“Staying Out” Rather than “Cracking In”: Asymmetric Membrane Insertion of 12:0 Lysophosphocholine

Interactions between detergents and model membranes are well described by the three-stage model: saturation and solubilization boundaries divide bilayer-only, bilayer–micelle coexistence, and micelle-only ranges. An underlying assumption of the model is the equilibration of detergent between the two membrane leaflets. However, many detergents partition asymmetrically at room temperature due to slow flip-flop, such as sodium dodecyl sulfate (SDS) and lysolipids. In this work, we use isothermal titration calorimetry (ITC) and dynamic light scattering (DLS) to investigate the solubilization of unilamellar POPC vesicles by 12:0 lysophosphocholine (12:0 LPC). Flip-flop of 12:0 LPC occurs beyond the time scale of our experiments, which establish a characteristic nonequilibrated state with asymmetric distribution: 12:0 LPC partitions primarily into the outer leaflet. Increasing asymmetry stress in the membrane does not lead to membrane failure, i.e., “cracking in” as seen for alkyl maltosides and other surfactants; instead, it reduces further membrane insertion which leads to the “staying out” of 12:0 LPC in solution. At above the critical micellar concentration of 12:0 LPC in the presence of the membrane, micelles persist and accommodate further LPC but take up lipid from vesicles only very slowly. Ultimately, solubilization proceeds via the micellar mechanism (Kragh-Hansen et al., 1995). With a combination of demicellization and solubilization experiments, we quantify the molar ratio partition coefficient (0.6 ± 0.1 mM^–1) and enthalpy of partitioning (6.1 ± 0.3 kJ·mol^–1) and estimate the maximum detergent/lipid ratio reached in the outer leaflet (<0.13). Despite the inapplicability of the three-stage model to 12:0 LPC at room temperature, we are able to extract quantitative information from ITC solubilization experiments and DLS that are important for the understanding of asymmetry-dependent processes such as endocytosis and the gating of mechanosensitive channels <i>in vitro</i>

Effect of Hydrophobic Interactions on Volume and Thermal Expansivity as Derived from Micelle Formation

Volumetric parameters have long been used to elucidate the phenomena governing the stability of protein structures, ligand binding, or transitions in macromolecular or colloidal systems. In spite of much success, many problems remain controversial. For example, hydrophobic groups have been discussed to condense adjacent water to a volume lower than that of bulk water, causing a negative contribution to the volume change of unfolding. However, expansivity data were interpreted in terms of a structure-making effect that expands the water interacting with the solute. We have studied volume and expansivity effects of transfer of alkyl chains into micelles by pressure perturbation calorimetry and isothermal titration calorimetry. For a series of alkyl maltosides and glucosides, the methylene group contribution to expansivity was obtained as 5 uL/(mol K) in a micelle (mimicking bulk hydrocarbon) but 27 uL/(mol K) in water (20 °C). The latter value is virtually independent of temperature and similar to that obtained from hydrophobic amino acids. Methylene contributions of micellization are about −60 J/(mol K) to heat capacity and 2.7 mL/mol to volume. Our data oppose the widely accepted assumption that water-exposed hydrophobic groups yield a negative contribution to expansivity at low temperature that would imply a structure-making, water-expanding effect

Efficacy as an Intrinsic Property of the M₂ Muscarinic Receptor in Its Tetrameric State

Muscarinic and other G protein-coupled receptors exhibit an agonist-specific heterogeneity that tracks efficacy and commonly is attributed to an effect of the G protein on an otherwise homogeneous population of sites. To examine this notion, M₂ muscarinic receptors were purified from <i>Sf</i>9 cells as monomers devoid of G protein and reconstituted as tetramers in phospholipid vesicles. In assays with <i>N</i>-[³H]­methylscopolamine, seven agonists revealed a dispersion of affinities indicative of two or more classes of sites. Unlabeled <i>N</i>-methylscopolamine and the antagonist quinuclidinylbenzilate recognized one class of sites; atropine recognized two classes with a preference that was the opposite of that of agonists, as indicated by the effects of <i>N</i>-ethylmaleimide. The data were inconsistent with an explicit model of constitutive asymmetry within a tetramer, and the fit improved markedly upon the introduction of cooperative interactions (<i>P</i> < 0.00001). Purified monomers appeared to be homogeneous or nearly so to all ligands except the partial agonists pilocarpine and McN-A-343, where heterogeneity emerged from intramolecular cooperativity between the orthosteric site and an allosteric site. The breadth of each dispersion was quantified empirically as the area between the fitted curve for two classes of sites and the theoretical curve for a single class of lower affinity, which approximates the expected effect of GTP if a G protein were present. The areas measured for 10 ligands at reconstituted tetramers correlated with similar measures of heterogeneity and with intrinsic activities reported previously for binding and response in natural membranes (<i>P</i> ≤ 0.00002). The data suggest that the GTP-sensitive heterogeneity typically revealed by agonists at M₂ receptors is intrinsic to the receptor in its tetrameric state. It exists independently of the G protein, and it appears to arise at least in part from cooperativity between linked orthosteric sites

Engineering Asymmetric Lipid Vesicles: Accurate and Convenient Control of the Outer Leaflet Lipid Composition

The asymmetric distribution of lipids between the two bilayer leaflets represents a typical feature of biological membranes. The loss of this asymmetry, for example the exposure of negatively charged lipids on the extracellular membrane leaflet of mammalian cells, is involved in apoptosis and occurs in tumor cells. Thus, the controlled production of asymmetric liposomes helps to better understand such crucial cellular processes. Here, we present an approach that allows us to design asymmetric model-membrane experiments on a rational basis and predict the fraction of exchanged lipid. In addition, we developed a label-free and nondestructive assay to quantify the asymmetric uptake of negatively charged lipids in terms of the zeta potential. This significantly enhances the applicability, impact, and predictive power of model membranes