The membrane insertion of helical antimicrobial peptides from the N-terminus of Helicobacter pylori ribosomal protein L1

The interaction of two helical antimicrobial peptides, HPA3 and HPA3P with planar supported lipid membranes was quantitatively analysed using two complementary optical biosensors. The peptides are analogues of Hp(2-20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RpL1). The binding of these two peptide analogues to zwitterionic dimyristoyl-phosphatidylcholine (DMPC) and negatively charged membranes composed of DMPC/dimyristoylphosphatidylglycerol (DMPG) (4:1) was determined using surface plasmon resonance (SPR) and dual polarisation interferometry (DPI). Using SPR analysis, it was shown that the proline substitution in HPA3P resulted in much lower binding for both zwitterionic and anionic membranes than HPA3. Structural changes in the planar DMPC and DMPC/DMPG (4:1) bilayers induced by the binding of both Hp(2-20) analogues were then resolved in real-time with DPI. The overall process of peptide-induced changes in membrane structure was analysed by the real-time changes in bound peptide mass as a function of bilayer birefringence. The insertion of both HPA3 and HPA3P into the supported lipid bilayers resulted in a decrease in birefringence with increasing amounts of bound peptide which reflects a decrease in the order of the bilayer. The binding of HPA3 to each membrane was associated with a higher level of bound peptide and greater membrane lipid disordering and a faster and higher degree of insertion into the membrane than HPA3P. Furthermore, the binding of both HPA3 and HPA3P to negatively charged DMPC/DMPG bilayers also leads to a greater disruption of the lipid ordering. These results demonstrate the geometrical changes in the membrane upon peptide insertion and the extent of membrane structural changes can be obtained quantitatively. Moreover, monitoring the effect of peptides on a structurally characterised bilayer has provided further insight into the role of membrane structure changes in the molecular basis of peptide selectivity and activity and may assist in defining the mode of antimicrobial action.     

Lipid-induced conformation and lipid-binding properties of cytolytic and antimicrobial peptides: determination and biological specificity

While antimicrobial and cytolytic peptides exert their effects on cells largely by interacting with the lipid bilayers of their membranes, the influence of the cell membrane lipid composition on the specificity of these peptides towards a given organism is not yet understood. The lack of experimental model systems that mimic the complexity of natural cell membranes has hampered efforts to establish a direct correlation between the induced conformation of these peptides upon binding to cell membranes and their biological specificities. Nevertheless, studies using model membranes reconstituted from lipids and a few membrane-associated proteins, combined with spectroscopic techniques (i.e. circular dichroism, fluorescence spectroscopy, Fourier transform infra red spectroscopy, etc.), have provided information on specific structure-function relationships of peptide-membrane interactions at the molecular level. Reversed phase-high performance chromatography (RP-HPLC) and surface plasmon resonance (SPR) are emerging techniques for the study of the dynamics of the interactions between cytolytic and antimicrobial peptides and lipid surfaces. Thus, the immobilization of lipid moieties onto RP-HPLC sorbent now allows the investigation of peptide conformational transition upon interaction with membrane surfaces, while SPR allows the observation of the time course of peptide binding to membrane surfaces. Such studies have clearly demonstrated the complexity of peptide-membrane interactions in terms of the mutual changes in peptide binding, conformation, orientation, and lipid organization, and have, to a certain extent, allowed correlations to be drawn between peptide conformational properties and lytic activity.     

Intra-Golgi protein transport depends on a cholesterol balance in the lipid membrane

Transport of proteins between intracellular membrane compartments is mediated by a protein machinery that regulates the budding and fusion processes of individual transport steps. Although the core proteins of both processes are defined at great detail, much less is known about the involvement of lipids. Here we report that changing the cellular balance of cholesterol resulted in changes of the morphology of the Golgi apparatus, accompanied by an inhibition of protein transport. By using a well characterized cell-free intra-Golgi transport assay, these observations were further investigated, and it was found that the transport reaction is sensitive to small changes in the cholesterol content of Golgi membranes. Addition as well as removal of cholesterol (10 +/- 6%) to Golgi membranes by use of methyl-beta-cyclodextrin specifically inhibited the intra-Golgi transport assay. Transport inhibition occurred at the fusion step. Modulation of the cholesterol content changed the lipid raft partitioning of phosphatidylcholine and heterotrimeric G proteins, but not of other (non) lipid raft proteins and lipids. We suggest that the cholesterol balance in Golgi membranes plays an essential role in intra-Golgi protein transport and needs to be carefully regulated to maintain the structural and functional organization of the Golgi apparatus.     

Surface plasmon resonance spectroscopy studies of membrane proteins: transducin binding and activation by rhodopsin monitored in thin membrane films.

Surface plasmon resonance (SPR) spectroscopy can provide useful information regarding average structural properties of membrane films supported on planar solid substrates. Here we have used SPR spectroscopy for the first time to monitor the binding and activation of G-protein (transducin or Gt) by bovine rhodopsin incorporated into an egg phosphatidylcholine bilayer deposited on a silver film. Rhodopsin incorporation into the membrane, performed by dilution of a detergent solution of the protein, proceeds in a saturable manner. Before photolysis, the SPR data show that Gt binds tightly (Keq approximately equal to 60 nM) and with positive cooperativity to rhodopsin in the lipid layer to form a closely packed film. A simple multilayer model yields a calculated average thickness of about 57 A, in good agreement with the structure of Gt. The data also demonstrate that Gt binding saturates at a Gt/rhodopsin ratio of approximately 0.6. Moreover, upon visible light irradiation, characteristic changes occur in the SPR spectrum, which can be modeled by a 6 A increase in the average thickness of the lipid/protein film caused by formation of metarhodopsin II (MII). Upon subsequent addition of GTP, further SPR spectral changes are induced. These are interpreted as resulting from dissociation of the alpha-subunit of Gt, formation of new MII-Gt complexes, and possible conformational changes of Gt as a consequence of complex formation. The above results clearly demonstrate the ability of SPR spectroscopy to monitor interactions among the proteins associated with signal transduction in membrane-bound systems.     

Substrate-supported phospholipid membranes studied by surface plasmon resonance and surface plasmon fluorescence spectroscopy

Substrate-supported planar lipid bilayer membranes are attractive model cellular membranes for biotechnological applications such as biochips and sensors. However, reliable fabrication of the lipid membranes on solid surfaces still poses significant technological challenges. In this study, simultaneous surface plasmon resonance (SPR) and surface plasmon fluorescence spectroscopy (SPFS) measurements were applied to the monitoring of adsorption and subsequent reorganization of phospholipid vesicles on solid substrates. The fluorescence intensity of SPFS depends very sensitively on the distance between the gold substrate and the fluorophore because of the excitation energy transfer to gold. By utilizing this distance dependency, we could obtain information about the topography of the adsorbed membranes: Adsorbed vesicles could be clearly distinguished from planar bilayers due to the high fluorescence intensity. SPSF can also incorporate various analytical techniques to evaluate the physicochemical properties of the adsorbed membranes. As an example, we demonstrated that the lateral mobility of lipid molecules could be estimated by observing the recovery of fluorescence after photobleaching. Combined with the film thickness information obtained by SPR, SPR-SPFS proved to be a highly informative technique to monitor the lipid membrane assembly processes on solid substrates.     

Dynamics of synaptosomal membranes in experimental neurosis in animals using donor-acceptor pairs of luminophores

Dynamic of synaptosomal membrane's structural parameters (fluidity, protein clusterization, thickness of lipid bilayer) during chronic (15 days) psychogenic stress was compared with kinetic of membrane bound enzyme--Na,K-ATPase. For evaluation of structural changes in membranes a special multiprobe method, based on the use of fluorescent probes ANSA and pyrene, fluorescence of endogenous tryptophane and inductive-resonance energy transfer was designed. The data obtained indicates correlation between structural and functional changes in synaptosomal membranes. It was also concluded that the multiprobe procedure used is a sensitive and adequate tool for investigation of structural changes in biomembranes.     

Vesicle fusion studied by surface plasmon resonance and surface plasmon fluorescence spectroscopy

Substrate-supported planar lipid bilayers are generated most commonly by the adsorption and transformation of phospholipid vesicles (vesicle fusion). We have recently demonstrated that simultaneous measurements of surface plasmon resonance (SPR) and surface plasmon fluorescence spectroscopy (SPFS) are highly informative for monitoring lipid membranes on solid substrates. SPR and SPFS provide information on the amount and topography of adsorbed lipid membranes, respectively. In this study, the vesicle fusion process was studied in detail by measuring SPR-SPFS at a higher rate and plotting the obtained fluorescence intensity versus film thickness. We could track the initial adsorption of vesicles, the onset of vesicle rupture occurring at certain vesicle coverage of the surface, and the autocatalytic transformation into planar bilayers. We also monitored vesicle fusion of the same vesicle suspensions by quartz crystal microbalance with dissipation monitoring (QCM-D). We compared the results obtained from SPR-SPFS and QCM-D to highlight the unique information provided by SPR-SPFS.     

Two-step membrane binding by Equinatoxin II,a pore-forming toxin from the sea anemone,involves an exposed aromatic cluster and a flexible helix

Equinatoxin II (EqtII) belongs to a unique family of 20-kDa pore-forming toxins from sea anemones. These toxins preferentially bind to membranes containing sphingomyelin and create cation-selective pores by oligomerization of 3-4 monomers. In this work we have studied the binding of EqtII to lipid membranes by the use of lipid monolayers and surface plasmon resonance (SPR). The binding is a two-step process, separately mediated by two regions of the molecule. An exposed aromatic cluster involving tryptophans 112 and 116 mediates the initial attachment that is prerequisite for the next step. Steric shielding of the aromatic cluster or mutation of Trp-112 and -116 to phenylalanine significantly reduces the toxin-lipid interaction. The second step is promoted by the N-terminal amphiphilic helix, which translocates into the lipid phase. The two steps were distinguished by the use of a double cysteine mutant having the N-terminal helix fixed to the protein core by a disulfide bond. The kinetics of membrane binding derived from the SPR experiments could be fitted to a two-stage binding model. Finally, by using membrane-embedded quenchers, we showed that EqtII does not insert deeply in the membrane. The first step of the EqtII binding is reminiscent of the binding of the evolutionarily distant cholesterol-dependant cytolysins, which share a similar structural motif in the membrane attachment domain.     

Differential thermal effects on the energy distribution between photosystem II and photosystem I in thylakoid membranes of a psychrophilic and a mesophilic alga

Sensitivity of the photosynthetic thylakoid membranes to thermal stress was investigated in the psychrophilic Antarctic alga Chlamydomonas subcaudata. C. subcaudata thylakoids exhibited an elevated heat sensitivity as indicated by a temperature-induced rise in F(o) fluorescence in comparison with the mesophilic species, Chlamydomonas reinhardtii. This was accompanied by a loss of structural stability of the photosystem (PS) II core complex and functional changes at the level of PSI in C. reinhardtii, but not in C. subcaudata. Lastly, C. subcaudata exhibited an increase in unsaturated fatty acid content of membrane lipids in combination with unique fatty acid species. The relationship between lipid unsaturation and the functioning of the photosynthetic apparatus under elevated temperatures is discussed.     

Structural changes in erythrocyte membranes during cooling studied by a spin probe method

Peculiarities of structural changes in erythrocyte membranes during freezing (from -20 degrees to -50 degrees) were studied by electron paramagnetic resonance method using spin-labelled derivative of stearic acid-5-doxylstearate. It was established that membranes underwent a number of structural reconstructions due to the temperature decrease and water freezing-out. Differences were found in temperature dependences that characterize lipid ordering during probe insertion into membranes of native erythrocytes, white ghosts, and liposomes from total lipids of erythrocyte membranes. The data obtained indicate the impairment in the structure of lipid components and lipid-protein interactions in erythrocyte membranes during cooling.     

Vasopressin and water permeability of artificial lipid membranes

Vasopressin markedly stimulated the water permeability of bilayer lipid membranes: a two-fold increase was measured at 25° in presence of 1.7·10⁻⁹ M (50 μunits/ml) vasopressin. Oxytocin and a mixture of the amino acids comprising the vasopressin molecule could not substitute for vasopressin at comparable concentration. The experimental activation energy of water transport was reduced in the presence of vasopressin from 14 to 4 kcal/mole, in agreement with the effect of the hormone on water permeability of toad bladder.     

Light-induced reduction of protochlorophyllide in angiosperms and chloroplast development

One of the final reactions of chlorophyll (Chl) biosynthesis, e.g: photoreduction of protochlorophyllide (Pchlid) to chlorophyllide (Chlid) is a light-induced process in Angiosperm plants and it is catalyzed by light-dependent NADPH-Pchlid oxidoreductase (1.3.1.33; LPOR). In darkness, Chl biosynthesis is stopped at the stage of Pchlid formation. Seedlings and plastids develop according to a different pattern than that observed in the light. Moreover, synthesis of some proteins of the photosynthetic apparatus is inhibited. Light triggers the Pchlid photoreduction to Chlid, which induces the cascade of biochemical reactions and structural changes leading to the assembly of thylakoid membranes. In the present paper, the current knowledge on LPOR protein, mechanism of Pchlid to Chlid photoreduction, the role of lipid structure in etioplasts as well as spectral properties of Pchlid in etiolated seedlings and model systems is summarized.     

Structural changes in lipid membranes and collagen irradiated with UV light and the protective effect of plant extracts

The results of experimental studies on the effect of UV irradiation on collagen, artificial lipid membranes, and rat skin, as well as the protective effect of plant extracts from UV radiation are presented. The irradiation of collagen and lipid membranes with solar and artificial UV light leads to structural changes in these objects. In particular, collagen molecules denature and transfer into a new conformational state. The effect of UV light on lipid membranes and liposomes leads to a disturbance of membrane structure, which is connected with a decrease in the number of lipid molecules involved in the cooperative transition from gel into a liquid crystal state. The components of plant extracts (mainly flavonoids) absorb UV radiation in the erythem-forming spectral area and block the destructive processes occurring in collagen and lipids.     

Thermodynamic profiling of peptide membrane interactions by isothermal titration calorimetry: a search for pores and micelles

Antimicrobial peptides are known to interact strongly with negatively charged lipid membranes, initially by peripheral insertion of the peptide into the bilayer, which for some antimicrobial peptides will be followed by pore formation, and successive solubilization of the membranes resulting in mixed peptide-lipid micelles. We have investigated the mode of action of the antimicrobial peptide mastoparan-X using isothermal titration calorimetry (ITC) and cryo-transmission electron microscopy (cryo-TEM). The results show that mastoparan-X induces a range of structural transitions of POPC/POPG (3:1) lipid membranes at different peptide/lipid ratios. It has been established that ITC can be used as a fast method for localizing membrane transitions and when combined with DLS and cryo-TEM can elucidate structural changes, including the threshold for pore formation and micellation. Cryo-TEM was employed to confirm the structural changes associated with the thermodynamic transitions found by ITC. The pore-formation process has furthermore been investigated in detail and the thermodynamic parameters of pore formation have been characterized using a system-specific temperature where the enthalpy of peptide partitioning becomes zero (T_zero). This allows for an exclusive study of the pore-formation process. The use of ITC to find T_zero allows for characterization of the thermodynamic parameters of secondary processes on lipid membranes.     

Interactions of acid sphingomyelinase and lipid bilayers in the presence of the tricyclic antidepressant desipramine

The tricyclic antidepressant desipramine causes a decrease in cellular acid sphingomyelinase (A-SMase, EC 3.1.4.12) activity when added to culture medium of human fibroblasts. This effect can be prevented by incubation of the cells with the protease inhibitor leupeptin, which suggests that desipramine induces proteolytic degradation of the lysosomal enzyme. By using surface plasmon resonance (SPR, Biacore) we were able to monitor the interactions of A-SMase and substrate-containing lipid bilayers immobilized on the surface of a Pioneer trade mark L1 sensor chip. SPR binding curves show that the enzyme hardly dissociates from the lipid surface at acidic pH values. On the other hand, a drop in binding signals (resonance units, RU) of approximately 50% occurred after injection of 20 mM desipramine. Our findings indicate that desipramine interferes with the binding of A-SMase to the lipid bilayers and thereby displaces the enzyme from its membrane-bound substrate. The application of control substances suggests a key role for the cationic moiety of desipramine. We hypothesize that the displacement of the glycoprotein A-SMase from the inner membranes of late endosomes and lysosomes by desipramine renders it susceptible to proteolytic cleavage by lysosomal proteases.     

Effects of 2-hydroxyoleic acid on the structural properties of biological and model plasma membranes

Genetic hypertension is associated with alterations in lipid metabolism, membrane lipid composition and membrane-protein function. 2-Hydroxyoleic acid (2OHOA) is a new antihypertensive molecule that regulates the structure of model membranes and their interaction with certain peripheral signalling proteins in vitro. While the effect of 2OHOA on elevated blood pressure is thought to arise through its influence on signalling proteins, its effects on membrane lipid composition remain to be assessed. 2OHOA administration altered the lipid membrane composition of hypertensive and normotensive rat plasma membranes, and increased the fluidity of reconstituted liver membranes from hypertensive rats. In spontaneously hypertensive rats (SHR), treatment with 2OHOA increased the cholesterol and sphingomyelin content while decreasing that of phosphatidylserine-phosphatidylinositol lipids. In addition, monounsaturated fatty acid levels increased as well as the propensity of reconstituted membranes to form HII-phases. These data suggest that 2OHOA regulates lipid metabolism that is altered in hypertensive animals, and that it affects the structural properties of liver plasma membranes in SHR. These changes in the structural properties of the plasma membrane may modulate the activity of signalling proteins that associate with the cell membrane such as the Galphaq/11 protein and hence, signal transduction.     

Weak Glycolipid Binding of a Microdomain-Tracer Peptide Correlates with Aggregation and Slow Diffusion on Cell Membranes

Organized assembly or aggregation of sphingolipid-binding ligands, such as certain toxins and pathogens, has been suggested to increase binding affinity of the ligand to the cell membrane and cause membrane reorganization or distortion. Here we show that the diffusion behavior of the fluorescently tagged sphingolipid-interacting peptide probe SBD (Sphingolipid Binding Domain) is altered by modifications in the construction of the peptide sequence that both result in a reduction in binding to ganglioside-containing supported lipid membranes, and at the same time increase aggregation on the cell plasma membrane, but that do not change relative amounts of secondary structural features. We tested the effects of modifying the overall charge and construction of the SBD probe on its binding and diffusion behavior, by Surface Plasmon Resonance (SPR; Biacore) analysis on lipid surfaces, and by Fluorescence Correlation Spectroscopy (FCS) on live cells, respectively. SBD binds preferentially to membranes containing the highly sialylated gangliosides GT1b and GD1a. However, simple charge interactions of the peptide with the negative ganglioside do not appear to be a critical determinant of binding. Rather, an aggregation-suppressing amino acid composition and linker between the fluorophore and the peptide are required for optimum binding of the SBD to ganglioside-containing supported lipid bilayer surfaces, as well as for interaction with the membrane. Interestingly, the strength of interactions with ganglioside-containing artificial membranes is mirrored in the diffusion behavior by FCS on cell membranes, with stronger binders displaying similar characteristic diffusion profiles. Our findings indicate that for aggregation-prone peptides, aggregation occurs upon contact with the cell membrane, and rather than giving a stronger interaction with the membrane, aggregation is accompanied by weaker binding and complex diffusion profiles indicative of heterogeneous diffusion behavior in the probe population.     

The fusogenic properties of the cytotoxins of cobra venom in a model membrane system

By the methods of EPR spinal probes, energy migration of triplet excitation and NMR spectroscopy, the structural changes on hydrocarbon region of membranes, the changes in dynamic state of water of lipid hydrate jacket, the intermembrane lipid material exchange and fusion of membranes induced by cytotoxins of cobra venom have been studied. The sequence of events preceded the membrane fusion is suggested. The probability of membrane fusion has been shown not to be determined by fusogenic agent structure only, but much it depends on lipid composition of membranes.     

How can Ca2+ selectively activate recoverin in the presence of Mg2+? Surface plasmon resonance and FT-IR spectroscopic studies

We investigated the relationship between metal ion selective conformational changes of recoverin and its metal-bound coordination structures. Recoverin is a 23 kDa heterogeneously myristoylated Ca(2+)-binding protein that inhibits rhodopsin kinase. Upon accommodating two Ca(2+) ions, recoverin extrudes a myristoyl group and associates with the lipid bilayer membrane, which was monitored by the surface plasmon resonance (SPR) technique. Large changes in SPR signals were observed for Sr(2+), Ba(2+), Cd(2+), and Mn(2+) as well as Ca(2+), indicating that upon binding to these ions, recoverin underwent a large conformational change to extrude the myristoyl group, and thereby interacted with lipid membranes. In contrast, no SPR signal was induced by Mg(2+), confirming that even though it accommodates two Mg(2+) ions, recoverin does not induce the large conformational change. To investigate the coordination structures of metal-bound Ca(2+) binding sites, FT-IR studies were performed. The EF-hands, Ca(2+)-binding regions each comprising 12 residues, arrange to coordinate Ca(2+) with seven oxygen ligands, two of which are provided by a conserved bidentate Glu at the 12th relative position in the EF-hand. FT-IR analysis confirmed that Sr(2+), Ba(2+), Cd(2+), and Mn(2+) were coordinated to COO(-) of Glu by a bidentate state as well as Ca(2+), while coordination of COO(-) with Mg(2+) was a pseudobridging state with six-coordinate geometry. These SPR and FT-IR results taken together reveal that metal ions with seven-coordinate geometry in the EF-hands induce a large conformational change in recoverin so that it extrudes the myristoyl group, while metal ions with six-coordinate geometry in the EF-hands such as Mg(2+) remain the myristoyl group sequestered in recoverin.     

The effect of rimantadine on the structure of model and biological membranes

The action of the antiviral drug rimantadine on the structure of bilayer lipid membranes (BLM) and RBC membranes was investigated. Structural changes in BLM were recorded by ionophore conductivity changes and by changes in the third harmonic of capacity current signal due to lateral compression of BLM in an electric field. It was shown that the adsorption of rimantadine on BLM results in an increase in ionophore mobility in bilayer membranes of dioleolyllecithin (DOL) and common lipids of bovine brain (CL) and in a decrease in those of azolectin (A). Relative changes in the third harmonic signal also depend on the membrane composition and have different signs. The results may be explained by the rimantadine action on the lipid bilayer structure: "rigidification" of A-membranes and "fluidization" of BLM from DOL and CL. Structural reorganization of RBC membranes as investigated by the ability of the cells to enter a micropipette (inner diameter greater than or equal to 3 microns) thereby undergoing deformation. It was shown that rimantadine influences RBC deformability due to drug induced inhomogenous mechanical membrane properties. Also, rimantadine accelerated the process of artificially induced aggregation of erythrocytes. The relation of the effects on artificial and biological membranes, and the structural changes in the lipid phase of membrane are discussed.