Mechanisms of Formation, Structure, and Dynamics of Lipoprotein Discs Stabilized by Amphiphilic Copolymers: A Comprehensive Review.

Amphiphilic copolymers consisting of alternating hydrophilic and hydrophobic units account for a major recent methodical breakthrough in the investigations of membrane proteins. Styrene-maleic acid (SMA), diisobutylene-maleic acid (DIBMA), and related copolymers have been shown to extract membrane proteins directly from lipid membranes without the need for classical detergents. Within the particular experimental setup, they form disc-shaped nanoparticles with a narrow size distribution, which serve as a suitable platform for diverse kinds of spectroscopy and other biophysical techniques that require relatively small, homogeneous, water-soluble particles of separate membrane proteins in their native lipid environment. In recent years, copolymer-encased nanolipoparticles have been proven as suitable protein carriers for various structural biology applications, including cryo-electron microscopy (cryo-EM), small-angle scattering, and conventional and single-molecule X-ray diffraction experiments. Here, we review the current understanding of how such nanolipoparticles are formed and organized at the molecular level with an emphasis on their chemical diversity and factors affecting their size and solubilization efficiency.

Lipid Dynamics in Diisobutylene-Maleic Acid (DIBMA) Lipid Particles in Presence of Sensory Rhodopsin II.

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 Natronomonas pharaonis (NpSRII) results in a slight increase in the lipid dynamics compared to empty DIBMALPs. The light-induced photocycle shows full functionality of DIBMALPs-embedded NpSRII and a significant effect of the protein-to-lipid ratio during preparation on the NpSRII dynamics. This study indicates a possible expansion of the applicability of the DIBMALP technology on studies of membrane protein-protein interaction and oligomerization in a constraining environment.

Evaluation of DIBMA nanoparticles of variable size and anionic lipid content as tools for the structural and functional study of membrane proteins.

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.

A Three-Dimensional Model of the Yeast Transmembrane Sensor Wsc1 Obtained by SMA-Based Detergent-Free Purification and Transmission Electron Microscopy.

The cell wall sensor Wsc1 belongs to a small family of transmembrane proteins, which are crucial to sustain cell integrity in yeast and other fungi. Wsc1 acts as a mechanosensor of the cell wall integrity (CWI) signal transduction pathway which responds to external stresses. Here we report on the purification of Wsc1 by its trapping in water-soluble polymer-stabilized lipid nanoparticles, obtained with an amphipathic styrene-maleic acid (SMA) copolymer. The latter was employed to transfer tagged sensors from their native yeast membranes into SMA/lipid particles (SMALPs), which allows their purification in a functional state, i.e., avoiding denaturation. The SMALPs composition was characterized by fluorescence correlation spectroscopy, followed by two-dimensional image acquisition from single particle transmission electron microscopy to build a three-dimensional model of the sensor. The latter confirms that Wsc1 consists of a large extracellular domain connected to a smaller intracellular part by a single transmembrane domain, which is embedded within the hydrophobic moiety of the lipid bilayer. The successful extraction of a sensor from the yeast plasma membrane by a detergent-free procedure into a native-like membrane environment provides new prospects for in vitro structural and functional studies of yeast plasma proteins which are likely to be applicable to other fungi, including plant and human pathogens.

Lipid dynamics in nanoparticles formed by maleic acid-containing copolymers: EPR spectroscopy and molecular dynamics simulations.

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.

MitoCLox: A Novel Mitochondria-Targeted Fluorescent Probe for Tracing Lipid Peroxidation.

Peroxidation of cardiolipin (CL) in the inner mitochondrial membrane plays a key role in the development of various pathologies and, probably, aging. The four fatty acid tails of CL are usually polyunsaturated, which makes CL particularly sensitive to peroxidation. Peroxidation of CL is involved in the initiation of apoptosis, as well as in some other important cellular signaling chains. However, the studies of CL peroxidation are strongly limited by the lack of methods for its tracing in living cells. We have synthesized a new mitochondria-targeted fluorescent probe sensitive to lipid peroxidation (dubbed MitoCLox), where the BODIPY fluorophore, carrying a diene-containing moiety (as in the C11-BODIPY (581/591) probe), is conjugated with a triphenylphosphonium cation (TPP+) via a long flexible linker that contains two amide bonds. The oxidation of MitoCLox could be measured either as a decrease of absorbance at 588 nm or as an increase of fluorescence in the ratiometric mode at 520/590 nm (emission). In CL-containing liposomes, MitoCLox oxidation was induced by cytochrome c and developed in parallel with cardiolipin oxidation. TPP+-based mitochondria-targeted antioxidant SkQ1, in its reduced form, inhibited oxidation of MitoCLox concurrently with the peroxidation of cardiolipin. Molecular dynamic simulations of MitoCLox in a cardiolipin-containing membrane showed affinity of positively charged MitoCLox to negatively charged CL molecules; the oxidizable diene moiety of MitoCLox resided on the same depth as the cardiolipin lipid peroxides. We suggest that MitoCLox could be used for monitoring CL oxidation in vivo and, owing to its flexible linker, also serve as a platform for producing peroxidation sensors with affinity to particular lipids.

Proton leakage across lipid bilayers: Oxygen atoms of phospholipid ester linkers align water molecules into transmembrane water wires.

Up to half of the cellular energy gets lost owing to membrane proton leakage. The permeability of lipid bilayers to protons is by several orders of magnitude higher than to other cations, which implies efficient proton-specific passages. The nature of these passages remains obscure. By combining experimental measurements of proton flow across phosphatidylcholine vesicles, steered molecular dynamics (MD) simulations of phosphatidylcholine bilayers and kinetic modelling, we have analyzed whether protons could pass between opposite phospholipid molecules when they sporadically converge. The MD simulations showed that each time, when the phosphorus atoms of the two phosphatidylcholine molecules got closer than 1.6 nm, the eight oxygen atoms of their ester linkages could form a transmembrane 'oxygen passage' along which several water molecules aligned into a water wire. Proton permeability along such water wires would be limited by rearrangement of oxygen atoms, which could explain the experimentally shown independence of the proton permeability of pH, H2O/D2O substitution, and membrane dipole potential. We suggest that protons can cross lipid bilayers by moving along short, self-sustaining water wires supported by oxygen atoms of lipid ester linkages.

Conformational Dynamics of Sensory Rhodopsin II in Nanolipoprotein and Styrene-Maleic Acid Lipid Particles.

Styrene-maleic acid lipid particles (SMALPs) provide stable water-soluble nanocontainers for lipid-encased membrane proteins. Possible effects of the SMA-stabilized lipid environment on the interaction dynamics between functionally coupled membrane proteins remain to be elucidated. The photoreceptor sensory rhodopsin II, NpSRII and its cognate transducer, NpHtrII, of Natronomonas pharaonis form a transmembrane complex, NpSRII2 /NpHtrII2 that plays a key role in negative phototaxis and provides a unique model system to study the light-induced transfer of a conformational signal between two integral membrane proteins. Photon absorption induces transient structural changes in NpSRII comprising an outward movement of helix F that cause further conformational alterations in NpHtrII. We applied site-directed spin labeling and time-resolved optical and EPR spectroscopy to compare the conformational dynamics of NpSRII2 /NpHtrII2 reconstituted in SMALPs with that of nanolipoprotein particle and liposome preparations. NpSRII and NpSRII2 /NpHtrII2 show similar photocycles in liposomes and nanolipoprotein particles. An accelerated decay of the M photointermediate found for SMALPs can be explained by a high local proton concentration provided by the carboxylic groups of the SMA polymer. Light-induced large-scale conformational changes of NpSRII2 /NpHtrII2 observed in liposomes and nanolipoprotein particles are affected in SMALPs, indicating restrictions of the protein's conformational freedom.

Styrene/Maleic Acid Copolymers Form SMALPs by Pulling Lipid Patches out of the Lipid Bilayer.

Amphiphilic copolymers composed of styrene and maleic acid (SMA) monomers caused a major methodical breakthrough in the study of membrane proteins. They were found to directly release phospholipids and membrane proteins both from artificial and natural lipid bilayers, yielding stable water-soluble discoidal SMA/lipid particles (SMALPs) of uniform size. Although many empirical studies indicate the great potency of SMALPs for membrane protein research, the mechanisms of their formation remain obscure. It is unknown which factors account for the very assembly of SMALPs and govern their uniform size. We have developed a coarse-grained (CG) molecular model of SMA copolymers based on the MARTINI CG force field and used it to probe the behavior of SMA copolymers with varying composition/charge/concentration in solution as well as their interaction with lipid membranes. First, we found that SMA copolymers tend to aggregate in solution into clusters, which could account for the uniform size of SMALPs. Next, molecular dynamics (MD) simulations showed that periodic SMA copolymers with styrene/maleic acid ratios of 2:1 ([SSM] n) and 3:1 ([SSSM] n) differently interacted with lipid bilayers. While clusters of 2:1 SMA copolymers induced membrane poration, the clusters of 3:1 SMA copolymers extracted lipid patches from the membrane yielding SMALP-like structures. Extraction of lipid patches was also observed when we simulated the behavior of 3:1 copolymers with varying lengths and statistical distribution of styrene and MA units. Analysis of MD simulation trajectories and comparison with experimental data indicate that the formation of SMALPs requires copolymer molecules with a sufficient number of units made of more than two sequential styrene monomers.

Impact of Antioxidants on Cardiolipin Oxidation in Liposomes: Why Mitochondrial Cardiolipin Serves as an Apoptotic Signal?

Molecules of mitochondrial cardiolipin (CL) get selectively oxidized upon oxidative stress, which triggers the intrinsic apoptotic pathway. In a chemical model most closely resembling the mitochondrial membrane-liposomes of pure bovine heart CL-we compared ubiquinol-10, ubiquinol-6, and alpha-tocopherol, the most widespread naturally occurring antioxidants, with man-made, quinol-based amphiphilic antioxidants. Lipid peroxidation was induced by addition of an azo initiator in the absence and presence of diverse antioxidants, respectively. The kinetics of CL oxidation was monitored via formation of conjugated dienes at 234 nm. We found that natural ubiquinols and ubiquinol-based amphiphilic antioxidants were equally efficient in protecting CL liposomes from peroxidation; the chromanol-based antioxidants, including alpha-tocopherol, were 2-3 times less efficient. Amphiphilic antioxidants, but not natural ubiquinols and alpha-tocopherol, were able, additionally, to protect the CL bilayer from oxidation by acting from the water phase. We suggest that the previously reported therapeutic efficiency of mitochondrially targeted amphiphilic antioxidants is owing to their ability to protect those CL molecules that are inaccessible to natural hydrophobic antioxidants, being trapped within respiratory supercomplexes. The high susceptibility of such occluded CL molecules to oxidation may have prompted their recruitment as apoptotic signaling molecules by nature.

Clustering and dynamics of phototransducer signaling domains revealed by site-directed spin labeling electron paramagnetic resonance on SRII/HtrII in membranes and nanodiscs.

In halophilic archaea the photophobic response is mediated by the membrane-embedded 2:2 photoreceptor/-transducer complex SRII/HtrII, the latter being homologous to the bacterial chemoreceptors. Both systems bias the rotation direction of the flagellar motor via a two-component system coupled to an extended cytoplasmic signaling domain formed by a four helical antiparallel coiled-coil structure. For signal propagation by the HAMP domains connecting the transmembrane and cytoplasmic domains, it was suggested that a two-state thermodynamic equilibrium found for the first HAMP domain in NpSRII/NpHtrII is shifted upon activation, yet signal propagation along the coiled-coil transducer remains largely elusive, including the activation mechanism of the coupled kinase CheA. We investigated the dynamic and structural properties of the cytoplasmic tip domain of NpHtrII in terms of signal transduction and putative oligomerization using site-directed spin labeling electron paramagnetic resonance spectroscopy. We show that the cytoplasmic tip domain of NpHtrII is engaged in a two-state equilibrium between a dynamic and a compact conformation like what was found for the first HAMP domain, thus strengthening the assumption that dynamics are the language of signal transfer. Interspin distance measurements in membranes and on isolated 2:2 photoreceptor/transducer complexes in nanolipoprotein particles provide evidence that archaeal photoreceptor/-transducer complexes analogous to chemoreceptors form trimers-of-dimers or higher-order assemblies even in the absence of the cytoplasmic components CheA and CheW, underlining conservation of the overall mechanistic principles underlying archaeal phototaxis and bacterial chemotaxis systems. Furthermore, our results revealed a significant influence of the NpHtrII signaling domain on the NpSRII photocycle kinetics, providing evidence for a conformational coupling of SRII and HtrII in these complexes.

Light-induced switching of HAMP domain conformation and dynamics revealed by time-resolved EPR spectroscopy.

HAMP domains are widely abundant signaling modules. The putative mechanism of their function comprises switching between two distinct states. To unravel these conformational transitions, we apply site-directed spin labeling and time-resolved EPR spectroscopy to the phototactic receptor/transducer complex NpSRII/NpHtrII. We characterize the kinetic coupling of NpHtrII to NpSRII along with the activation period of the transducer and follow the transient conformational signal. The observed transient shift towards a more compact state of the HAMP domain upon light-activation agrees with structure-based calculations. It thereby validates the two modeled signaling states and integrates the domain's dynamics into the current model.

Chromatophore vesicles of Rhodobacter capsulatus contain on average one F(O)F(1)-ATP synthase each.

ATP synthase is a unique rotary machine that uses the transmembrane electrochemical potential difference of proton (Delta(H(+))) to synthesize ATP from ADP and inorganic phosphate. Charge translocation by the enzyme can be most conveniently followed in chromatophores of phototrophic bacteria (vesicles derived from invaginations of the cytoplasmic membrane). Excitation of chromatophores by a short flash of light generates a step of the proton-motive force, and the charge transfer, which is coupled to ATP synthesis, can be spectrophotometrically monitored by electrochromic absorption transients of intrinsic carotenoids in the coupling membrane. We assessed the average number of functional enzyme molecules per chromatophore vesicle. Kinetic analysis of the electrochromic transients plus/minus specific ATP synthase inhibitors (efrapeptin and venturicidin) showed that the extent of the enzyme-related proton transfer dropped as a function of the inhibitor concentration, whereas the time constant of the proton transfer changed only marginally. Statistical analysis of the kinetic data revealed that the average number of proton-conducting F(O)F(1)-molecules per chromatophore was approximately one. Thereby chromatophores of Rhodobacter capsulatus provide a system where the coupling of proton transfer to ATP synthesis can be studied in a single enzyme/single vesicle mode.