Interaction of the Pore-Forming Protein Equinatoxin II with Model Lipid Membranes: A Calorimetric and Spectroscopic Study † (original) (raw)
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Solid-state NMR study of membrane interactions of the pore-forming cytolysin, equinatoxin II
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2010
Equinatoxin II (EqtII) is a pore-forming protein from Actinia equina that lyses red blood cell and model membranes. Lysis is dependent on the presence of sphingomyelin (SM) and is greatest for vesicles composed of equimolar SM and phosphatidylcholine (PC). Since SM and cholesterol (Chol) interact strongly, forming domains or "rafts" in PC membranes, 31 P and 2 H solid-state NMR were used to investigate changes in the lipid order and bilayer morphology of multilamellar vesicles comprised of different ratios of dimyristoylphosphatidylcholine (DMPC), SM and Chol following addition of EqtII. The toxin affects the phase transition temperature of the lipid acyl chains, causes formation of small vesicle type structures with increasing temperature, and changes the T 2 relaxation time of the phospholipid headgroup, with a tendency to order the liquid disordered phases and disorder the more ordered lipid phases. The solid-state NMR results indicate that Chol stabilizes the DMPC bilayer in the presence of EqtII but leads to greater disruption when SM is in the bilayer. This supports the proposal that EqtII is more lytic when both SM and Chol are present as a consequence of the formation of domain boundaries between liquid ordered and disordered phases in lipid bilayers leading to membrane disruption.
The Journal of Physical Chemistry B, 2010
Avian liver bile acid-binding protein (L-BABP) binds peripherically to anionic lipid membranes. We previously showed that in the absence of added salt the binding to 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) occurs with changes in the secondary structure, the extent of which depends on the phase state of the lipid. In the present work, we used Fourier transform infrared spectroscopy to study the conformations of L-BABP bound to lipids with phosphoglycerol and phosphatidic acid polar head groups and with different transition temperatures in an aqueous medium with high ionic strength (0.1 M NaCl). When L-BABP was bound to the lipids with saturated acyl chains, DMPG, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2dimyristoyl-sn-glycero-3-phosphate (DMPA), and 1,2-dilauroyl-sn-glycero-3-phosphate (DLPA), the conformation shifted from a native-like secondary structure to an unfolded state at the temperature of lipid chain melting. The protein was in the native-like conformation when it was bound to the unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) in the liquid-crystalline phase. We also measured the electrokinetic surface potential of POPG and DMPG vesicles in the gel and in the liquid-crystalline phase and the protein binding constant to these lipid membranes. We found a correlation indicating that protein unfolding in the interface was due to the increase in the electrostatic surface potential that occurs in the lipid phase transition.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2007
We applied precise densimetry and ultrasound velocimetry methods to study the interaction of a synthetic α-helical transmembrane peptide, acetyl-K 2 -L 24 -K 2 -amide (L 24 ), with model bilayer lipid membranes. The large unilamellar vesicles (LUVs) utilized were composed of a homologous series of n-saturated diacylphosphatidylcholines (PCs). PCs whose hydrocarbon chains contained from 13 to 16 carbon atoms, thus producing phospholipid bilayers of different thicknesses and gel to liquid-crystalline phase transition temperatures. This allowed us to analyze how the difference between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer influences the thermodynamical and mechanical properties of the membranes. We showed that the incorporation of L 24 decreases the temperature and cooperativity of the main phase transition of all LUVs studied. The presence of L 24 in the bilayer also caused an increase of the specific volume and of the volume compressibility in the gel state bilayers. In the liquid crystalline state, the peptide decreases the specific volume at relatively higher peptide concentration (mole ratio L 24 :PC = 1:50). The overall volume compressibility of the peptide-containing lipid bilayers in the liquidcrystalline state was in general higher in comparison with pure membranes. There was, however, a tendency for the volume compressibility of these lipid bilayers to decrease with higher peptide content in comparison with bilayers of lower peptide concentration. For one lipid composition, we also compared the thermodynamical and mechanical properties of LUVs and large multilamellar vesicles (MLVs) with and without L 24 . As expected, a higher cooperativity of the changes of the thermodynamical and mechanical parameters took place for MLVs in comparison with LUVs. These results are in agreement with previously reported DSC and 2 H NMR spectroscopy study of the interaction of the L 24 and structurally related peptides with phosphatidylcholine bilayers. An apparent discrepancy between 2 H NMR spectroscopy and compressibility data in the liquid crystalline state may be connected with the complex and anisotropic nature of macroscopic mechanical properties of the membranes. The observed changes in membrane mechanical properties induced by the presence of L 24 suggest that around each peptide a distorted region exists that involves at least 2 layers of lipid molecules.
Effect of cytochrome c on the phase behavior of charged multicomponent lipid membranes
Biochimica et biophysica acta, 2014
We studied the effect of submicromolar concentrations of cytochrome c (cyt c) on the phase behavior of ternary lipid membranes composed of charged dioleoylphosphatidylglycerol, egg sphingomyelin and cholesterol. The protein was found to induce micron-sized domains in membranes belonging to the single-fluid-phase region of the protein-free ternary mixture and, as a result, to expand the region of coexistence of liquid ordered (Lo) and liquid disordered (Ld) phases. Direct observations on individual vesicles revealed that protein adsorption increases the area of Ld domains. Measurements using a fluorescent analog of cyt c showed that the protein preferentially adsorbs onto domains belonging to the Ld phase. The adsorption was quantitatively characterized in terms of partitioning ratios between the Ld and the Lo phases. The protein was also found to induce vesicle leakage even at relatively low concentrations. In eukaryotic cells under normal physiological conditions, cyt c is localize...
Biochimica Et Biophysica Acta - Biomembranes, 1982
Glycophorin-dimyristoylphosphatidylcholine bilayers were studied at the limit of low protein concentrations by a number of different techniques. These included freeze-fracture electron microscopy, calorimetry, electron paramagnetic resonance spectroscopy, the photobleaching technique and energy transfer measurements. The liquidus and solidus lines of the dimyristoylphosphatidylcholine-glycophorin system were determined by differential thermal analysis. Structural information was obtained from changes in the texture of the freeze-fracture micrographs. Electron microscopy, together with diffusion measurements, established the important role of linear defects in the solid lipid phases for the incorporation of small amounts of glycophorin and for the fast transport of the protein. Particles were observed above 1 mol%o of protein content, below 10°C, which form due to the immiscibility of glycophorin in the crystalline lipid phase. Lipid-protein aggregates are expelled from the bilayer into the aqueous phase. The concentration dependence of the energy transfer between fluorescein-and eosin-carrying protein headgroups is interpreted in terms of a two-conformation model: At low concentrations (xp <0.8 mol~) the carhnhydrate-carrying beadgroup spreads at the lipid-water interlace forming a two-dimensional (pancake-like) structure, while at higher concentrations the headgroup starts to assume a three-dimensional conformation protruding into the aqueous phase. The concentration dependence of the heat of transition led to the conclusion that one protein molecule interacts with about 300 lipid molecules in the first conformation and with about 100 lipids in the second one. The high number of protein-bound lipids is explained by hydrophilic lipid-protein interaction. All experiments demonstrated that the protein concentration of 0.8 mol~ plays a critical role. (I) The solidus line starts to exhibit a horizontal deflection between 0.4 and 3.2 mol%~ demonstrating solid state phase separation. (2) The spin label order parameter in the fluid lipid phase exhibits a minimum, indicating a maximum in the lipid packing density. (3) The regular ripple phase completely vanishes at 0.4 ~ while it reappears at higher concentrations. This is related to a headgroup conformational change. The results are summarized in a tentative phase diagram of the dimyristoylpbosphatidylcboline-glycophorin system.
2016
Aquaporin-0 (AQP0) is a lens-specific water channel that also forms membrane junctions. Reconstitution of AQP0 with dimyristoyl phosphatidylcholine (DMPC) and E. coli polar lipids (EPL) yielded well-ordered, double-layered two-dimensional (2D) crystals that allowed electron crystallographic structure determination of the AQP0-mediated membrane junction. The interacting tetramers in the two crystalline layers are exactly in register, resulting in crystals with p422 symmetry. The high-resolution density maps also allowed modeling of the annular lipids surrounding the tetramers. Comparison of the DMPC and EPL bilayers suggested that the lipid head groups do not play an important role in the interaction of annular lipids with AQP0. We now reconstituted AQP0 with the anionic lipid dimyristoyl phosphatidylglycerol (DMPG), which yielded a mixture of 2D crystals with different symmetries. The different crystal symmetries result from shifts between the two crystalline layers, suggesting that the negatively charged PG head group destabilizes the interaction between the extracellular AQP0 surfaces. Reconstitution of AQP0 with dimyristoyl phosphatidylserine (DMPS), another anionic lipid, yielded crystals that had the usual p422 symmetry, but the crystals showed a pH-dependent tendency to stack through their cytoplasmic surfaces. Finally, AQP0 failed to reconstitute into membranes that were composed of more than 40% dimyristoyl phosphatidic acid (DMPA). Hence, although DMPG, DMPS, and DMPA are all negatively charged lipids, they have very different effects on AQP0 2D crystals, illustrating the importance of the specific lipid head group chemistry beyond its mere charge.
Lipid conformation in model membranes and biological membranes
Quarterly Reviews of Biophysics, 1980
Protein molecules in solution or in protein crystals are characterized by rather well-defined structures in which α-helical regions, β-pleated sheets, etc., are the key features. Likewise, the double helix of nucleic acids has almost become the trademark of molecular biology as such. By contrast, the structural analysis of lipids has progressed at a relatively slow pace. The early X-ray diffraction studies by V. Luzzati and others firmly established the fact that the lipids in biological membranes are predominantly organized in bilayer structures (Luzzati, 1968). V. Luzzati was also the first to emphasize the liquid-like conformation of the hydrocarbon chains, similar to that of a liquid paraffin, yet with the average orientation of the chains perpendicular to the lipid–water interface. This liquid–crystalline bilayer is generally observed in lipid–water systems at sufficiently high temperature and water content, as well as in intact biological membranes under physiological conditio...
Solid‐state NMR and simulation studies of equinatoxin II N‐terminus interaction with lipid bilayers
2010
Abstract The interaction with model membranes of a peptide, EqtII 1–32, corresponding to the N-terminal region of the pore-forming toxin equinatoxin II (EqtII) has been studied using solid-state NMR and molecular dynamics (MD) simulations. The distances between specifically labeled nuclei in [19 F-para] Phe16-[1-13 C] Leu19 and [19 F-para] Phe16-[15 N] Leu23 analogs of EqtII 1–32 measured by REDOR in lyophilized peptide were in agreement with published crystal and solution structures.