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.

Protein and solutes freeze-concentration in water/glycerol mixtures revealed by pulse EPR.

Lyophilization can extend protein drugs stability and shelf life, but it also can lead to protein degradation in some cases. The development of safe freeze-drying approaches for sensitive proteins requires a better understanding of lyophilization on the molecular level. The evaluation of the freezing process and its impact on the protein environment in the nm scale is challenging because feasible experimental methods are scarce. In the present work we apply pulse EPR as a tool to study the local concentrations of the solute in the 20 nm range and of the solvent in the 1 nm range for a spin labeled 27 kDa monomeric green fluorescent protein, mEGFP, and the 172 Da TEMPOL spin probe, frozen in different water/glycerol-d5 mixtures. For average glycerol volume fractions, φgly-d5avg, ≥ 0.4 we observed transparent glassy media; the local concentration and the 1 nm solvent shell of TEMPOL and the protein correspond to those of a uniform vitrified glass. At φgly-d5avg ≤ 0.3 we observed partial ice crystallization, which led to ice exclusion of glycerol and TEMPOL with freeze-concentration up to the glycerol maximal-freeze local volume fraction, φgly-d5loc, of 0.64. The protein concentration and its shell behavior was similar except for the lowest φgly-d5avg (0.1), which showed a 4.7-fold freeze-concentration factor compared to sevenfold for TEMPOL, and also a smaller φgly-d5loc. We explain this behavior with an increased probability for proteins to get stuck in the ice phase during fast freezing at higher freeze-concentration and the related large-scale mass transfer.

Interaction kinetics and accessibility of sulfadiazine in model clay-humic acid suspension: Electron spin resonance investigations with nitroxide spin label.

The characterization of the interaction of sulfonamides with soil is of particular interest in environmental risk and persistence assessment. In the present work electron spin resonance spectroscopy (ESR) was used to investigate the interaction kinetics of spin labelled sulfadiazine (SL-SDZ) with model clay-humic acid suspensions. The ESR spectra showed that SL-SDZ incubated with Leonardite humic acid (LHA) and Ca-hectorite as model clay was immobilized due to covalent binding of its aniline moiety to LHA. From the immobilization kinetics measured over a period of 1200 h a pseudo-first order reaction with a time constant of 82.6 ± 25.0 h of covalent binding was determined. Additionally, SL-SDZ was strongly sorbed by LHA immediately after incubation but not durably sequestered. Compared to incubation without Ca-hectorite the covalent binding kinetics of SL-SDZ as well as its strong sorption were retarded.

Spatiotemporal Resolution of Conformational Changes in Biomolecules by Combining Pulsed Electron-Electron Double Resonance Spectroscopy with Microsecond Freeze-Hyperquenching.

The function of proteins is linked to their conformations that can be resolved with several high-resolution methods. However, only a few methods can provide the temporal order of intermediates and conformational changes, with each having its limitations. Here, we combine pulsed electron-electron double resonance spectroscopy with a microsecond freeze-hyperquenching setup to achieve spatiotemporal resolution in the angstrom range and lower microsecond time scale. We show that the conformational change of the Cα-helix in the cyclic nucleotide-binding domain of the Mesorhizobium loti potassium channel occurs within about 150 µs and can be resolved with angstrom precision. Thus, this approach holds great promise for obtaining 4D landscapes of conformational changes in biomolecules.

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.

In cell Gd3+-based site-directed spin labeling and EPR spectroscopy of eGFP.

Label-based functional studies of biomolecules in their native environment require labeling reactions inside living cells. In cell spin labeling using alkyne-azide click chemistry with a Gd3+-DOTAM-azide complex is shown to provide high spin label stability and narrow EPR lines for EPR spectroscopic detection of a spin labeled protein in living cells at ambient temperatures.

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.

Dynamic interactions of CbiN and CbiM trigger activity of a cobalt energy-coupling-factor transporter.

Energy-coupling factor (ECF) transporters for uptake of vitamins and transition-metal ions into prokaryotic cells share a common architecture consisting of a substrate-specific integral membrane protein (S), a transmembrane coupling protein (T) and two cytoplasmic ATP-binding-cassette-family ATPases. S components rotate within the membrane to expose their binding pockets alternately to the exterior and the cytoplasm. In contrast to vitamin transporters, metal-specific systems rely on additional proteins with essential but poorly understood functions. CbiN, a membrane protein composed of two transmembrane helices tethered by an extracytoplasmic loop of 37 amino-acid residues represents the auxiliary component that temporarily interacts with the CbiMQO2 Co2+ transporter. CbiN was previously shown to induce significant Co2+ transport activity in the absence of CbiQO2 in cells producing the S component CbiM plus CbiN or a Cbi(MN) fusion. Here we analyzed the mode of interaction between the two protein domains. Any deletion in the CbiN loop abolished transport activity. In silico predicted protein-protein contacts between segments of the CbiN loop and loops in CbiM were confirmed by cysteine-scanning mutagenesis and crosslinking. Likewise, an ordered structure of the CbiN loop was observed by electron paramagnetic resonance analysis after site-directed spin labeling. The N-terminal loop of CbiM containing three of four metal ligands was partially immobilized in wild-type Cbi(MN) but completely immobile in inactive variants with CbiN loop deletions. Decreased dynamics of the inactive form was also detected by solid-state nuclear magnetic resonance of isotope-labeled protein in proteoliposomes. In conclusion, CbiM-CbiN loop-loop interactions facilitate metal insertion into the binding pocket.

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.

Relationship of Catalysis and Active Site Loop Dynamics in the (ßα)8-Barrel Enzyme Indole-3-glycerol Phosphate Synthase.

It is important to understand how the catalytic activity of enzymes is related to their conformational flexibility. We have studied this activity-flexibility correlation using the example of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (ssIGPS), which catalyzes the fifth step in the biosynthesis of tryptophan. ssIGPS is a thermostable representative of enzymes with the frequently encountered and catalytically versatile (ßα)8-barrel fold. Four variants of ssIGPS with increased catalytic turnover numbers were analyzed by transient kinetics at 25 °C, and wild-type ssIGPS was likewise analyzed both at 25 °C and at 60 °C. Global fitting with a minimal three-step model provided the individual rate constants for substrate binding, chemical transformation, and product release. The results showed that in both cases, namely, the application of activating mutations and temperature increase, the net increase in the catalytic turnover number is afforded by acceleration of the product release rate relative to the chemical transformation steps. Measurements of the solvent viscosity effect at 25 °C versus 60 °C confirmed this change in the rate-determining step with temperature, which is in accordance with a kink in the Arrhenius diagram of ssIGPS at ∼40 °C. When rotational diffusion rates of electron paramagnetic spin-labels attached to active site loop ß1α1 are plotted in the form of an Arrhenius diagram, kinks are observed at the same temperature. These findings, together with molecular dynamics simulations, demonstrate that a different degree of loop mobility correlates with different rate-limiting steps in the catalytic mechanism of ssIGPS.

Helical jackknives control the gates of the double-pore K+ uptake system KtrAB.

Ion channel gating is essential for cellular homeostasis and is tightly controlled. In some eukaryotic and most bacterial ligand-gated K+ channels, RCK domains regulate ion fluxes. Until now, a single regulatory mechanism has been proposed for all RCK-regulated channels, involving signal transduction from the RCK domain to the gating area. Here, we present an inactive ADP-bound structure of KtrAB from Vibrio alginolyticus, determined by cryo-electron microscopy, which, combined with EPR spectroscopy and molecular dynamics simulations, uncovers a novel regulatory mechanism for ligand-induced action at a distance. Exchange of activating ATP to inactivating ADP triggers short helical segments in the K+-translocating KtrB dimer to organize into two long helices that penetrate deeply into the regulatory RCK domains, thus connecting nucleotide-binding sites and ion gates. As KtrAB and its homolog TrkAH have been implicated as bacterial pathogenicity factors, the discovery of this functionally relevant inactive conformation may advance structure-guided drug development.

Sensory Rhodopsin I and Sensory Rhodopsin II Form Trimers of Dimers in Complex with their Cognate Transducers.

Archaeal photoreceptors consist of sensory rhodopsins in complex with their cognate transducers. After light excitation, a two-component signaling chain is activated, which is homologous to the chemotactic signaling cascades in enterobacteria. The latter system has been studied in detail. From structural and functional studies, a picture emerges which includes stable signaling complexes, which assemble to receptor arrays displaying hexagonal structural elements. At this higher order structural level, signal amplification and sensory adaptation occur. Here, we describe electron microscopy data, which show that also the archaeal phototaxis receptors sensory rhodopsin I and II in complex with their cognate transducers can form hexagonal lattices even in the presence of a detergent. This result could be confirmed by molecular dynamics calculations, which revealed similar structural elements. Calculations of the global modes of motion displayed one mode, which resembles the "U"-"V" transition of the NpSRII:NpHtrII complex, which was previously argued to represent a functionally relevant global conformational change accompanying the activation process [Ishchenko et al. (2013) J. Photochem. Photobiol. B 123, 55-58]. A model of cooperativity at the transmembrane level is discussed.

Conformational Changes and Competitive Adsorption between Serum Albumin and Hemoglobin on Bioceramic Substrates.

Traditional methods to analyze interactions and conformational changes of proteins adsorbed onto biomaterials are limited by the protein's associations with the substrate material and the complexity of the surrounding media. We have used EPR spectroscopy in combination with site-directed spin labeling (SDSL) to investigate single protein and competitive adsorption kinetics of horse hemoglobin (Hgb) and bovine serum albumin (BSA) on a silica-calcium-phosphate bioceramic substrate. Combined continuous wave and pulsed (DEER) EPR techniques were employed to monitor local mobility/flexibility changes within the proteins and tertiary structure dynamics upon adsorption. An alternate labeling technique was introduced to allow for specific quantification of each protein adsorbed to the bioceramic surface. We show that at buffer pH 7.4 and 4.7 the amount of adsorbed hemoglobin was increased by a factor of 4-5 compared with BSA. The tertiary structure of hemoglobin was strongly affected upon adsorption, leading to a dissociation of the tetrameric molecule into monomers or αß dimers. When the bioceramic substrate was previously functionalized with a layer of BSA, dissociation was reduced by 71 % compared with the untreated surface, indicating a "primer" effect of BSA for better adhesion of the globular hemoglobin.

Orthogonal spin labeling using click chemistry for in vitro and in vivo applications.

Site-directed spin labeling for EPR- and NMR spectroscopy has mainly been achieved exploiting the specific reactivity of cysteines. For proteins with native cysteines or for in vivo applications, an alternative coupling strategy is required. In these cases click chemistry offers major benefits by providing a fast and highly selective, biocompatible reaction between azide and alkyne groups. Here, we establish click chemistry as a tool to target unnatural amino acids in vitro and in vivo using azide- and alkyne-functionalized spin labels. The approach is compatible with a variety of labels including reduction-sensitive nitroxides. Comparing spin labeling efficiencies from the copper-free with the strongly reducing copper(I)-catalyzed azide-alkyne click reaction, we find that the faster kinetics for the catalyzed reaction outrun reduction of the labile nitroxide spin labels and allow quantitative labeling yields within short reaction times. Inter-spin distance measurements demonstrate that the novel side chain is suitable for paramagnetic NMR- or EPR-based conformational studies of macromolecular complexes.

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.