Interactions of surfactants and fatty acids with lipids

The recent studies on the interaction of surfactants and fatty acids with lipids are inspired by the want of knowledge from several research fields of highest activity: identification of membrane rafts; membrane protein crystallization; formation of non-lamellar phases in membranes; membrane fusion. Detailed phase diagrams for lipid–surfactant and lipid–fatty acid mixtures, obtained in the last few years, reveal complicated mesomorphic and polymorphic behavior, including miscibility gaps and compound formation. Surfactant-induced non-lamellar to lamellar transitions in lipids and specific temperature-driven bilayer–micelle transitions represent extensions of the general three-stage model of membrane solubilization. Further research is required to construct phase diagrams as to delineate the principles of lipid–surfactant and lipid–fatty acid interactions for the variety of membrane lipid classes.     

Electrostatic properties of membrane lipids coupled to metarhodopsin II formation in visual transduction

Changes in lipid composition have recently been shown to exert appreciable influences on the activities of membrane-bound proteins and peptides. We tested the hypothesis that the conformational states of rhodopsin linked to visual signal transduction are related to biophysical properties of the membrane lipid bilayer. For bovine rhodopsin, the meta I-meta II conformational transition was studied in egg phosphatidylcholine (PC) recombinants versus the native rod outer segment (ROS) membranes by means of flash photolysis. Formation of metarhodopsin II was observed by the change in absorbance at 478 nm after a single actinic flash was delivered to the sample. The meta I/meta II ratio was investigated as a function of both temperature and pH. The data clearly demonstrated thermodynamic reversibility of the transition for both the egg PC recombinants and the native ROS membranes. A significant shift of the apparent pK(a) for the acid-base equilibrium to lower values was evident in the egg PC recombinant, with little meta II produced under physiological conditions. Calculations of the membrane surface pH using a Poisson-Boltzmann model suggested the free energies of the meta I and meta II states were significantly affected by electrostatic properties of the bilayer lipids. In the ROS membranes, phosphatidylserine (PS) is needed for full formation of meta II, in combination with phosphatidylethanolamine (PE) and polyunsaturated docosahexaenoic acid (DHA; 22:6omega3) chains. We propose that the PS surface potential leads to an accumulation of hydronium ions, H(3)O(+), in the electrical double layer, which drive the reaction together with the large negative spontaneous curvature (H(0)) conferred by PE plus DHA chains. The elastic stress/strain of the bilayer arises from an interplay of the approximately zero H(0) from PS and the negative H(0) due to the PE headgroups and polyunsaturated chains. The lipid influences are further explained in terms of matching of the bilayer spontaneous curvature to the curvature at the lipid/rhodopsin interface, as formulated by the Helfrich bending energy. These new findings guide current ideas as to how bilayer properties govern the conformational energetics of integral membrane proteins. Moreover, they yield knowledge of how membrane lipid-protein interactions involving acidic phospholipids such as PS and neutral polyunsaturated DHA chains are implicated in key biological functions such as vision.     

Interfacial enzyme activation, non-lamellar phase formation and membrane fusion. Is there a conducting thread?

Previous studies from this laboratory have shown that the enzymic generation of diacylglycerol in bilayers by phospholipase C may lead to membrane fusion through the formation of transient non-lamellar lipidic intermediates. The present paper intends to explore the correlations existing among the three main processes involved, namely (a) the induction (or inhibition) of lamellar-to-non-lamellar phase transitions in lipid mixtures through the addition of small (< 5 mol%) proportions of other lipids, (b) the promotion, by the latter lipids, of fusion in otherwise stable phospholipid vesicles (large unilamellar liposomes) under conditions leading to inverted hexagonal/inverted cubic phase formation in bulk lipid systems, and (c) the modulation, by the same small proportions of lipids, of phospholipase C hydrolysis of phosphatidylcholine in liposome bilayers. It is concluded that phospholipase C may give rise to non-lamellar lipidic structures that in turn permit liposomal fusion to occur, but neither enzyme activity is directly modulated by non-lamellar phase formation, nor will whatever kind of enzyme-induced non-lamellar structure give rise to fusion. Moreover, only under certain kinetic conditions will the enzyme give rise to the organization of non-lamellar structures that are conducive to the fusion event.     

Bilayer hydrophobic thickness and integral membrane protein function

The influence of the lipid environment on the function of membrane proteins is increasingly recognized as crucial. Nevertheless, the molecular mechanisms underlying protein-lipid interactions remain obscure. Membrane lipid composition has a regulatory effect on membrane protein activity, and for a number of membrane proteins a clear correlation was found between protein activity and properties of the membrane bilayer such as fluidity. Membrane thickness is an important property of a lipid bilayer. It is expected that hydrophobic thickness match the hydrophobic thickness of transmembrane segments of integral membrane proteins. Any mismatch between the hydrophobic thicknesses of the lipid bilayer and the protein would lead to some modification in either the structure of the protein or the structure of the bilayer, or both. Consequent rearrangements may result in changes in protein activity. Here we review the behavior of several transmembrane proteins whose activity is altered by hydrophobic core thickness.     

Probing the Lipid Annular Belt by Gas‐Phase Dissociation of Membrane Proteins in Nanodiscs

Interactions between membrane proteins and lipids are often crucial for structure and function yet difficult to define because of their dynamic and heterogeneous nature. Here, we use mass spectrometry to demonstrate that membrane protein oligomers ejected from nanodiscs in the gas phase retain large numbers of lipid interactions. The complex mass spectra that result from gas‐phase dissociation were assigned using a Bayesian deconvolution algorithm together with mass defect analysis, allowing us to count individual lipid molecules bound to membrane proteins. Comparison of the lipid distributions measured by mass spectrometry with molecular dynamics simulations reveals that the distributions correspond to distinct lipid shells that vary according to the type of protein–lipid interactions. Our results demonstrate that nanodiscs offer the potential for native mass spectrometry to probe interactions between membrane proteins and the wider lipid environment.     

The cell-penetrating peptide TAT(48-60) induces a non-lamellar phase in DMPC membranes.

Cell-penetrating peptides (CPPs) are short polycationic sequences that can translocate into cells without disintegrating the plasma membrane. CPPs are useful tools for delivering cargo, but their molecular mechanism of crossing the lipid bilayer remains unclear. Here we study the interaction of the HIV-derived CPP TAT (48-60) with model membranes by solid-state NMR spectroscopy and electron microscopy. The peptide induces a pronounced isotropic (31)P NMR signal in zwitterionic DMPC, but not in anionic DMPG bilayers. Octaarginine and to a lesser extent octalysine have the same effect, in contrast to other cationic amphiphilic membrane-active peptides. The observed non-lamellar lipid morphology is attributed to specific interactions of polycationic peptides with phosphocholine head groups, rather than to electrostatic interactions. Freeze-fracture electron microscopy indicates that TAT(48-60) induces the formation of rodlike, presumably inverted micelles in DMPC, which may represent intermediates during the translocation across eukaryotic membranes.     

Alteration of Lipid Membrane Rigidity by Cholesterol and Its Metabolic Precursors

Caused by biosynthesis defects, cholesterol deficiency can lead to developmental disorders and malformations, with possible implication of lipid membrane properties. We show that modification of sterol chemical structure alters membrane physical properties significantly. By X-ray diffraction and osmotic stress, we measure changes in the bending rigidity of bilayers containing either cholesterol or one of its metabolic precursors. Membrane elasticity differs dramatically between slightly different sterols and varies in the sequence lanosterol < 7-dehydrocholesterol < lathosterol < cholesterol. We interpret the results in terms of sterol location within lipid structures and modification of lateral stress, a structural feature relevant to interactions within biological membranes. We find that cholesterol is most efficient in enhancing membrane rigidity, a possible clue to why depletion or replacement with other sterols can affect cellular structures.     

Analysis of lipid surface area in protein-membrane systems combining Voronoi tessellation and Monte Carlo integration methods

All-atom molecular dynamics (MD) simulation has become a powerful research tool to investigate structural and dynamical properties of biological membranes and membrane proteins. The lipid structures of simple membrane systems in recent MD simulations are in good agreement with those obtained by experiments. However, for protein-membrane systems, the complexity of protein-lipid interactions makes investigation of lipid structure difficult. Although the area per lipid is one of the essential structural properties in membrane systems, the area in protein-membrane systems cannot be computed easily by conventional approaches like the Voronoi tessellation method. To overcome this limitation, we propose a new method combining the two-dimensional Voronoi tessellation and Monte Carlo integration methods. This approach computes individual surface areas of lipid molecules not only in bulk lipids but also in proximity to membrane proteins. We apply the method to all-atom MD trajectories of the sarcoplasmic reticulum Ca(2+)-pump and the SecY protein-conducting channel. The calculated lipid surface area is in agreement with experimental values and consistent with other structural parameters of lipid bilayers. We also observe changes in the average area per lipid induced by the conformational transition of the SecY channel. Our method is particularly useful for examining equilibration of lipids around membrane proteins and for analyzing the time course of protein-lipid interactions.     

Screening molecular associations with lipid membranes using natural abundance 13C cross-polarization magic-angle spinning NMR and principal component analysis

We describe an NMR approach for detecting the interactions between phospholipid membranes and proteins, peptides, or small molecules. First, 1H-13C dipolar coupling profiles are obtained from hydrated lipid samples at natural isotope abundance using cross-polarization magic-angle spinning NMR methods. Principal component analysis of dipolar coupling profiles for synthetic lipid membranes in the presence of a range of biologically active additives reveals clusters that relate to different modes of interaction of the additives with the lipid bilayer. Finally, by representing profiles from multiple samples in the form of contour plots, it is possible to reveal statistically significant changes in dipolar couplings, which reflect perturbations in the lipid molecules at the membrane surface or within the hydrophobic interior.     

Lipid-protein interactions in membranes: effect of lipid composition on mobility of spin-labeled cysteine residues in yeast plasma membrane.

In order to gain direct evidence for lipid-dependent protein conformation in membrane, effects of modification of lipid composition on mobility of spin-labeled cysteine residues were investigated in the plasma membrane of the yeast Saccharomyces cerevisiae. Conversion of the bulk of phospholipids to diglycerides by treatment of the membrane with phospholipase C substantially enhanced spectral anisotropy. However, alterations of the viscosity of the lipid-bilayer by enriching the membrane with palmitelaidic or oleic acid had no effect on mobility of spin-labeled cysteine residues. These observations indicate that while the spin-labeled residues are not in direct contact with the lipid core of the membrane, there are lipid-protein interactions to the extent that removal of the polar portion of the bulk of phospholipids induces conformational changes in proteins, which in turn restrict mobility of these residues. It is concluded that conformation of membrane proteins on lipid structure and that phospholipids have a role in preserving the native conformation of proteins.     

Aligning nanodiscs at the air-water interface, a neutron reflectivity study

Nanodiscs are self-assembled nanostructures composed of a belt protein and a small patch of lipid bilayer, which can solubilize membrane proteins in a lipid bilayer environment. We present a method for the alignment of a well-defined two-dimensional layer of nanodiscs at the air-water interface by careful design of an insoluble surfactant monolayer at the surface. We used neutron reflectivity to demonstrate the feasibility of this approach and to elucidate the structure of the nanodisc layer. The proof of concept is hereby presented with the use of nanodiscs composed of a mixture of two different lipid (DMPC and DMPG) types to obtain a net overall negative charge of the nanodiscs. We find that the nanodisc layer has a thickness or 40.9 ± 2.6 ? with a surface coverage of 66 ± 4%. This layer is located about 15 ? below a cationic surfactant layer at the air-water interface. The high level of organization within the nanodiscs layer is reflected by a low interfacial roughness (~4.5 ?) found. The use of the nanodisc as a biomimetic model of the cell membrane allows for studies of single membrane proteins isolated in a confined lipid environment. The 2D alignment of nanodiscs could therefore enable studies of high-density layers containing membrane proteins that, in contrast to membrane proteins reconstituted in a continuous lipid bilayer, remain isolated from influences of neighboring membrane proteins within the layer.     

Noncovalent keystone interactions controlling biomembrane structure

There is a biomedical need to develop molecular recognition systems that selectively target the interfaces of protein and lipid aggregates in biomembranes. This is an extremely challenging problem in supramolecular chemistry because the biological membrane is a complex dynamic assembly of multifarious molecular components with local inhomogeneity. Two simplifying concepts are presented as a framework for basing molecular design strategies. The first generalization is that association of two binding partners in a biomembrane will be dominated by one type of non-covalent interaction which is referred to as the keystone interaction. Structural mutations in membrane proteins that alter the strength of this keystone interaction will likely have a major effect on biological activity and often will be associated with disease. The second generalization is to view the structure of a cell membrane as three spatial regions, that is, the polar membrane surface, the midpolar interfacial region and the non-polar membrane interior. Each region has a distinct dielectric, and the dominating keystone interaction between binding partners will be different. At the highly polar membrane surface, the keystone interactions between charged binding partners are ion-ion and ion-dipole interactions; whereas, ion-dipole and ionic hydrogen bonding are very influential at the mid-polar interfacial region. In the non-polar membrane interior, van der Waals forces and neutral hydrogen bonding are the keystone interactions that often drive molecular association. Selected examples of lipid and transmembrane protein association systems are described to illustrate how the association thermodynamics and kinetics are dominated by these keystone noncovalent interactions.     

Lipid/protein interactions and the membrane/water interfacial region

Despite the importance of lipid/protein interactions in the folding, assembly, stability, and function of membrane proteins, information at an atomic level on how such proteins interact with the lipids that surround them remains sparse. The dynamics and flexible nature of the protein/bilayer interaction make it difficult to study, for example, by crystallographic means. However, based on recent progress in molecular simulations of membranes it is possible to address this problem computationally. This communication reports one of the first attempts to use multiple ns molecular simulations to establish a qualitative picture of the intermolecular interactions between the lipids of a bilayer and two topologically different membrane proteins for which a high resolution (2 A or better) X-ray structure is available.     

Bead-linked proteoliposomes: a reconstitution method for nmr analyses of membrane protein-ligand interactions

Structural information about the interactions between membrane proteins and their ligands provides insights into the membrane protein functions. A variety of surfactants have been used for structural analyses of membrane proteins, and in some cases, they yielded successful results. However, the use of surfactants frequently increases the conformational instability of membrane proteins and distorts their normal function. Here, we propose a new strategy of membrane protein reconstitution into lipid bilayers on affinity beads, which maintains the native conformation and function of the protein for NMR studies. The reconstituted membrane proteins are suitable for NMR analyses of interactions, by using the transferred cross-saturation method. The strategy was successfully applied to the interaction between a potassium ion channel, KcsA, and a pore-blocker, agitoxin2 (AgTx2). This strategy would be useful for analyzing the interactions between various membrane proteins and their ligands.     

Quantifying the Initial Unfolding of Bacteriorhodopsin Reveals Retinal Stabilization

The forces that stabilize membrane proteins remain elusive to precise quantification. Particularly important, but poorly resolved, are the forces present during the initial unfolding of a membrane protein, where the most native set of interactions is present. A high‐precision, atomic force microscopy assay was developed to study the initial unfolding of bacteriorhodopsin. A rapid near‐equilibrium folding between the first three unfolding states was discovered, the two transitions corresponded to the unfolding of five and three amino acids, respectively, when using a cantilever optimized for 2 μs resolution. The third of these states was retinal‐stabilized and previously undetected, despite being the most mechanically stable state in the whole unfolding pathway, supporting 150 pN for more than 1 min. This ability to measure the dynamics of the initial unfolding of bacteriorhodopsin provides a platform for quantifying the energetics of membrane proteins under native‐like conditions.     

Bioactive Structure of Membrane Lipids and Natural Products Elucidated by a Chemistry‐Based Approach

Determining the bioactive structure of membrane lipids is a new concept, which aims to examine the functions of lipids with respect to their three‐dimensional structures. As lipids are dynamic by nature, their “structure” does not refer solely to a static picture but also to the local and global motions of the lipid molecules. We consider that interactions with lipids, which are completely defined by their structures, are controlled by the chemical, functional, and conformational matching between lipids and between lipid and protein. In this review, we describe recent advances in understanding the bioactive structures of membrane lipids bound to proteins and related molecules, including some of our recent results. By examining recent works on lipid‐raft‐related molecules, lipid–protein interactions, and membrane‐active natural products, we discuss current perspectives on membrane structural biology.     

beta-Barrel membrane bacterial proteins: structure, function, assembly and interaction with lipids

Membrane proteins, although constituting about one-third of all proteins encoded by the genomes of living organisms, are still strongly underrepresented in the database of 3D protein structures, which reflects the big challenge presented by this class of proteins. Structural biologists, by employing electron and x-ray approaches, are continuously revealing new and fundamental insights into the structure, function, assembly and interaction with lipids of membrane proteins. To date, two structural motifs, alpha-helices and beta-sheets, have been found in membrane proteins and interestingly these two structural motives correlate with the location: while alpha-helical bundles are most often found in the receptors and ion channels of plasma and endoplasmic reticulum membranes, beta-barrels are restricted to the outer membrane of Gram-negative bacteria and in the mitochondrial membrane, and represent the structural motif used by several microbial toxins to form cytotoxic transmembrane channels. The beta-barrel, while being a rigid and stable motif is a versatile scaffold, having a wide variation in the size of the barrel, in the mechanism to open or close the gate and to impose selectivity on substrates. Even if the number of x-ray structures of integral membrane proteins has greatly increased in recent years, only a few of them provide information at a molecular level on how proteins interact with lipids that surround them in the membrane. The detailed mechanism of protein lipid interactions is of fundamental importance for understanding membrane protein folding, membrane adsorption, insertion and function in lipid bilayers. Both specific and unspecific interactions with lipids may participate in protein folding and assembly.     

Lipid packing stress and polypeptide aggregation: alamethicin channel probed by proton titration of lipid charge

Lipid membranes are not passive, neutral scaffolds to hold membrane proteins. In order to examine the influence of lipid packing energetics on ion channel expression, we study the relative probabilities of alamethicin channel formation in dioleoylphosphatidylserine (DOPS) bilayers as a function of pH. The rationale for this strategy is our earlier finding that the higher-conductance states, corresponding to larger polypeptide aggregates, are more likely to occur in the presence of lipids prone to hexagonal HII-phase formation (specifically DOPE), than in the presence of lamellar L alpha-forming lipids (DOPC). In low ionic strength NaCl solutions at neutral pH, the open channel in DOPS membranes spends most of its time in states of lower conductance and resembles alamethicin channels in DOPC; at lower pH, where the lipid polar groups are neutralized, the channel probability distribution resembles that in DOPE. X-Ray diffraction studies on DOPS show a progressive decrease in the intrinsic curvature of the constituent monolayers as well as a decreased probability of HII-phase formation when the charged lipid fraction is increased. We explore how proton titration of DOPS affects lipid packing energetics, and how these energetics couple titration to channel formation.     

Fluorescence wavelength, intensity and lifetime for multidimensional transduction of selective interactions of acetylcholine receptor by lipid membranes

Concurrent analysis of the fluorescence intensity, at different emission wavelengths, of lipid vesicles containing acetylcholine receptor (AChR) labelled with a nitrobenzoxadiazole (NBD) moiety shows that selective interactions with the agonist carbamylcholine can be detected reproducibly by a self-calibration method with muM detection limits. Concurrent analysis of the fluorescence intensity and lifetime of the new probe 4-dicyanomethylene-1,2,3,4-tetrahydromethylquinoline (DCQ) shows that general alterations of lipid membrane structure induced by temperature variation in the head-group region of lipid vesicles can be determined. A general approach to detection of selective interactions is introduced by observation of fluorescence intensity and lifetime changes of the probe NBD-phosphatidyl ethanolamine dispersed in lipid membranes containing unlabelled AChR. Detection and differentiation of selective interactions between carbamylcholine and the antagonist alpha-bungarotoxin are possible by correlation with intensity and lifetime at different emission wavelengths.     

Molecular dynamics simulation study of the interaction of trehalose with lipid membranes

The interactions of the cryoprotective agent trehalose with a lipid membrane made of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine at 323 K were studied by means of molecular dynamics simulations. It was observed that trehalose binds to the phospholipid headgroups with its main axis parallel to the membrane normal. Trehalose establishes hydrogen bonds with the carbonyl and phosphate groups and replaces water molecules from the lipid headgroup. Notably, the number of hydrogen bonds (HBs) that the membrane made with its environment was conserved after trehalose binding. The HBs between lipid and trehalose have a longer lifetime than those established between lipid and water. The binding of the sugar does not produce changes either in the lipid area or in the lipid order parameter. The effect of trehalose on the dipole potential is in agreement with experimental results. The contribution of the different components to the membrane dipole potential was analyzed. It was observed that the binding of trehalose produces changes in the different components and the sugar itself contributes to the surface potential due to the polarization of its hydroxyl in the interface.