Lipid Solvation Research Articles

The role of the membrane in driving CLC-ec1 dimerization.

Most membrane proteins oligomerize in the membrane. In the case of the bacterial chloride/proton antiporter CLC-ec1, the role of highly stable homodimers is not understood since individual CLC-ec1 protomers can maintain the chloride/proton transport stoichiometry. In a previous study, we used experimental and computational approaches to show that in C16:0/C18:1 palmitoyl-oleoyl lipid membranes, the monomeric state of CLC-ec1 creates a membrane defect that is ≈ 8 Å thinner than the bulk. However, in the dimer state, the membrane is restored to a near-native bilayer thickness and structure. We hence proposed that the dimerization is driven by the larger free-energy cost of lipid solvation of the dissociated monomers. Here, we carry out coarse-grained molecular dynamics simulations with umbrella sampling and free energy perturbation methods to quantify the membrane's contribution to free energy of dimerization. In the case of CLC-ec1, addition of 30% C12:0 di-lauryl chain lipids stabilizes the free energy of the monomeric state by ≈ 2 kcal/mole, in qualitative agreement with our experimental observations. Therefore, we have a physical model of how changes in lipid composition can regulate self-assembly reactions of membrane proteins in membranes. This work was partially performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. LLNL-ABS-840782.

Sampling protein-lipid interactions with swarms of trajectories and a transition matrix analysis

Lipid-protein interactions are critical for integral membrane protein function, where lipids can play the role of both solvent and ligand. Because resolving the lipids that solvate a membrane protein is experimentally challenging, simulations have come to play a prominent role in linking lipid solvation and membrane protein function. However, converging the lipid distribution around a membrane protein can require surprisingly long simulations, especially in cholesterol-rich mixtures where lipid diffusion and exchange at the surface of the protein can be quite slow.

Membrane transporter dimerization driven by differential lipid solvation energetics of dissociated and associated states.

Over two-thirds of integral membrane proteins of known structure assemble into oligomers. Yet, the forces that drive the association of these proteins remain to be delineated, as the lipid bilayer is a solvent environment that is both structurally and chemically complex. In this study, we reveal how the lipid solvent defines the dimerization equilibrium of the CLC-ec1 Cl-/H+ antiporter. Integrating experimental and computational approaches, we show that monomers associate to avoid a thinned-membrane defect formed by hydrophobic mismatch at their exposed dimerization interfaces. In this defect, lipids are strongly tilted and less densely packed than in the bulk, with a larger degree of entanglement between opposing leaflets and greater water penetration into the bilayer interior. Dimerization restores the membrane to a near-native state and therefore, appears to be driven by the larger free-energy cost of lipid solvation of the dissociated protomers. Supporting this theory, we demonstrate that addition of short-chain lipids strongly shifts the dimerization equilibrium toward the monomeric state, and show that the cause of this effect is that these lipids preferentially solvate the defect. Importantly, we show that this shift requires only minimal quantities of short-chain lipids, with no measurable impact on either the macroscopic physical state of the membrane or the protein's biological function. Based on these observations, we posit that free-energy differentials for local lipid solvation define membrane-protein association equilibria. With this, we argue that preferential lipid solvation is a plausible cellular mechanism for lipid regulation of oligomerization processes, as it can occur at low concentrations and does not require global changes in membrane properties.

Activation of G-protein-coupled receptors is thermodynamically linked to lipid solvation

Preferential lipid solvation of the G-protein-coupled A2A adenosine receptor (A2AR) is evaluated from 35 μs of all-atom molecular dynamics simulation. A coarse-grained transition matrix algorithm is developed to overcome slow equilibration of the first solvation shell, obtaining estimates of the free energy of solvation by different lipids for the receptor in different activation states. Results indicate preference for solvation by unsaturated chains, which favors the active receptor. A model for lipid-dependent G-protein-coupled receptor activity is proposed in which the chemical potential of lipids in the bulk membrane modulates receptor activity. The entropies associated with moving saturated and unsaturated lipids from bulk to A2AR’s first solvation shell are evaluated. Overall, the acyl chains are more disordered (i.e., obtain a favorable entropic contribution) when partitioning to the receptor surface, and this effect is augmented for the saturated chains, which are relatively more ordered in bulk.

Activation of G-Protein Coupled Receptors Is Thermodynamically Linked to Lipid Solvation

Preferential lipid solvation of the G-protein coupled A2A adenosine receptor (A2AR) is evaluated from 35 μsec of all-atom molecular dynamics simulation. A coarse-grained transition matrix algorithm is developed to overcome slow equilibration of the first solvation shell, obtaining statistically robust estimates of the free energy of solvation by different lipids for the receptor in different activation states. Results indicate preference for solvation by unsaturated chains, which favors the active receptor.

Structural cavities are critical to balancing stability and activity of a membrane-integral enzyme

Packing interaction is a critical driving force in the folding of helical membrane proteins. Despite the importance, packing defects (i.e., cavities including voids, pockets, and pores) are prevalent in membrane-integral enzymes, channels, transporters, and receptors, playing essential roles in function. Then, a question arises regarding how the two competing requirements, packing for stability vs. cavities for function, are reconciled in membrane protein structures. Here, using the intramembrane protease GlpG of Escherichiacoli as a model and cavity-filling mutation as a probe, we tested the impacts of native cavities on the thermodynamic stability and function of a membrane protein. We find several stabilizing mutations which induce substantial activity reduction without distorting the active site. Notably, these mutations are all mapped onto the regions of conformational flexibility and functional importance, indicating that the cavities facilitate functional movement of GlpG while compromising the stability. Experiment and molecular dynamics simulation suggest that the stabilization is induced by the coupling between enhanced protein packing and weakly unfavorable lipid desolvation, or solely by favorable lipid solvation on the cavities. Our result suggests that, stabilized by the relatively weak interactions with lipids, cavities are accommodated in membrane proteins without severe energetic cost, which, in turn, serve as a platform to fine-tune the balance between stability and flexibility for optimal activity.

Local Enrichment of Unsaturated Chains around the A2A Adenosine Receptor.

Two 15 μs all-atom simulations of the A2A adenosine receptor were obtained in a ternary mixture of cholesterol, saturated phosphatidylcholine lipids, and unsaturated phosphatidylcholine lipids. An analysis of local lipid solvation is reported on the basis of a Voronoi tessellation of the upper and lower leaflets, identifying first and second solvation shells. The local environments of both the inactive state and the partially active state of the receptor are significantly enriched with unsaturated chains but depleted of cholesterol and saturated chains, relative to the bulk membrane composition. In spite of the local depletion of cholesterol, the partially active receptor binds cholesterol at three locations during the entire simulation trajectory. These long-lived interactions represent the extreme of a very broad distribution of first-solvation shell lipid lifetimes, confounding sharp distinctions between lipid interactions. The broad distributions of lifetimes also make equilibrating the local lipid environment difficult, necessitating long simulation times.

Molecular dynamics study of homo-oligomeric ion channels: Structures of the surrounding lipids and dynamics of water movement

Molecular dynamics simulations were used to study the structural perturbations of lipids surrounding transmembrane ion channel forming helices/helical bundles and the movement of water within the pores of the ion-channels/bundles. Specifically, helical monomers to hexameric helical bundles embedded in palmitoyl-oleoyl-phosphatidyl-choline (POPC) lipid bilayer were studied. Two amphipathic α-helices with the sequence Ac-(LSLLLSL)₃-NH₂ (LS2), and Ac-(LSSLLSL)₃-NH₂ (LS3), which are known to form ion channels, were used. To investigate the surrounding lipid environment, we examined the hydrophobic mismatch, acyl chain order parameter profiles, lipid head-to-tail vector projection on the membrane surface, and the lipid headgroup vector projection. We find that the lipid structure is perturbed within approximately two lipid solvation shells from the protein bundle for each system (~15.0 Å). Beyond two lipid “solvation” shells bulk lipid bilayer properties were observed in all systems. To understand water flow, we enumerated each time a water molecule enters or exited the channel, which allowed us to calculate the number of water crossing events and their rates, and the residence time of water in the channel. We correlate the rate of water crossing with the structural properties of these ion channels and find that the movements of water are predominantly governed by the packing and pore diameter, rather than the topology of each peptide or the pore (hydrophobic or hydrophilic). We show that the crossing events of water fit quantitatively to a stochastic process and that water molecules are traveling diffusively through the pores. These lipid and water findings can be used for understanding the environment within and around ion channels. Furthermore, these findings can benefit various research areas such as rational design of novel therapeutics, in which the drug interacts with membranes and transmembrane proteins to enhance the efficacy or reduce off-target effects.

α-Synuclein Bound to Mitochondrial Membranes—Changes in Lipid Bilayer Structure and Mechanics

The association between α-Synuclein and mitochondrial membranes is believed to be an important event in the neuronal degeneration that leads to Parkinson Disease (PD). In particular,α-Synuclein binding to mitochondrial membranes has recently been seen to cause organelle fragmentation and suppression of mitochondrial fusion. The partitioning of monomeric α-Synuclein in the lipid bilayer and subsequent bilayer remodeling has been previously described for simple, model lipid bilayers that lack the complexity of mitochondrial membranes. Here, we ran a series of coarse-grain molecular dynamics MARTINI simulations of α-Synuclein absorbed in either pure cardiolipin bilayers or in mixed lipid bilayers that reflect the lipid composition of inner mitochondrial membrane of rat brain mitochondria. We investigated changes in bilayer structure and variations in lipid solvation near α-synuclein. A thinning of the bilayer is observed in simulations having higher levels of added protein. Other aspects studied include α-Synuclein partition depth (investigated by potential of mean force calculations), bilayer bending rigidities and curvature as a function of protein and/or cardiolipin density.

Membrane Depth-Dependent Energetic Contribution of the Tryptophan Side Chain to the Stability of Integral Membrane Proteins

Lipid solvation provides the primary driving force for the insertion and folding of integral membrane proteins. Although the structure of the lipid bilayer is often simplified as a central hydrophobic core sandwiched between two hydrophilic interfacial regions, the complexity of the liquid-crystalline bilayer structure and the gradient of water molecules across the bilayer fine-tune the energetic contributions of individual amino acid residues to the stability of membrane proteins at different depths of the bilayer. The tryptophan side chain is particularly interesting because despite its widely recognized role in anchoring membrane proteins in lipid bilayers, there is little consensus about its hydrophobicity among various experimentally determined hydrophobicity scales. Here we investigated how lipid-facing tryptophan residues located at different depths in the bilayer contribute to the stability of integral membrane proteins using outer membrane protein A (OmpA) as a model. We replaced all lipid-contacting residues of the first transmembrane β-strand of OmpA with alanines and individually incorporated tryptophans in these positions along the strand. By measuring the thermodynamic stability of these proteins, we found that OmpA is slightly more stable when tryptophans are placed in the center of the bilayer and that it is somewhat destabilized as tryptophans approach the interfacial region. However, this trend may be partially reversed when a moderate concentration of urea rather than water is taken as the reference state. The measured stability profiles are driven by similar profiles of the m-value, a parameter that reflects the shielding of hydrophobic surface area from water. Our results indicate that knowledge of the free energy level of the protein's unfolded reference state is important for quantitatively assessing the stability of membrane proteins, which may explain differences in observed profiles between in vivo and in vitro scales.

Influence of the lipid environment on valinomycin structure and cation complex formation

Carrier-type molecular ionophores, such as the cyclic dodecadepsipeptide valinomycin, often must undergo structural changes during the binding and transport of a cation across the lipid membrane. Observing the structural fluctuations that occur during this process experimentally has proven extremely difficult due to the complexities of spectroscopic analysis of protein structure/dynamics in native lipid bilayer environments. Currently, our understanding of how valinomycin selectively transports ions across membranes is derived from atomic structures solved of the cyclic macromolecule solvated in various organic solvents and complimentary in silico dynamics experiments. We have shown recently that deep-UV excited resonance Raman spectroscopy (DUVRR) has a unique ability to characterize secondary structure content and simultaneously provide information about the relative solvation of the probed peptide backbone C.M. Halsey, J. Xiong, O. Oshokoya, J.A. Johnson, S. Shinde, J.T. Beatty, G. Ghirlanda, R.D. JiJi, J.W. Cooley, Simultaneous observation of peptide backbone lipid solvation and a-helical structure by deep-UV resonance Raman spectroscopy, ChemBioChem 12 (2011) 2125–2128, [16]. Interpretation of DUVRR spectra of valinomycin in swelled lipid and unilamellar lipid bilayer environments indicate that the uncomplexed valinomycin molecule dynamically samples both the open and closed conformations as described for the structures derived from polar and non-polar organic solvents, respectively. Upon introduction of potassium, the structure of valinomycin in swelled lipid environments resembles more closely that of the open conformation. The shift in structure upon complexation is accompanied by a significant decrease in the valinomycin DUVRR spectral amide I intensity, indicating that the open conformation is more water solubilized and is seemingly “trapped” or predominantly located close to the lipid–water interface. The trapping of the valinomycin in the act of complex of potassium at the bilayer-solvent interface and its analysis by DUVRR represents the first spectroscopic description of this state. Conversely, an opposite trend is observed in the amide I intensity upon potassium complexation in unilamellar (or extruded) vesicles, implying the predominant conformation upon potassium binding in native bilayers is one where the peptide backbone of valinomycin is desolvated as would be expected if the molecule were more readily able to traverse a bilayer interior. Interpretation of the DUVRR spectral features is also consistent with the loss or formation of hydrogen bonds observed in the open and closed structures, respectively. Valinomycin must then sample several conformations in the absence of appropriate ions depending upon its locale in the lipid bilayer until potassium causes a greater degree of closure of the open conformer and an increased residency within the more non-polar interior. The potassium induced decreased solubility enables diffusion across the membrane where potassium release can occur by equilibration at the opposite lipid water interface.

A New Putative Cholesterol-Recognition Motif in Transmembrane Proteins

We report the finding of a new putative cholesterol-recognition motif in the transmembrane region of membrane proteins like the nicotinic acetylcholine receptor (AChR) and other members of the Cys-loop superfamily of neurotransmitters involved in rapid synaptic transmission, as well as in members of the important superfamily of G protein coupled receptors (GPCRs) involved in Alzheimer's disease. The motif is an inverted CRAC domain (cholesterol recognition/interaction amino acid consensus) which we have named CARC and is present in the transmembrane regions of the receptor subunits having extensive contact with the surrounding lipid, predominantly in the first (TM1) segment in the case of the AChR, and optimally suited to convey cholesterol-mediated signaling. Three cholesterol molecules could be docked on the transmembrane regions of the five human AChR subunits, rendering a total of 15 cholesterol molecules per AChR molecule, adding up to a total of about 200 kJ.mol-1 per receptor molecule, i.e. ∼40% of the total lipid solvation free energies for the whole AChR molecule. The novel motif is remarkably conserved among Cys-loop receptors along the phylogenetic scale, from prokaryotes to humans, its high degree of conservation suggesting that it could be responsible for some of the structural/functional properties of these transduction macromolecules.Reference: Baier, C.J., Fantini, J. and Barrantes, F.J. (2011). Sci. Rep. 1:00069.

Disclosure of cholesterol recognition motifs in transmembrane domains of the human nicotinic acetylcholine receptor

Cholesterol influences ion-channel function, distribution and clustering in the membrane, endocytosis, and exocytic sorting of the nicotinic acetylcholine receptor (AChR). We report the occurrence of a cholesterol recognition motif, here coined “CARC”, in the transmembrane regions of AChR subunits that bear extensive contact with the surrounding lipid, and are thus optimally suited to convey cholesterol-mediated signaling from the latter. Three cholesterol molecules could be docked on the transmembrane segments of each AChR subunit, rendering a total of 15 cholesterol molecules per AChR molecule. The CARC motifs contribute each with an energy of interaction between 35 and 52 kJ.mol−1, adding up to a total of about 200 kJ.mol−1 per receptor molecule, i.e. ∼40% of the lipid solvation free energy/ AChR molecule. The CARC motif is remarkably conserved along the phylogenetic scale, from prokaryotes to human, suggesting that it could be responsible for some of the above structural/functional properties of the AChR.

Simultaneous Observation of Peptide Backbone Lipid Solvation and α‐Helical Structure by Deep‐UV Resonance Raman Spectroscopy

Currently no structurally sensitive spectroscopic techniques are capable of co-determining ensemble structural content and localized lipid versus aqueous solvation information. Here, we describe the first deep-UV (λex<210 nm) resonance Raman (dUVRR) spectra of a model α-helical peptide embedded in a membrane-mimetic environment, confirming sensitivity to secondary structure content and revealing sensitivity of dUVRR to the lipid solvation of the peptide backbone. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Composition fluctuations, correlated response, and protein solvation in membranes

Membrane composition fluctuations are deduced from the deuterium NMR relaxation data of S. L. Veatch et al. [Proc. Natl. Acad. Sci. U.S.A. 104, 17650 (2007)]. A theoretical model for these fluctuations is used to determine the parameters of a correlation function. A fluctuation-response relation is then derived to infer the response of a lipid bilayer membrane to perturbations, such as the presence of a protein. The energy of the correlated response is shown to decrease as a bilayer miscibility critical point is approached from higher temperatures. Near the critical temperature the low energy of the composition response facilitates the lipid solvation of membrane proteins and minimizes lipid-mediated nonspecific protein-protein interactions. This facilitated lipid solvation of membrane proteins may be the basis of reports that at the growth temperature, the lipids of animal cell membranes have compositions such that they are within approximately 10 degrees of a miscibility critical point.

The lipid/protein interface as a target site for general anesthetics: a multiple-site kinetic analysis of synaptosomal Ca 2+-ATPase

There is a long-standing controversy on whether membrane lipids or proteins are the target for general anesthetics. The plasma membrane-associated Ca 2+-ATPase of synaptosomes has recently been established as a model system for general anesthesia, the protein interior being the proposed target site (M.M. Lopez, D. Kosk-Kosicka, J. Biol. Chem. 270 (1995) 28239–28245). Multiple-site kinetics is now applied as a mechanistic tool to analyze inhibition by organic solvents and general anesthetics. A close fit to the experimental data points was achieved using the complex equations for a competitive displacement of lipid activators from multiple sites on the protein surface. Inhibitor dissociation constants were about 1.6×10 5-fold higher than the microscopic lipid dissociation binding constants that are derived here for the first time. Binding of lipid therefore is by −7.1 kcal/mole favored over that of the tested inhibitors. The latter are nevertheless effective because in the model used displacement of only few of the lipid solvation molecules cause complete inhibition. The lipid/protein interface rather than protein or lipid alone appeared to be the anesthetic target site.

Spin label EPR study of lipid solvation of supramolecular photosynthetic protein complexes in thylakoids

Lipid-protein association in the chloroplast membrane and its various thylakoid fractions from higher plants, namely pea and maize, rich in Photosystem I (PSI) and Photosystem II (PSII), respectively, were studied using EPR spectroscopy of spin-labelled lipid molecules. All the EPR spectra consisted of two spectral components corresponding to bulk fluid lipids and solvation lipids motionally restricted at the hydrophobic surface of membrane proteins. Spin-labelled stearic acid and phosphatidylglycerol exhibited marked selectivity towards the supramolecular protein complexes of both PSI and PSII although to different extent. In addition, lipid-protein titration experiments are described for partially delipidated PSII-enriched membrane fractions of pea chloroplasts, incorporating unlabelled egg phosphatidylcholine prior to or after the incorporation of spin-labelled lipids. Two sets of solvation sites were resolved by timed labelling experiments and a significant result of these studies was that a well-defined population of solvation sites (approx. 100 mol lipids/820 kDa protein) was rapidly exchanged by laterally diffusing membrane lipids, while other solvation sites (approx. 50 mol lipids/820 kDa protein) were exchanged much slower or not exchanged at all.

An Optimized Freeze-Fracture Replication Procedure for Human Skin

This in vitro study aimed at substantial modification of the freeze-fracture replication technique (FFRT) which should result in an optimal visualization of the ultrastructure of human skin. The technique was modified in two ways: firstly, the conventional sample holders such as gold cups and copper plates were replaced by silver cylinders (83.5% silver, 16.5% copper) resulting in almost perpendicular cross fractures through the skin. Secondly, the replica cleaning procedure was optimized through the following sequence of treatments. Firstly, a mild tissue destruction was obtained by simultaneous lipid solvation and water extraction with absolute methanol (20 h), followed by protein denaturation with dimethyl sulfoxide (DMSO, 24 h). Subsequently, a final treatment was given using an alkaline sodium hypochlorite solution (20% KOH/13% NaClO; 1:3 v/v, 4 d). After rinsing the replicas for 45 min in aqua bidest, they were mounted on copper grids and examined in the transmission electron microscope (TEM). The combination of the unorthodox fracturing method and the optimized cleaning procedure yielded large, practically undamaged and very clean replicas of near perpendicular cross fractures through human skin. Common handicaps related to current freeze-fracture procedures when applied to skin, such as incomplete cleaning and fragmentation of replicas and oblique or irregular fracturing planes, can largely be avoided in this way. In this paper a complete description of the method is given, and a number of advantages are illustrated with the aid of TEM micrographs.

Lipid solvation of the aqueous form of the myelin proteolipid apoprotein: evidence and characterization of two lipid populations by fluorescence polarization, differential calorimetry, and sucrose gradient centrifugation.

The interaction between dipalmitoylphosphatidylcholine (DPPC) and the aqueous form of the myelin proteolipid apoprotein (PLA) has been investigated. Lyophilization was found to be an efficient and nonperturbing method for membrane reconstitution. Mixtures of different lipid/protein ratios were analyzed by means of differential calorimetry, fluorescence polarization, and sucrose gradient centrifugation. The presence of two coexisting lipid populations, termed "bulk" and "interacting" lipids, was demonstrated by these three techniques. By differential calorimetry, 23 DPPC molecules per molecule of protein (30 kDa) were shown to be excluded from the lipid phase transition. By fluorescence polarization, we detected above the phase-transition temperature a large perturbation of the lipid acyl chain dynamics induced by the aqueous form of PLA. Increasing the protein content above 35% by weight within the recombinants caused drastic changes in both delta H values and the fluorescence anisotropy parameter, which could stem from protein aggregation.

Lipid solvation of cytochrome c oxidase. Deuterium, nitrogen-14, and phosphorus-31 nuclear magnetic resonance studies on the phosphocholine head group and on cis-unsaturated fatty acyl chains.

Lipid solvation of cytochrome c oxidase. Deuterium, nitrogen-14, and phosphorus-31 nuclear magnetic resonance studies on the phosphocholine head group and on cis-unsaturated fatty acyl chains.