Neutron reflectivities of phosphatidylcholine monolayers in the liquid condensed (LC) phase on ultrapure H(2)O and D(2)O subphases have been measured on a Langmuir film balance. Using a dedicated liquid surface reflectometer, reflectivities down to R = 10(-6) in the momentum transfer range Q(z) = 0-0.4 A(-1) were accessed.In a new approach, by refining neutron reflectivity data from chain-perdeuterated DPPC-d(62) in combination with x-ray measurements on the same monolayer under similar conditions it is shown that the two techniques mutually complement one another. This analysis leads to a detailed conception of the interface structure. It is found that in the LC phase (which is analogous to the L(beta), phase in vesicle dispersions) the head group is interpenetrated with subphase water (4 +/- 2.5 molecules per lipid) and the average tilt angle of the hydrophobic chains from the surface normal is 33 +/- 3 degrees.
The structure and function of the PTEN phosphatase is investigated by studying its membrane affinity and localization on in-plane fluid, thermally disordered synthetic membrane models. The membrane association of the protein depends strongly on membrane composition, where phosphatidylserine (PS) and phosphatidylinositol diphosphate (PI(4,5)P2) act pronouncedly synergistic in pulling the enzyme to the membrane surface. The equilibrium dissociation constants for the binding of wild type (wt) PTEN to PS and PI(4,5)P2 were determined to be Kd∼12 µM and 0.4 µM, respectively, and Kd∼50 nM if both lipids are present. Membrane affinities depend critically on membrane fluidity, which suggests multiple binding sites on the protein for PI(4,5)P2. The PTEN mutations C124S and H93R show binding affinities that deviate strongly from those measured for the wt protein. Both mutants bind PS more strongly than wt PTEN. While C124S PTEN has at least the same affinity to PI(4,5)P2 and an increased apparent affinity to PI(3,4,5)P3, due to its lack of catalytic activity, H93R PTEN shows a decreased affinity to PI(4,5)P2 and no synergy in its binding with PS and PI(4,5)P2. Neutron reflection measurements show that the PTEN phosphatase “scoots" along the membrane surface (penetration <5 Å) but binds the membrane tightly with its two major domains, the C2 and phosphatase domains, as suggested by the crystal structure. The regulatory C-terminal tail is most likely displaced from the membrane and organized on the far side of the protein, ∼60 Å away from the bilayer surface, in a rather compact structure. The combination of binding studies and neutron reflection allows us to distinguish between PTEN mutant proteins and ultimately may identify the structural features required for membrane binding and activation of PTEN.
The self-assembled monolayers (SAMs) of the new lipidic anchor molecule HC18 [Z 20-(Z-octadec-9-enyloxy)-3,6,9,12,15,18,22-heptaoxatetracont-31-ene-1-thiol], and mixed HC18/β-mercaptoethanol (βME) SAMs were studied by spectroscopic ellipsometry, contact angle measurements, reflection adsorption infrared spectroscopy (RAIRS), electrochemical impedance spectroscopy (EIS), and evaluated in tethered bilayer lipid membranes (tBLMs). Our data indicate that HC18, containing a double bond in the alkyl segments, forms highly disordered SAMs up to anchor/βME molar fraction ratios of 80/20 and result in tBLMs that exhibit higher lipid diffusion coefficients, relative to previous anchor compounds with saturated alkyl chains, as determined by fluorescence correlation spectroscopy. EIS data shows the HC18 tBLMs, completed by rapid solvent exchange (RSE) or vesicle fusion, form more easily than with saturated lipidic anchors, exhibit excellent electrical insulating properties indicating low defect densities, and readily incorporate the pore forming toxin, α-hemolysin. Neutron reflectivity measurements on HC18 tBLMs confirm the formation of complete tBLMs, even at low tether compositions and high ionic lipid compositions. Our data indicates HC18 results in tBLMs with improved physical properties for incorporation of integral membrane proteins (IMPs) and that 80% HC18 tBLMs appear to be optimal for practical applications such as biosensors where high electrical insulation and IMP/peptide reconstitution is imperative.
The coupling of bacterial surface (S)-layer proteins to lipid membranes is studied in molecular detail for proteins from Bacillus sphaericus CCM2177 and B. coagulans E38-66 recrystallized at dipalmitoylphosphatidylethanolamine (DPPE) monolayers on aqueous buffer. A comparison of the monolayer structure before and after protein recrystallization shows minimal reorganization of the lipid chains. By contrast, the lipid headgroups show major rearrangements. For the B. sphaericus CCM2177 protein underneath DPPE monolayers, x-ray reflectivity data suggest that amino acid side chains intercalate the lipid headgroups at least to the phosphate moieties, and probably further beyond. The number of electrons in the headgroup region increases by more than four per lipid. Analysis of the changes of the deduced electron density profiles in terms of a molecular interpretation shows that the phosphatidylethanolamine headgroups must reorient toward the surface normal to accommodate such changes. In terms of the protein structure (which is as yet unknown in three dimensions), the electron density profile reveals a thickness lz approximately 90 A of the recrystallized S-layer and shows water-filled cavities near its center. The protein volume fraction reaches maxima of >60% in two horizontal sections of the S-layer, close to the lipid monolayer and close to the free subphase. In between it drops to approximately 20%. Four S-layer protein monomers are located within the unit cell of a square lattice with a spacing of approximately 131 A.
To many biophysical characterisation techniques, biological membranes appear as twodimensional structures with details of their third dimension hidden within a 5 nm profile. Probing this structure requires methods able to discriminate multiple layers a few Ångströms thick. Given sufficient resolution, neutron methods can provide the required discrimination between different biochemical components, especially when selective deuteration is employed. We have used stateof-the-art neutron reflection methods, with resolution enhancement via magnetic contrast variation to study an oriented model membrane system. The model is based on the Escherichia coli outer membrane protein OmpF fixed to a gold surface via an engineered cysteine residue. Below the gold is buried a magnetic metal layer which, in a magnetic field, displays different scattering strengths to spin-up and spin-down neutrons. This provides two independent datasets from a single biological sample. Simultaneous fitting of the two datasets significantly refines the resulting model. A β-mercaptoethanol (βME) passivating surface, applied to the gold to prevent protein denaturation, is resolved for the first time as an 8.2 ± 0.6 Å thick layer, demonstrating the improved resolution and confirming that this layer remains after OmpF assembly. The thiolipid monolayer (35.3 ± 0.5 Å), assembled around the OmpF is determined and finally a fluid DMPC layer is added (total lipid thickness 58.7 ± 0.9 Å). The dimensions of trimeric OmpF in isolation (53.6 ± 2.5 Å), after assembly of lipid monolayer (57.5 ± 0.9 Å) and lipid bilayer (58.7 ± 0.9 Å), are precisely determined and show little variation.
The influence of the hydrophobic proteins SP-B and SP-C, isolated from pulmonary surfactant, on the morphology of binary monomolecular lipid films containing phosphocholine and phosphoglycerol (DPPC and DPPG) at the air-water interface has been studied using epifluorescence and dark-field microscopy. In contrast to previously published studies, the monolayer experiments used the entire hydrophobic surfactant protein fraction (containing both the SP-B and SP-C peptides) at physiologically relevant concentrations (approximately 1 wt %). Even at such low levels, the SP-B/C peptides induce the formation of a new phase in the surface monolayer that is of lower intrinsic order than the liquid condensed (LC) phase that forms in the pure lipid mixture. This presumably leads to a higher structural flexibility of the surface monolayer at high lateral pressure. Variation of the subphase pH indicates that electrostatic interaction dominates the association of the SP-B/C peptides with the lipid monolayer. As evidenced from dark-field microscopy, monolayer material is excluded from the DPPC/DPPG surface film on compression and forms three-dimensional, surface-associated structures of micron dimensions. Such exclusion bodies formed only with SP-B/C peptides. This observation provides the first direct optical evidence for the squeeze-out of pulmonary surfactant material in situ at the air-water interface upon increasing monolayer surface pressures.
Excess chemical potential of small solutes across water-membrane and water-hexane interfaces An experimental setup is described to microscopically observe surfactant monolayers at the air/ water interface. It basically consists of a film balance, incorporated into a fluorescence microscope, detecting the emission of dye probes. The apparatus is stable enough to observe dynamical processes over more than 5 h, with a spatial resolution ofless than 2 J-lm and is sensitive enough to use probe concentrations as low as 0.1 mol%. Examples of surface textures appearing during lipid phase transitions demonstrate the good performance characteristics.
An improved technique to study transferred lipid monolayers by electron microscopy and electron diffraction is presented. In combination with a microfluorescence technique it provides a powerful technique to study the microstructure of monolayers. It is demonstrated that the non-horizontal slope of the isotherms at the fluid-to-crystalline phase transition is due to the coexistence of fluid and crystalline phases up to the second order like transition to the completely condensed crystalline state. An electrostatic model is presented which explains (1) the thermodynamic stability of the fluid-solid coexistence, (2) the non-horizontal slope of the transition region, (3) the existence of a pressure dependent but uniform size of the crystalline platelets at the initial slope of the transition region
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