sodium-dodecyl-sulfate and 1-palmitoyl-2-oleoylphosphatidylcholine

Sodium dodecyl sulfate (SDS) is one of the extensively used surfactants in bioprocesses. Present work analyzes the penetration of SDS in pure and mixed Langmuir monolayers consisting of 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol. A series of isotherms are obtained by compressing the monolayers over subphase containing varying concentrations of SDS. The extent of penetration of SDS is quantified in terms of its mole percentage in the monolayer by applying various thermodynamic equations derived from Gibbs adsorption equation. Analysis of pure monolayers shows that SDS penetration is directly correlated with the monolayer fluidity and the lipid packing density. We find that POPC monolayers, which do not pack closely on compression due to a kink in one of the acyl chains, exhibit higher SDS penetration than DPPC monolayers which are more rigid due to two saturated acyl chains that pack very efficiently. The SDS penetration is also analyzed in mixed lipid monolayers in which the lipids are present in molar proportions similar to that in the plasma membranes of mammalian cells. Thermodynamic quantification shows that mixed monolayers of DPPC with POPC (in 2:1 molar ratio) exhibit penetration similar to pure POPC monolayers indicating dominating effect of unsaturated phospholipids on membrane packing. We find lower SDS penetration in mixed cholesterol-POPC monolayers than pure POPC monolayers at all surface pressures and for all SDS subphase concentrations used. This confirms with the well-known condensation effect of cholesterol on POPC monolayers. Cholesterol is known to have a rigidifying effect on DPPC monolayers at low surface pressures and a fluidizing effect at high surface pressures. Consistent with these experimental observations, our thermodynamic quantification predicts lower SDS penetration at low pressures and higher penetration at high pressures when compared to pure DPPC monolayers. Thus, thermodynamic theories are able to accurately predict the experimentally observed trends for penetration in mixed monolayers of cholesterol with POPC and DPPC. Our work demonstrates that thermodynamic quantification is a reliable technique to estimate extent of penetration of various additives in pure and mixed lipid monolayers.

Topics: Adsorption; Cholesterol; Phosphatidylcholines; Phospholipids; Pressure; Sodium Dodecyl Sulfate; Surface Properties; Thermodynamics

Membrane proteins adopt two fundamental types of folds in nature: membranes in all organisms harbor α-helical bundles linked by extramembranous loops of varying length, while β-barrel structures are found in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. Here we report that turn-inducing loop mutations in a transmembrane hairpin induce the conversion of an α-helical hairpin to β-sheet oligomers in membrane environments. On the basis of an observation of a sequence bias toward Pro and Gly in the turns of native β-barrel membrane proteins, we characterized in sodium dodecyl sulfate (SDS) micelles and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers several "hairpin" constructs of cystic fibrosis transmembrane conductance regulator transmembrane segments 3 and 4 (TM3-loop-TM4; loop region being (215)IWELLQASA(223)) in which Pro-Gly residues were either inserted or substituted at several positions. Remarkably, suitable positioning of the Pro-Gly doublet caused the adoption of stable β-sheet structures by several mutants in SDS micelles, as shown by circular dichroism spectroscopy, concurrent with a ladder of discrete oligomers observed via SDS-polyacrylamide gel electrophoresis. Reconstitution of wild-type (WT) TM3/4 into POPC vesicles studied by Trp fluorescence, in conjunction with positional quenchers in brominated phospholipids, indicated a transbilayer position for helical WT TM3/4, but likely a largely surface-embedded conformation for the β-sheet mutant with loop region IWPGELLQASA. To the best of our knowledge, such a complete change in the fold with a minimal number of mutations has not been previously observed for a membrane protein. These facile α-helix to β-sheet conversions highlight the contribution of loops to membrane protein structure.

Topics: Amino Acid Sequence; Cystic Fibrosis Transmembrane Conductance Regulator; Glycine; Humans; Lipid Bilayers; Micelles; Models, Molecular; Molecular Sequence Data; Mutation; Phosphatidylcholines; Proline; Protein Folding; Protein Structure, Secondary; Sodium Dodecyl Sulfate

Production of helical integral membrane proteins (IMPs) in a folded state is a necessary prerequisite for their functional and structural studies. In many cases large-scale expression of IMPs in cell-based and cell-free systems results in misfolded proteins, which should be refolded in vitro. Here using examples of the bacteriorhodopsin ESR from Exiguobacterium sibiricum and full-length homotetrameric K(+) channel KcsA from Streptomyces lividans we found that the efficient in vitro folding of the transmembrane domains of the polytopic and multimeric IMPs could be achieved during the protein encapsulation into the reconstructed high-density lipoprotein particles, also known as lipid-protein nanodiscs. In this case the self-assembly of the IMP/nanodisc complexes from a mixture containing apolipoprotein, lipids and the partially denatured protein solubilized in a harsh detergent induces the folding of the transmembrane domains. The obtained folding yields showed significant dependence on the properties of lipids used for nanodisc formation. The largest recovery of the spectroscopically active ESR (~60%) from the sodium dodecyl sulfate (SDS) was achieved in the nanodiscs containing anionic saturated lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPG) and was approximately twice lower in the zwitterionic DMPC lipid. The reassembly of tetrameric KcsA from the acid-dissociated monomer solubilized in SDS was the most efficient (~80%) in the nanodiscs containing zwitterionic unsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The charged and saturated lipids provided lower tetramer quantities, and the lowest yield (<20%) was observed in DMPC. The overall yield of the ESR and KcsA folding was mainly restricted by the efficiency of the protein encapsulation into the nanodiscs.

Topics: Bacteria; Bacterial Proteins; Bacteriorhodopsins; Cell Membrane; Detergents; Dimerization; Dimyristoylphosphatidylcholine; Lipids; Membrane Proteins; Nanostructures; Nanotechnology; Phosphatidylcholines; Phosphatidylglycerols; Potassium Channels; Protein Structure, Secondary; Protein Structure, Tertiary; Proteins; Sodium Dodecyl Sulfate; Streptomyces lividans

The partition of the amphiphile sodium dodecyl sulfate (SDS) between an aqueous solution and a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer was followed by isothermal titration calorimetry (ITC) as a function of the total concentration of SDS. It was found that the obtained partition coefficient is strongly affected by the ligand concentration, even after correction for the charge imposed in the bilayer by the bound SDS. The partition coefficient decreased as the total concentration of SDS increased, with this effect being significant for local concentrations of SDS in the lipid bilayer above 5 molar%. At those high local concentrations, the properties of the lipid bilayer are strongly affected, leading to nonideal behavior and concentration-dependent apparent partition coefficients. It is shown that with the modern ITC instruments available, the concentrations of SDS can be drastically reduced while maintaining a good signal-to-noise ratio. The intrinsic parameters of the interaction with unperturbed membranes can be obtained from the asymptotic behavior of the apparent parameters as a function of the ligand concentration for both nonionic and ionic solutes. A detailed analysis is performed, and a spreadsheet is provided to obtain the interaction parameters with and without correction for electrostatics.

Topics: Calorimetry; Lipid Bilayers; Phosphatidylcholines; Sodium Dodecyl Sulfate

DD K, a peptide first isolated from the skin secretion of the Phyllomedusa distincta frog, has been prepared by solid-phase chemical peptide synthesis and its conformation was studied in trifluoroethanol/water as well as in the presence of sodium dodecyl sulfate and dodecylphosphocholine micelles or small unilamellar vesicles. Multidimensional solution NMR spectroscopy indicates an alpha-helical conformation in membrane environments starting at residue 7 and extending to the C-terminal carboxyamide. Furthermore, DD K has been labeled with (15)N at a single alanine position that is located within the helical core region of the sequence. When reconstituted into oriented phosphatidylcholine membranes the resulting (15)N solid-state NMR spectrum shows a well-defined helix alignment parallel to the membrane surface in excellent agreement with the amphipathic character of DD K. Proton-decoupled (31)P solid-state NMR spectroscopy indicates that the peptide creates a high level of disorder at the level of the phospholipid headgroup suggesting that DD K partitions into the bilayer where it severely disrupts membrane packing.

Topics: Animals; Antimicrobial Cationic Peptides; Anura; Circular Dichroism; Lipid Bilayers; Micelles; Models, Molecular; Nitrogen Isotopes; Nuclear Magnetic Resonance, Biomolecular; Phosphatidylcholines; Phosphorus Isotopes; Phosphorylcholine; Protein Conformation; Sodium Dodecyl Sulfate; Trifluoroethanol; Unilamellar Liposomes; Water

The region 35-43 of human alpha-Synuclein bound to small unilamellar lipid vesicles and to sodium dodecyl sulfate micelles has been investigated by site-directed spin labeling and electron paramagnetic resonance spectroscopy. The distance distributions obtained from spectral fitting have been analyzed on the basis of the allowed rotamers of the spin-label side-chain. Very similar results have been obtained in the two environments: an unbroken helical structure of the investigated region can be ruled out. The distance distributions are rather compatible with the presence of conformational disorder, in agreement with previous findings for micelle-bound alpha-Synuclein. The propensity for helix breaking is confirmed by molecular dynamics simulations.

Topics: alpha-Synuclein; Amino Acid Sequence; Computer Simulation; Electron Spin Resonance Spectroscopy; Humans; Lipid Bilayers; Micelles; Models, Molecular; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Phosphatidylcholines; Protein Structure, Secondary; Sodium Dodecyl Sulfate; Spin Labels

We establish high-sensitivity isothermal titration calorimetry (ITC) as a fast, reliable, and versatile tool for assessing membrane translocation of charged compounds. A combination of ITC uptake and release titrations can discriminate between the two extreme cases of half-sided binding and complete transbilayer equilibration on the experimental time scale. To this end, we derive a general fit function for both assays that allows for incorporation of different membrane partitioning models. Electrostatic effects are taken into account with the aid of Gouy-Chapman theory, thus rendering uptake and release experiments amenable to the investigation of charged solutes. This is exemplified for the flip-flop of the anionic detergent sodium dodecyl sulfate (SDS) across the membranes of 100-nm-diameter unilamellar vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in aqueous solution (10 mM phosphate buffer, 154 mM NaCl, pH 7.4). If repulsive electrostatic forces are accounted for adequately, SDS binding to POPC membranes can be evaluated on the basis of ideal mixing in all phases. At 25 degrees C, the intrinsic partition coefficient between the interfacial aqueous phase and the membrane amounts to 3.5 x 10(6); however, detergent flip-flop is negligibly slow under these conditions. Raising the temperature to 65 degrees C lowers the intrinsic partition coefficient to 1.4 x 10(6) but enables rapid transbilayer distribution of the detergent and, therefore, binding to or desorption from both membrane leaflets. Thus, combining a surface partition equilibrium with simple electrostatic theory appears highly useful in monitoring transmembrane movement of ionic compounds by ITC, thereby eliminating the need for specific reporter groups.

Topics: Calorimetry; Lipid Bilayers; Phosphatidylcholines; Sodium Dodecyl Sulfate; Solubility; Static Electricity; Temperature; Thermodynamics

We have studied the thermodynamic, surface, and structural properties of alphaM1 transmembrane sequence of the nicotinic acetylcholine receptor (nAChR) by using Langmuir monolayer, FT-IR spectroscopy and molecular dynamics simulation techniques in membrane-mimicking environments. M1 spontaneously incorporates into a lipid-free air-water interface, showing a favourable adsorption free energy of -7.2 kcal/mol. A cross-sectional molecular area of 210 A(2)/molecule, a surface potential of 4.2 fV/molecule and a high stability of the film were deducted from pure M1 monolayers. FT-IR experiments and molecular dynamics simulations in membrane-mimicking environments (sodium-dodecyl-sulfate and CCl(4), respectively) indicate coexistence between helical and non-helical structures. Furthermore, mixed peptide-lipid monolayers and monolayer penetration experiments were performed in order to study the peptide-lipid interaction. Mixed with condensed lipids (dipalmitoyl-phosphocholine, and dipalmitoyl-phosphoglycerol), M1 shows immiscible/miscible behaviour at low/high peptide concentration, respectively. Conversely, a complete miscible peptide-lipid interface is observed with liquid-expanded lipids (palmitoyl-oleoyl-phosphocholine, and palmitoyl-oleoyl-phosphoglycerol). Peptide penetration experiments demonstrate that the M1 peptide preferentially interacts with zwitterionic phosphocholine interfaces.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Adsorption; Amino Acid Sequence; Cell Membrane; Computer Simulation; Micelles; Models, Molecular; Molecular Sequence Data; Peptide Fragments; Phosphatidylcholines; Phosphatidylglycerols; Protein Conformation; Protein Subunits; Receptors, Nicotinic; Sodium Dodecyl Sulfate; Spectroscopy, Fourier Transform Infrared; Surface Properties; Thermodynamics; Water

We provide a comprehensive thermodynamic description of lipid membrane dissolution by a charged detergent. To this end, we have studied the interactions between the anionic detergent sodium dodecyl sulfate (SDS) and the zwitterionic phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in dilute aqueous solution (10 mM phosphate buffer, 154 mM NaCl, pH 7.4). Thermodynamic parameters of vesicle solubilization and reconstitution, membrane partitioning, and micelle formation were assessed by right-angle light scattering and isothermal titration calorimetry. Membrane translocation and dissolution proceed very slowly at 25 degrees C but are considerably accelerated at 65 degrees C. At this temperature, a simple SDS/POPC phase diagram (comprising vesicular, coexistence, and micellar ranges) and a complete set of partition coefficients and transfer enthalpies were obtained. Electrostatic repulsion effects at the membrane surface were implemented by combining Gouy-Chapman theory with a Langmuir adsorption isotherm to account for Na+ binding to membrane-incorporated DS-. This approach offered a quantitative understanding of solubilization and reconstitution processes, which were interpreted in terms of partition equilibria between and ideal mixing in all phases. More than any other property, the transbilayer flip-flop rate under given experimental conditions hence appears to dictate a detergent's suitability for thermodynamically controlled lipid membrane solubilization and reconstitution.

Topics: Lipid Bilayers; Liposomes; Membrane Fluidity; Membranes, Artificial; Molecular Conformation; Phase Transition; Phosphatidylcholines; Sodium Dodecyl Sulfate; Solubility; Temperature; Thermodynamics

Vesicle leakage experiments were carried out to establish how cholesterol content regulates membrane permeability as induced by surfactant exposure. Vesicles containing up to 50% cholesterol were examined. Four different surfactants were chosen as membrane perturbants, including nonoxynol-9 which is commonly used in spermicidal formulations. As part of this study, we establish that the extrusion procedure commonly used to fabricate unilamellar vesicles does not unintentionally alter the desired composition of these model membrane systems. The kinetics of the leakage process is well characterized by a single exponential rate of release, similar to the form seen in the absence of membrane cholesterol. Our leakage experiments show that membranes become more resistant toward surfactant attack, in direct proportion with cholesterol content. This rise in resistance is surfactant specific. Above 30%, all membranes show positive deviation from the linear increase in resistance with increasing cholesterol content. Two other sterols, dihydrocholesterol and coprostanol, were also found to increase membrane resistance and behaved similarly despite a key difference in molecular structure. A peculiar leakage response was observed when membranes were exposed to the surfactant sodium dodecyl sulfate (SDS) above its critical micelle concentration. Our findings support the hypothesis that SDS micelles solubilize phospholipid molecules, creating a membrane with higher cholesterol content that is extremely resistant to perturbation.

Topics: Animals; Cholestanol; Cholesterol; Detergents; Dose-Response Relationship, Drug; Membranes; Phosphatidylcholines; Phospholipids; Sodium Dodecyl Sulfate; Spectrometry, Fluorescence; Spermatocidal Agents; Sterols; Surface-Active Agents; Time Factors

The new fluorescent membrane probe Fluorazophore-L, a lipophilic derivative of the azoalkane 2,3-diazabicyclo[2.2.2]oct-2-ene, is employed to study the quenching of alpha-tocopherol (alpha-Toc) by time-resolved fluorescence in the microheterogeneous environments of Triton XR-100 and SDS micelles, as well as POPC liposomes. Fluorazophore-L has a small nonaromatic fluorescent polar headgroup and an exceedingly long-lived fluorescence (e.g., 140 ns in aerated SDS micelles), which is efficiently quenched by alpha-Toc (3.9 x 10(9) M(-1) s(-1) in benzene). Based on solvatochromic effects and the accessibility by water-soluble quenchers, the reactive headgroup of Fluorazophore-L, along with the chromanol group of alpha-Toc, resides at the water-lipid interface, which allows for a diffusion-controlled quenching in the lipidic environments. The quenching experiments represent an immobile or stationary case; that is, interparticle probe or quencher exchange during the excited-state lifetime is insignificant. Different quenching models are used to characterize the dynamics and antioxidant action of alpha-Toc in terms of diffusion coefficients or, where applicable, rate constants. The ideal micellar quenching model is suitable to describe the fluorescence quenching in SDS micelles and affords a pseudo-unimolecular quenching rate constant of 2.4 (+/- 0.4) x 10(7) s(-1) for a single quencher per micelle along with a mean aggregation number of 63 +/- 3. In Triton micelles as well as in unilamellar POPC liposomes, a two-dimensional (lateral) diffusion model is most appropriate. The mutual lateral diffusion coefficient D(L) for alpha-Toc and Fluorazophore-L in POPC liposomes is found to be 1.8 (+/- 0.1) x 10(-7) cm(2) s(-1), about a factor of 2 larger than for mutual diffusion of POPC, but more than 1 order of magnitude lower than a previously reported value. The comparison of the different environments suggests a quenching efficiency in the order benzene >> SDS micelles > Triton micelles > POPC liposomes, in line with expectations from microviscosity. The kinetic measurements provide important benchmark values for the modeling of oxidative stress in membranes and other lipidic assemblies. The special case of small lipidic assemblies (SDS micelles), for which the net antioxidant efficacy of alpha-Toc may be lower than expected on the grounds of its diffusional behavior, is discussed.

Topics: alpha-Tocopherol; Antioxidants; Cell Membrane; Diffusion; Fluorescence; Kinetics; Liposomes; Micelles; Models, Biological; Molecular Structure; Octoxynol; Phosphatidylcholines; Sodium Dodecyl Sulfate; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Time Factors

Human Calcitonin (hCt) is a peptide hormone which has a regulatory action in calcium-phosphorus metabolism. It is currently used as a therapeutic tool in bone pathologies such as osteoporosis and Paget's disease. However, due to its amphiphilic property tends to form a gelatinous solution in water which consists of fibrils that limits its therapeutic use. Here we show that sodium dodecyl sulfate (SDS), an anionic detergent able to induce and stabilize alpha-helices in polypeptides, at a monomeric concentration ranging between 0.26 mM-5 pM (all concentrations are below the CMC), increases the rate and number of hCt channel formation in planar lipid membranes, at both high and low hCt concentrations, with a maximum increase at a molecular hCt/SDS ratio of 1000:1. This effect could be interpreted as a counteraction to the fibrillation process of hCt molecules by removing molecules available for aggregation from the fluid; furthermore, this action, independently of channel formation in the cell membrane, could improve the peptide-receptor interaction. The action of SDS could be attributable to the strength of the sulfate negative charge and the hydrophobic chain; in fact, a similar effect was obtained with lauryl sarcosine and not with a neutral detergent such as n-dodecyl-beta-D-maltoside. The very low molecular ratio between SDS and peptide is suggestive of a possible catalytic action of SDS that could induce alpha-helices, the appropriate structures for interacting with the membrane. Moreover, in the experimental conditions investigated, the addition of SDS does not modify the membrane's electrical properties and most of the channel properties. This finding may contribute to the knowledge of environment-folding diseases due to protein and peptides.

Topics: Calcitonin; Catalysis; Electric Conductivity; Humans; Ion Channel Gating; Ion Channels; Lipid Bilayers; Membrane Potentials; Membranes, Artificial; Microchemistry; Nanotechnology; Phosphatidylcholines; Phosphatidylglycerols; Protein Conformation; Protein Structure, Secondary; Sodium Dodecyl Sulfate

The reactivity of the water-soluble antioxidant vitamin C (l-ascorbic acid) depends on pH. It is generally recognized that the ascorbate monoanion, which predominates at neutral physiological pH, acts as a stronger antioxidant than the protonated form, ascorbic acid. Fluorazophore-L, a long-lived fluorescent probe, was employed as a mimic for lipid peroxyl radicals. The experiments with micellar and bilayer membrane models demonstrate that vitamin C becomes, in fact, a more powerful antioxidant at low pH. This phenomenon may be general for the interception of reactive oxidizing species at the lipid/water interface.

Topics: Antioxidants; Ascorbic Acid; Bridged Bicyclo Compounds, Heterocyclic; Fluorescence; Fluorescent Dyes; Hydrogen-Ion Concentration; Kinetics; Liposomes; Membranes; Micelles; Microscopy, Fluorescence; Octoxynol; Phosphatidylcholines; Sodium Dodecyl Sulfate