Measurement of the affinity of melittin for zwitterionic and anionic membranes using immobilized lipid biosensors.

The binding of melittin to zwitterionic dimyristyphosphatidylcholine (DMPC) and anionic dimyristylphosphatidylglycerol (DMPG) was analysed using two different immobilized model membrane systems. The first system used surface plasmon resonance (SPR), which monitors the real-time binding of peptides to an immobilized hybrid bilayer. SPR experiments reflected a stronger binding of melittin for DMPG than for DMPC, while kinetic analysis suggested the existence of at least two distinct binding steps. The second lipid biosensor system involved an immobilized phospholipid monolayer covalently attached to a microporous silica surface. The binding of melittin to the immobilized monolayer was then monitored using dynamic elution chromatography with varied methanol concentrations to analyse the binding of melittin to DMPC and DMPG. The nonlinear binding behaviour observed for melittin with the phosphatidylcholine (PC) and phosphatidylglycerol (PG) monolayers compared with the linear retention plots and Gaussian peak shapes observed for the control molecule demonstrated that melittin undergoes significant conformational and orientational changes upon binding to the immobilized PC and PG ligands. The dependence of log k' on per cent methanol also demonstrated a bimodal interaction whereby hydrophobic forces predominated at higher temperatures and methanol concentrations, while other forces, presumably electrostatic in nature, also made a contribution to the affinity of the peptides for the lipid monolayer, particularly at lower temperatures. The complementary use of these two lipid biosensors thus allows the role of hydrophobic and electrostatic forces in peptide-membrane interactions to be studied.     

The membrane-binding properties of a class A amphipathic peptide

The membrane-binding properties of a class A amphipathic peptide (18D) were investigated using two different immobilized model membrane systems. The first system involved the use of surface plasmon resonance (SPR) to study the binding of 18D to dimyristylphosphatidylcholine (DMPC) and dimyristylphosphatidylglycerol (DMPG), which allowed peptide binding to be monitored in real time. The SPR experiments indicated stronger binding of 18D to DMPG than DMPC, which kinetic analysis revealed was due to a faster on-rate. The second model membrane system involved immobilized membrane chromatography in which the binding of 18D to either DMPC or DMPG monolayers covalently linked to silica particles was analysed by elution chromatography. Stronger binding affinity of 18D was also obtained with the negatively charged phosphatidylglycerol (PG) monolayer compared to the phosphatidylcholine (PC) monolayer, which was consistent with the SPR results. Non-linear binding behaviour of 18D to the immobilized lipid monolayers was also observed, which suggests that the peptide undergoes conformational and orientational changes upon binding to the immobilized PC and PG ligands. Significant band broadening was also observed on both monolayers, with larger bandwidths obtained on the PC surface, indicating slower binding and orientation kinetics with the zwitterionic surface. The dependence of logk' on the percentage of methanol also demonstrated a bimodal interaction whereby hydrophobic forces predominated at higher temperatures and methanol concentrations, while at lower temperatures, electrostatic and other polar forces also made a contribution to the affinity of the peptides for the lipid monolayer particularly. Overall, these results demonstrate the complementary use of these two lipid biosensors which allows the role of hydrophobic and electrostatic forces in peptide–membrane interactions to be studied and insight gained into the kinetic factors associated with these interactions.     

Interaction of amphipathic peptides with an immobilised model membrane

Summary The interaction of melittin and a truncated analogue of melittin with an immobilised phosphatidylcholine monolayer has been studied using dynamic elution techniques. The melittin analogue (21Q analogue) had five amino acids omitted from the C-terminal region of melittin. The influence of temperature and methanol concentration on the binding affinity of the two peptides was determined and compared to the binding behaviour of two control moleculesN-acetyltryptophanamide and diphenylalanine. Both peptides exhibited non-linear dependence of affinity on % methanol at different temperatures, whileN-acetyltryptophanamide and diphenylalanine exhibited linear behaviour. In addition, both melittin and the 21Q analogue exhibited significant band broadening under a range of experimental conditions, which was not evident forN-acetyltryptophanamide and diphenylalanine. As melittin is known to adopt a significant degree of α-helical conformation in the presence of lipids, the results suggest that melittin and the 21Q analogue adopt different conformations and orientations upon binding to the immobilised phosphatidylcholine surface. Overall, the results of this study demonstrate that the immobilised lipid monolayer provides a powerful system to rapidly assess the affinity of peptides for different lipid surfaces.     

Interaction of amphipathic peptides with an immobilised model membrane

The interaction of melittin and a truncated analogue of melittin with an immobilised phosphatidylcholine monolayer has been studied using dynamic elution techniques. The melittin analogue (21Q analogue) had five amino acids omitted from the C-terminal region of melittin. The influence of temperature and methanol concentration on the binding affinity of the two peptides was determined and compared to the binding behaviour of two control molecules N-acetyltryptophanamide and diphenylalanine. Both peptides exhibited non-linear dependence of affinity on % methanol at different temperatures, while N-acetyltryptophanamide and diphenylalanine exhibited linear behaviour. In addition, both melittin and the 21Q analogue exhibited significant band broadening under a range of experimental conditions, which was not evident for N-acetyltryptophanamide and diphenylalanine. As melittin is known to adopt a significant degree of -helical conformation in the presence of lipids, the results suggest that melittin and the 21Q analogue adopt different conformations and orientations upon binding to the immobilised phosphatidylcholine surface. Overall, the results of this study demonstrate that the immobilised lipid monolayer provides a powerful system to rapidly assess the affinity of peptides for different lipid surfaces.     

Statins (HMG-coenzyme A reductase inhibitors)-biomimetic membrane binding mechanism investigated by molecular chromatography

Many studies have demonstrated that the statin beneficial effects on cardiovascular diseases like coronary are linked to their hypocholesterolemic properties. These lipid-lowering drugs are the first-line pharmacologic therapy for hypercholesterolemia. In this paper, the interaction of a series of statin molecules STCOOH (pravastatin (prava), mevastatin (meva), simvastatin (simva) and fluvastatin (fluva)) with a phosphatidylcholine monolayer immobilized on to porous silica particles has been studied using a biochromatographic approach (molecular chromatography). The immobilized artificial membrane (IAM) provided a biophysical model system to study the binding of the statin molecules to a lipid membrane. For all the test statin molecules, linear retention plots were observed at all temperatures. An analysis of the thermodynamics (i.e., enthalpy (DeltaH(0)), entropy (DeltaS(0)*)) of the interaction of the statin molecules with the immobilized monolayer was also carried out. The DeltaH(0) and DeltaS(0)* values were negative due to van der Waals interactions and hydrogen bonding between the statin molecules with the polar head groups of the phospholipid monolayer (polar retention effect). The statin elution order was: Prava相似文献   

Peptide binding domains determined through chemical modification of the side-chain functional groups.

A clear understanding of the specific secondary structure and binding domain resulting from the interactions of proteins and peptides with lipid surfaces will provide insight into the specific functions of biologically active molecules. We have shown in earlier studies that the stationary phases used in reverse-phase high-performance liquid chromatography represent a model artificial lipid surface for the study of induced conformational states of peptides on lipid interaction. We have now used reverse-phase high-performance liquid chromatography to determine the binding domains of peptides and, by extension, of proteins to a lipid surface. This approach consists of performing chemical modifications of specific amino acid side-chain functionalities after the interaction of the peptides with the reverse-phase high-performance liquid chromatography C18 groups. The susceptibility to oxidation was also studied after binding of the same peptides to liposomes. Oxidation of a single methionine residue "walked" through an amphipathic alpha-helical 18-mer peptide was selected to illustrate this approach. The extent of oxidation was found to be clearly dictated by the accessibility of the methionine residue to the aqueous mobile phase. The binding domain found for the peptide in its lipid-induced conformational state was unequivocally the entire hydrophobic face of the amphipathic alpha-helix.     

Binding of cationic pentapeptides with modified side chain lengths to negatively charged lipid membranes: Complex interplay of electrostatic and hydrophobic interactions

Basic amino acids play a key role in the binding of membrane associated proteins to negatively charged membranes. However, side chains of basic amino acids like lysine do not only provide a positive charge, but also a flexible hydrocarbon spacer that enables hydrophobic interactions. We studied the influence of hydrophobic contributions to the binding by varying the side chain length of pentapeptides with ammonium groups starting with lysine to lysine analogs with shorter side chains, namely ornithine (Orn), α,γ-diaminobutyric acid (Dab) and α, β-diaminopropionic acid (Dap). The binding to negatively charged phosphatidylglycerol (PG) membranes was investigated by calorimetry, FT-infrared spectroscopy (FT-IR) and monolayer techniques. The binding was influenced by counteracting and sometimes compensating contributions. The influence of the bound peptides on the lipid phase behavior depends on the length of the peptide side chains. Isothermal titration calorimetry (ITC) experiments showed exothermic and endothermic effects compensating to a different extent as a function of side chain length. The increase in lipid phase transition temperature was more significant for peptides with shorter side chains. FTIR-spectroscopy revealed changes in hydration of the lipid bilayer interface after peptide binding. Using monolayer techniques, the contributions of electrostatic and hydrophobic effects could clearly be observed. Peptides with short side chains induced a pronounced decrease in surface pressure of PG monolayers whereas peptides with additional hydrophobic interactions decreased the surface pressure much less or even lead to an increase, indicating insertion of the hydrophobic part of the side chain into the lipid monolayer.     

Conformational flexibility of the hormonal peptide bombesin and its interaction with lipids

The conformational flexibility of the tetradecapeptide hormone bombesin has been studied using circular dichroism and fluorescence of its single tryptophan residue. The spectral changes observed indicate that the peptide changed from a random flexible coil in solution to a helical structure in lysolecithin micelles and dimyristoylphosphatidylserine vesicles. The tryptophan residue in the lipid complexes was located in a hydrophobic environment. The interaction with lipids was shown to involve both hydrophobic and electrostatic forces.     

Electrostatic and hydrophobic forces tether the proximal region of the angiotensin II receptor (AT1A) carboxyl terminus to anionic lipids

The carboxyl terminus of the type-1 angiotensin II receptor (AT(1A)) is a focal point for receptor activation and deactivation. Synthetic peptides corresponding to the membrane-proximal, first 20 amino acids of the carboxyl terminus adopt an alpha-helical conformation in organic solvents, suggesting that the secondary structure of this region may be sensitive to hydrophobic environments. Using surface plasmon resonance, immobilized lipid chromatography, and circular dichroism, we examined whether this positively charged, amphipathic alpha-helical region of the AT(1A) receptor can interact with lipid components in the cell membrane and thereby modulate local receptor attachment and structure. A synthetic peptide corresponding to the proximal region of the AT(1A) receptor carboxyl terminus (Leu(305) to Lys(325)) was shown by surface plasmon resonance to bind with high affinity to the negatively charged lipid, dimyristoyl L-alpha-phosphatidyl-DL-glycerol (DMPG), but poorly to the zwitterionic lipid, dimyristoyl L-alpha-phosphatidylcholine (DMPC). In contrast, a peptide analogue possessing substitutions at four lysine residues (corresponding to Lys(307,308,310,311)) displayed poor association with either lipid, indicating a crucial anionic component to the interaction. Circular dichroism analysis revealed that both the wild-type and substituted peptides possessed alpha-helical propensity in methanol and trifluoroethanol, while the wild-type peptide also adopted partially inserted helical structure in DMPG and DMPC liposomes. In contrast, the substituted peptide exhibited spectra that suggested the presence of beta-sheet and alpha-helical structure in both liposomes. Immobilized lipid chromatography was used to characterize the hydrophobic component of the membrane interaction, and the results demonstrated that hydrophobic and electrostatic interactions mediated the binding of the wild-type peptide but that the substituted peptide bound to the model membranes mainly via hydrophobic forces. We propose that, in intact AT(1A) receptors, the proximal carboxyl terminus associates with the cytoplasmic face of the cell membrane via a high-affinity, anionic phospholipid-specific tethering that serves to increase the amphipathic helicity of this region. Such associations may be important for receptor function and common for G protein-coupled receptors.     

Effect of hydrophobic surfactant peptides SP-B and SP-C on binary phospholipid monolayers. I. Fluorescence and dark-field microscopy.

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.     

The interaction of an anti-lipid antibody (TEPC 15) with a model biomembrane system (monolayer)

The interaction which occurs between an anti-lipid antibody (TEPC 15) and two phospholipids, phosphatidylcholine and phosphatidylethanolamine, when they are arranged in a lipid monolayer system has been studied. It is shown that the antibody is stabilised under the influence of a high lateral pressure when it is mixed with a lipid monolayer and that the behaviour of the antibody depends upon the lipid used. Measurements of the surface pressure and surface potential parameters of the lipid monolayers indicate that the antibody interacts differently with phosphatidylcholine compared with phosphatidylethanolamine. The antibody also exhibits a different interaction when it is pretreated with phosphorylcholine prior to being spread with a phosphatidylcholine monolayer. The interaction of the antibody with phosphatidylcholine-cholesterol monolayers has also been studied.     

Interaction of type-I collagen with phospholipid monolayer.

The effects of type-I collagen on dipalmitoyl phosphatidylcholine (DPPC) and dimyristoyl phosphatidylcholine (DMPC) monolayer films with different compositions were studied using monolayer technique. The addition of collagen in the subphase of different monolayer films induced a considerable shift towards larger area/molecule in the compression-isotherm curves. This is either referred to the insertion of collagen into the monolayer by its hydrophobic residues or to an adsorption process causing a protein layer to be located parallel to the lipid monolayer [1]. The variation of collagen interaction with different lipid compositions was also verified through the penetration-kinetics experiment. Comparing our results to the results of Pajean et al. [2] and Pajean and Herbage [3] on the effect of collagen on the stability of lipid vesicles implies that the collagen induced stability could be explained on the basis of collagen-lipid monolayer interaction.     

Binding to the lipid monolayer induces conformational transition in Aβ monomer

Using implicit solvent atomistic model and replica exchange molecular dynamics, we study binding of Aβ monomer to zwitterionic dimyristoylphosphatidylcholine (DMPC) lipid monolayer. Our results suggest that Aβ binding to the monolayer is governed primarily by positively charged and aromatic amino acids. Lysine residues tend to interact with surface choline and phosphorous lipid groups, whereas aromatic amino acids penetrate deeper into the monolayer, reaching its hydrophobic core. We show that binding to the DMPC monolayer causes a dramatic conformational transition in Aβ monomer, resulting in chain extension, loss of intrapeptide interactions, and formation of β-structure. This conformational transition is far more significant than that occurring during the initial stages of aggregation in water. We also found that Aβ binding perturbs surface ordering of lipids interacting with Aβ.     

Influence of vitamin C on alcohol binding to phospholipid monolayers

The simple model of the biological membrane is provided by well-controlled lipid monolayers at the air-water interface. The Maxwell displacement current technique (MDC) provides novel approach to conformation study of the membrane models. The effect of alcohols is interaction with membrane molecules, mainly with the lipid head group and consequent changes in physical-chemical properties of the membrane. The aim of study is to detect changes in structural, electrical and mechanical properties of dipalmitoyl-phosphatidylcholine (DPPC) monolayer on the subphase of methanol-water and ethanol-water mixtures before and after addition of antioxidant agent, vitamin C. Monolayers properties are investigated by a surface pressure analysis (including mechanical properties evaluation) and the Maxwell displacement current measurement, the dipole moment projection calculation. Surface pressure-area isotherms show similar behaviour of the DPPC monolayer on alcohol-water mixtures independently on presence of vitamin C. Binding/adsorption process induces change of electron density distribution across monolayer and thus the molecular dipole moment. We observe small or negligible binding of methanol molecules on oxygen bonds of DPPC. Thus the antioxidant, vitamin C, has no significant effect. For ethanol-water mixtures is observed recovery of electrical properties in presence of antioxidant agent. We suppose that vitamin C regulates DPPC-ethanol molecules interaction.     

Influence of stearyl and trifluoromethylquinoline modifications of the cell penetrating peptide TP10 on its interaction with a lipid membrane

The PepFect family of cell-penetrating peptides (CPPs) was designed to improve the delivery of nucleic acids across plasma membranes. We present here a comparative study of two members of the family, PepFect3 (PF3) and PepFect6 (PF6), together with their parental CPP transportan-10 (TP10), and their interactions with lipid membranes. We show that the addition of a stearyl moiety to TP10 increases the amphipathicity of these molecules and their ability to insert into a lipid monolayer composed of zwitterionic phospholipids. The addition of negatively charged phospholipids into the monolayer results in decreased binding and insertion of the stearylated peptides, indicating modification in the balance of hydrophobic versus electrostatic interactions of peptides with lipid bilayer, thus revealing some clues for the selective interaction of these CPPs with different lipids. The trifluoromethylquinoline moieties, in PF6 make no significant contribution to membrane binding and insertion. TP10 actively introduces pores into the bilayers of large and giant unilamellar vesicles, while PF3 and PF6 do so only at higher concentrations. This is consistent with the lower toxicity of PF3 and PF6 observed in previous studies.     

The lipid environment of the glucagon receptor regulates adenylate cyclase activity.

1. The lipids composition of rat liver plasma membranes was substantially altered by introducing synthetic phosphatidylcholines into the membrane by the techniques of lipid substitution or lipid fusion. 40-60% of the total lipid pool in the modified membranes consisted of a synthetic phosphatidylcholine. 2. Lipid substitution, using cholate to equilibrate the lipid pools, resulted in the irreversible loss of a major part of the adenylate cyclase activity stimulated by F-, GMP-P(NH)P or glucagon. However, fusion with presonicated vesicles of the synethic phosphatidylcholines causes only small losses in adenylate cyclase activity stimulated by the same ligands. 3. The linear form of the Arrhenius plots of adenylate cyclase activity stimulated by F- or GMP-(NH)P was unaltered in all of the membrane preparations modified by substitution or fusion, with very similar activation energies to those observed with the native membrane. The activity of the enzyme therefore appears to be very insensitive to its lipid environment when stimulated by F- or gmp-p(nh)p. 4. in contrast, the break at 28.5 degrees C in the Arrhenius plot of adenylate cyclase activity stimulated by glucagon in the native membrane, was shifted upwards by dipalmitoyl phosphatidylcholine, downwards by dimyristoyl phosphatidylcholine, and was abolished by dioleoyl phosphatidylcholine. Very similar shifts in the break point were observed for stimulation by glucagon or des-His-glucagon in combination with F- or GMP-P(NH)P. The break temperatures and activation energies for adenylate cyclase activity were the same in complexes prepared with a phosphatidylcholine by fusion or substitution. 5. The breaks in the Arrhenius plots of adenylate cyclase activity are attributed to lipid phase separations which are shifted in the modified membranes according to the transition temperature of the synthetic phosphatidylcholine. Coupling the receptor to the enzyme by glucagon or des-His-glucagon renders the enzyme sensitive to the lipid environment of the receptor. Spin-label experiments support this interpretation and suggest that the lipid phase separation at 28.5 degrees C in the native membrane may only occur in one half of the bilayer.     

Lipid-like behavior of signal sequence peptides at air–water interface

Several protein transport processes in the cell are mediated by signal sequence peptides located at the N-terminal side of the mature protein sequence. To date, the specific interaction and the stability of these peptides at the amphipathic interface of biological membranes and the relevance of the peptide conformation when they interact with lipids is not clear. We report the surface properties and the peptide–lipid interaction of three signal sequence peptides at the air–NaCl 145 mM interface by using the Langmuir monolayer approach. These synthetic peptides have a natural sequence with a non-periodic amphiphilicity, where hydrophobic and hydrophilic residues are located on opposed sides of the peptide primary sequence. We show that signal sequence peptides form insoluble monolayers of high stability against lateral compression. At close packing, peptide molecular area, surface potential and the high stability of the peptide monolayer are indicative that signal sequence peptides are compatible with a β-sheet conformation at the interface. Structure was confirmed with PM-IRRAS and transmission FT-IR studies. The peptides show lateral miscibility with either POPC (a liquid-expanded lipid) or DPPC (a liquid-condensed lipid) in mixed peptide–lipid monolayers. This indicates that signal sequence peptides studied are laterally miscible with phospholipids independent of the phase state of the lipid.     

Measuring adsorption of a hydrophobic probe with a surface plasmon resonance sensor to monitor conformational changes in immobilized proteins

Conformational changes of proteins immobilized on solid matrices were observed by measuring the adsorption of Triton X-100 (TX), a nonionic detergent, as a hydrophobic probe with BIACORE, a biosensor that utilizes the phenomenon of surface plasmon resonance (SPR). Two kinds of proteins, alpha-glucosidase and lysozyme, were covalently attached to dextran matrices on the sensor surface in the flow cell and then exposed to various concentrations of TX solution. We measured SPR signal changes derived from adsorption of TX to the immobilized proteins and calculated the monolayer adsorption capacity using the Brunauer-Emmett-Teller (BET) equation. The results demonstrated that monolayer adsorption capacity is proportional to the amount of immobilized proteins. Further, the unfolding process of immobilized proteins on the sensor surface induced by guanidine hydrochloride was investigated by monitoring SPR signal increases due to the adsorption of TX to the exposed hydrophobic region of the protein. Results strongly suggested that the increase in the SPR signal reflected the formation of the agglutinative unfolded state. We expect our measuring method using the SPR sensor and TX adsorption will be a novel tool to provide conformational information regarding various proteins on solid matrices.     

Thermodynamic assessment of the stability of thrombin receptor antagonistic peptides in hydrophobic environments

In this paper, a general procedure is described to determine thermodynamic parameters associated with the interaction of thrombin receptor antagonistic peptides (TRAPs) with immobilized nonpolar ligands. The results show that these interactions were associated with nonlinear van't Hoff dependencies over a wide temperature range. Moreover, changes in relevant thermodynamic parameters, namely the changes in Gibbs free energy of interaction, DeltaG(0)assoc, enthalpy of interaction, DeltaH(0)assoc, entropy of interaction, DeltaS(0)assoc, and heat capacity, DeltaC(0)p, have been related to the structural properties of these TRAP analogs. The implications of these investigations for the design of thrombin receptor agonists/antagonists with structures stabilized by intramolecular hydrophobic interactions are discussed.