phase-separated bilayers, SMA was found to have a strong preference for solubilization of lipids in the fluid phase as compared to those in either a gel phase or a liquid-ordered phase. Together the results suggest that (1) SMA is a reliable tool to characterize native interactions between membrane constituents, (2) any solubilization preference of SMA is not due to properties of individual lipids but rather due to properties of the membrane or membrane domains in which these lipids reside and (3) exploiting SMA resistance rather than detergent resistance may be an attractive approach for the isolation of ordered domains from biological membranes.

Detergent-free isolation, characterization, and functional reconstitution of a tetrameric K+ channel: the power of native nanodiscs.

A major obstacle in the study of membrane proteins is their solubilization in a stable and active conformation when using detergents. Here, we explored a detergent-free approach to isolating the tetrameric potassium channel KcsA directly from the membrane of Escherichia coli, using a styrene-maleic acid copolymer. This polymer self-inserts into membranes and is capable of extracting membrane patches in the form of nanosize discoidal proteolipid particles or "native nanodiscs." Using circular dichroism and tryptophan fluorescence spectroscopy, we show that the conformation of KcsA in native nanodiscs is very similar to that in detergent micelles, but that the thermal stability of the protein is higher in the nanodiscs. Furthermore, as a promising new application, we show that quantitative analysis of the co-isolated lipids in purified KcsA-containing nanodiscs allows determination of preferential lipid-protein interactions. Thin-layer chromatography experiments revealed an enrichment of the anionic lipids cardiolipin and phosphatidylglycerol, indicating their close proximity to the channel in biological membranes and supporting their functional relevance. Finally, we demonstrate that KcsA can be reconstituted into planar lipid bilayers directly from native nanodiscs, which enables functional characterization of the channel by electrophysiology without first depriving the protein of its native environment. Together, these findings highlight the potential of the use of native nanodiscs as a tool in the study of ion channels, and of membrane proteins in general.

Proton-Detected Solid-State NMR Spectroscopy of a Zinc Diffusion Facilitator Protein in Native Nanodiscs.

The structure, dynamics, and function of membrane proteins are intimately linked to the properties of the membrane environment in which the proteins are embedded. For structural and biophysical characterization, membrane proteins generally need to be extracted from the membrane and reconstituted in a suitable membrane-mimicking environment. Ensuring functional and structural integrity in these environments is often a major concern. The styrene/maleic acid co-polymer has recently been shown to be able to extract lipid/membrane protein patches directly from native membranes to form nanosize discoidal proteolipid particles, also referred to as native nanodiscs. In this work, we show that high-resolution solid-state NMR spectra can be obtained from an integral membrane protein in native nanodiscs, as exemplified by the 2×34 kDa bacterial cation diffusion facilitator CzcD.

Effect of Polymer Composition and pH on Membrane Solubilization by Styrene-Maleic Acid Copolymers.

The styrene-maleic acid (SMA) copolymer is rapidly gaining attention as a tool in membrane research, due to its ability to directly solubilize lipid membranes into nanodisk particles without the requirement of conventional detergents. Although many variants of SMA are commercially available, so far only SMA variants with a 2:1 and 3:1 styrene-to-maleic acid ratio have been used in lipid membrane studies. It is not known how SMA composition affects the solubilization behavior of SMA. Here, we systematically investigated the effect of varying the styrene/maleic acid on the properties of SMA in solution and on its interaction with membranes. Also the effect of pH was studied, because the proton concentration in the solution will affect the charge density and thereby may modulate the properties of the polymers. Using model membranes of 1,2-dimyristoyl-sn-glycero-3-phosphocholine lipids at pH > pHagg, we found that membrane solubilization is promoted by a low charge density and by a relatively high fraction of maleic acid units in the polymer. Furthermore, it was found that a collapsed conformation of the polymer is required to ensure efficient insertion into the lipid membrane and that efficient solubilization may be improved by a more homogenous distribution of the maleic acid monomer units along the polymer chain. Altogether, the results show large differences in behavior between the SMA variants tested in the various steps of solubilization. The main conclusion is that the variant with a 2:1 styrene-to-maleic acid ratio is the most efficient membrane solubilizer in a wide pH range.

The styrene-maleic acid copolymer: a versatile tool in membrane research.

A new and promising tool in membrane research is the detergent-free solubilization of membrane proteins by styrene-maleic acid copolymers (SMAs). These amphipathic molecules are able to solubilize lipid bilayers in the form of nanodiscs that are bounded by the polymer. Thus, membrane proteins can be directly extracted from cells in a water-soluble form while conserving a patch of native membrane around them. In this review article, we briefly discuss current methods of membrane protein solubilization and stabilization. We then zoom in on SMAs, describe their physico-chemical properties, and discuss their membrane-solubilizing effect. This is followed by an overview of studies in which SMA has been used to isolate and investigate membrane proteins. Finally, potential future applications of the methodology are discussed for structural and functional studies on membrane proteins in a near-native environment and for characterizing protein-lipid and protein-protein interactions.

Isolation of lipids from biological samples.

Isolation of the lipid fraction from biological samples has been a crucial part of countless studies over the last century. This considerable research interest has led to the development of a number of methods for isolating a range of molecular species that fall under the umbrella term "lipid". Such methods vary in popularity, complexity, specificity and even toxicity. In this review, we explore examples of published methods (1952-2014) for isolating lipids from biological samples and attempt to assess the limits of techniques both from a chemical and biological perspective. We also suggest how a suitable method might be chosen for a novel application.

Molecular model for the solubilization of membranes into nanodisks by styrene maleic Acid copolymers.

A recent discovery in membrane research is the ability of styrene-maleic acid (SMA) copolymers to solubilize membranes in the form of nanodisks allowing extraction and purification of membrane proteins from their native environment in a single detergent-free step. This has important implications for membrane research because it allows isolation as well as characterization of proteins and lipids in a near-native environment. Here, we aimed to unravel the molecular mode of action of SMA copolymers by performing systematic studies using model membranes of varying compositions and employing complementary biophysical approaches. We found that the SMA copolymer is a highly efficient membrane-solubilizing agent and that lipid bilayer properties such as fluidity, thickness, lateral pressure profile, and charge density all play distinct roles in the kinetics of solubilization. More specifically, relatively thin membranes, decreased lateral chain pressure, low charge density at the membrane surface, and increased salt concentration promote the speed and yield of vesicle solubilization. Experiments using a native membrane lipid extract showed that the SMA copolymer does not discriminate between different lipids and thus retains the native lipid composition in the solubilized particles. A model is proposed for the mode of action of SMA copolymers in which membrane solubilization is mainly driven by the hydrophobic effect and is further favored by physical properties of the polymer such as its relatively small cross-sectional area and rigid pendant groups. These results may be helpful for development of novel applications for this new type of solubilizing agent, and for optimization of the SMA technology for solubilization of the wide variety of cell membranes found in nature.

Lipid packing drives the segregation of transmembrane helices into disordered lipid domains in model membranes.

Cell membranes are comprised of multicomponent lipid and protein mixtures that exhibit a complex partitioning behavior. Regions of structural and compositional heterogeneity play a major role in the sorting and self-assembly of proteins, and their clustering into higher-order oligomers. Here, we use computer simulations and optical microscopy to study the sorting of transmembrane helices into the liquid-disordered domains of phase-separated model membranes, irrespective of peptide-lipid hydrophobic mismatch. Free energy calculations show that the enthalpic contribution due to the packing of the lipids drives the lateral sorting of the helices. Hydrophobic mismatch regulates the clustering into either small dynamic or large static aggregates. These results reveal important molecular driving forces for the lateral organization and self-assembly of transmembrane helices in heterogeneous model membranes, with implications for the formation of functional protein complexes in real cells.

Bacterial reaction centers purified with styrene maleic acid copolymer retain native membrane functional properties and display enhanced stability.

Integral membrane proteins often present daunting challenges for biophysical characterization, a fundamental issue being how to select a surfactant that will optimally preserve the individual structure and functional properties of a given membrane protein. Bacterial reaction centers offer a rare opportunity to compare the properties of an integral membrane protein in different artificial lipid/surfactant environments with those in the native bilayer. Here, we demonstrate that reaction centers purified using a styrene maleic acid copolymer remain associated with a complement of native lipids and do not display the modified functional properties that typically result from detergent solubilization. Direct comparisons show that reaction centers are more stable in this copolymer/lipid environment than in a detergent micelle or even in the native membrane, suggesting a promising new route to exploitation of such photovoltaic integral membrane proteins in device applications.

Fibril elongation by human islet amyloid polypeptide is the main event linking aggregation to membrane damage.

The aggregation of human islet amyloid polypeptide (hIAPP) is linked to the death of pancreatic ß-cells in type II diabetes. The process of fibril formation by hIAPP is thought to cause membrane damage, but the precise mechanisms are still unclear. Previously, we showed that the aggregation of hIAPP in the presence of membranes containing anionic lipids is dominated by secondary nucleation events, which occur at the interface between existing fibrils and the membrane surface. Here, we used vesicles with different lipid composition to explore the connection between hIAPP aggregation and vesicle leakage. We found that different anionic lipids promote hIAPP aggregation to the same extent, whereas remarkably stochastic behaviour is observed on purely zwitterionic membranes. Vesicle leakage induced by hIAPP consists of two distinct phases for any of the used membrane compositions: (i) an initial phase in which hIAPP binding causes a certain level of leakage that is strongly dependent on osmotic conditions, membrane composition and the used dye, and (ii) a main leakage event that we attribute to elongation of hIAPP fibrils, based on seeded experiments. Altogether, our results shed more light on the relationship between hIAPP fibril formation and membrane damage, and strongly suggest that oligomeric intermediates do not considerably contribute to vesicle leakage.

How lipid headgroups sense the membrane environment: an application of ¹4N NMR.

The orientation of lipid headgroups may serve as a powerful sensor of electrostatic interactions in membranes. As shown previously by (2)H NMR measurements, the headgroup of phosphatidylcholine (PC) behaves like an electrometer and varies its orientation according to the membrane surface charge. Here, we explored the use of solid-state (14)N NMR as a relatively simple and label-free method to study the orientation of the PC headgroup in model membrane systems of varying composition. We found that (14)N NMR is sufficiently sensitive to detect small changes in headgroup orientation upon introduction of positively and negatively charged lipids and we developed an approach to directly convert the (14)N quadrupolar splittings into an average orientation of the PC polar headgroup. Our results show that inclusion of cholesterol or mixing of lipids with different length acyl chains does not significantly affect the orientation of the PC headgroup. In contrast, measurements with cationic (KALP), neutral (Ac-KALP), and pH-sensitive (HALP) transmembrane peptides show very systematic changes in headgroup orientation, depending on the amount of charge in the peptide side chains and on their precise localization at the interface, as modulated by varying the extent of hydrophobic peptide/lipid mismatch. Finally, our measurements suggest an unexpectedly strong preferential enrichment of the anionic lipid phosphatidylglycerol around the cationic KALP peptide in ternary mixtures with PC. We believe that these results are important for understanding protein/lipid interactions and that they may help parametrization of membrane properties in computational studies.

Sterols have higher affinity for sphingomyelin than for phosphatidylcholine bilayers even at equal acyl-chain order.

The interaction between cholesterol and phospholipids in bilayer membranes is important for the formation and maintenance of membrane structure and function. However, cholesterol does not interact favorably with all types of phospholipids and, for example, prefers more ordered sphingomyelins (SMs) over phosphatidylcholines (PCs). The reason for this preference is not clear. Here we have studied whether acyl-chain order could be responsible for the preferred sterol interaction with SMs. Acyl-chain order was deduced from diphenylhexatriene anisotropy and from the deuterium order parameter obtained by (2)H-NMR on bilayers made from either 14:0/14:0((d27))-PC, or 14:0((d27))-SM. Sterol/phospholipid interaction was determined from sterol bilayer partitioning. Cholestatrienol (CTL) was used as a fluorescence probe for cholesterol, because its relative membrane partitioning is similar to cholesterol. When CTL was allowed to reach equilibrium partitioning between cyclodextrins and unilamellar vesicles made from either 14:0/14:0-PC or 14:0-SM, the molar-fraction partitioning coefficient (K(x)) was approximately twofold higher for SM bilayers than for PC bilayers. This was even the case when the temperature in the SM samples was raised to achieve equal acyl-chain order, as determined from 1,6-diphenyl-1,3,5-hexatriene (DPH) anisotropy and the deuterium order parameter. Although the K(x) did increase with acyl-chain order, the higher K(x) for SM bilayers was always evident. At equal acyl-chain order parameter (DPH anisotropy), the K(x) was also higher for 14:0-SM bilayers than for bilayers made from either 14:0/15:0-PC or 15:0-/14:0-PC, suggesting that minor differences in chain length or molecular asymmetry are not responsible for the difference in K(x). We conclude that acyl-chain order affects the bilayer affinity of CTL (and thus cholesterol), but that it is not the cause for the preferred affinity of sterols for SMs over matched PCs. Instead, it is likely that the interfacial properties of SMs influence and stabilize interactions with sterols in bilayer membranes.