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.     

Effect of Membrane Composition on Antimicrobial Peptides Aurein 2.2 and 2.3 From Australian Southern Bell Frogs

The effects of hydrophobic thickness and the molar phosphatidylglycerol (PG) content of lipid bilayers on the structure and membrane interaction of three cationic antimicrobial peptides were examined: aurein 2.2, aurein 2.3 (almost identical to aurein 2.2, except for a point mutation at residue 13), and a carboxy C-terminal analog of aurein 2.3. Circular dichroism results indicated that all three peptides adopt an α-helical structure in the presence of a 3:1 molar mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPC/DMPG), and 1:1 and 3:1 molar mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPC/POPG). Oriented circular dichroism data for three different lipid compositions showed that all three peptides were surface-adsorbed at low peptide concentrations, but were inserted into the membrane at higher peptide concentrations. The ³¹P solid-state NMR data of the three peptides in the DMPC/DMPG and POPC/POPG bilayers showed that all three peptides significantly perturbed lipid headgroups, in a peptide or lipid composition-dependent manner. Differential scanning calorimetry results demonstrated that both amidated aurein peptides perturbed the overall phase structure of DMPC/DMPG bilayers, but perturbed the POPC/POPG chains less. The nature of the perturbation of DMPC/DMPG bilayers was most likely micellization, and for the POPC/POPG bilayers, distorted toroidal pores or localized membrane aggregate formation. Calcein release assay results showed that aurein peptide-induced membrane leakage was more severe in DMPC/DMPG liposomes than in POPC/POPG liposomes, and that aurein 2.2 induced higher calcein release than aurein 2.3 and aurein 2.3-COOH from 1:1 and 3:1 POPC/POPG liposomes. Finally, DiSC₃5 assay data further delineated aurein 2.2 from the others by showing that it perturbed the lipid membranes of intact S. aureus C622 most efficiently, whereas aurein 2.3 had the same efficiency as gramicidin S, and aurein 2.3-COOH was the least efficient. Taken together, these data show that the membrane interactions of aurein peptides are affected by the hydrophobic thickness of the lipid bilayers and the PG content.     

A mechanistic investigation of cell-penetrating Tat peptides with supported lipid membranes

The multifarious Tat peptide derived from the HIV-1 virus exhibits antimicrobial activity. In this article, we use Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) to investigate the mechanisms of action of Tat (44-57) and Tat (49-57) on bacterial-mimetic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (sodium salt) (DMPG) membranes. The results reveal that both peptides disrupt DMPC/DMPG membranes via a surface-active (carpet-like) mechanism. The magnitude of this disruption is dependent on both membrane and peptide properties. Firstly, less disruption was observed on the more negatively charged membranes. Secondly, less disruption was observed for the longer and slightly more hydrophobic Tat (44-57) peptide. As a comparison, the behaviour of the two Tat peptides on mammalian-mimetic DMPC/cholesterol membranes was investigated. Consistent with the literature no membrane disruption was observed. These results suggest that both electrostatic and hydrophobic interactions, as well as peptide geometry, determine the antimicrobial activity of Tat. This should guide the development of more potent Tat antibiotics.     

Interaction study between maltose-modified PPI dendrimers and lipidic model membranes

The influence of maltose-modified poly(propylene imine) (PPI) dendrimers on dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) (3%) liposomes was studied. Fourth generation (G4) PPI dendrimers with primary amino surface groups were partially (open shell glycodendrimers — OS) or completely (dense shell glycodendrimers — DS) modified with maltose residues. As a model membrane, two types of 100 nm diameter liposomes were used to observe differences in the interactions between neutral DMPC and negatively charged DMPC/DMPG bilayers. Interactions were studied using fluorescence spectroscopy to evaluate the membrane fluidity of both the hydrophobic and hydrophilic parts of the lipid bilayer and using differential scanning calorimetry to investigate thermodynamic parameter changes. Pulsed-filed gradient NMR experiments were carried out to evaluate common diffusion coefficient of DMPG and DS PPI in D₂O when using below critical micelle concentration of DMPG. Both OS and DS PPI G4 dendrimers show interactions with liposomes. Neutral DS dendrimers exhibit stronger changes in membrane fluidity compared to OS dendrimers. The bilayer structure seems more rigid in the case of anionic DMPC/DMPG liposomes in comparison to pure and neutral DMPC liposomes. Generally, interactions of dendrimers with anionic DMPC/DMPG and neutral DMPC liposomes were at the same level. Higher concentrations of positively charged OS dendrimers induced the aggregation process with negatively charged liposomes. For all types of experiments, the presence of NaCl decreased the strength of the interactions between glycodendrimers and liposomes. Based on NMR diffusion experiments we suggest that apart from electrostatic interactions for OS PPI hydrogen bonds play a major role in maltose-modified PPI dendrimer interactions with anionic and neutral model membranes where a contact surface is needed for undergoing multiple H-bond interactions between maltose shell of glycodendrimers and surface membrane of liposome.     

Interaction of alpha-melanocyte stimulating hormone with binary phospholipid membranes: structural changes and relevance of phase behavior

The interaction of alpha-melanocyte stimulating hormone (alpha-MSH) with negatively charged binary membrane systems composed of either 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], (DMPC/DMPG) or DMPC/1,2-dimyristoyl-sn-glycero-3-phosphate (DMPC/DMPA), both at a 3:1 ratio, was studied using complementary techniques (differential scanning calorimetry, infrared and ultraviolet absorption spectroscopy, and steady-state and time-resolved fluorescence). The peptide structure in buffer, at medium to high concentrations, is a mixture of aggregated beta-strands and random coil, and upon increasing the temperature the random coil configuration becomes predominant. At low concentrations (micromolar) there are essentially no aggregates. When in interaction with the lipidic systems this transition is prevented and the peptide is stabilized in a specific conformation different from the one in solution. The incorporation of alpha-MSH into phosphatidic acid-containing systems produced a significant alteration of the calorimetric data. Lateral heterogeneity can be induced by the peptide in the DMPA-containing mixture, at variance with the one of DMPG. In addition, the lipid/water partition coefficient for the peptide in the presence of DMPC/DMPA is greater in the gel phase as compared to the fluid phase. From the high values of limiting anisotropies it can be concluded that the peptide presents a very reduced rotational dynamics when in interaction with the lipids, pointing out to a strong interaction. Overall, these results show that the structure and stability of alpha-MSH in a negatively charged membrane environment are substantially different from those of the peptide in solution, being stabilized in a specific conformation that could be important to eliciting its biological activity.     

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.     

Mechanism of penetration of Antp(43-58) into membrane bilayers

Antp(43-58) is one of many peptides with basic and aromatic residues capable of crossing cell membranes efficiently in a receptor-independent manner. The basic-aromatic motif is responsible for peptide binding to the negatively charged surface of membrane bilayers. However, the mechanism of membrane penetration is unclear. We use high-resolution (1)H solution NMR methods to establish the location of the Antp(43-58) peptide bound to membrane bicelles composed of DMPC, DMPG, and DHPC, and compare it to the location of an Antp(43-58) variant which is not able to cross cell membranes. Two critical tryptophans are substituted with phenylalanine in this variant (W48F and W56F). Additional (31)P and (2)H NMR measurements of membrane bicelles are used to probe the changes in orientation of the lipid headgroups and the changes in the mobility or segmental order of the lipid acyl chains upon peptide binding. We find that Trp48 and Trp56 of Antp(43-58) insert into the hydrophobic core of the membrane and that this induces a change in the orientation of the negatively charged DMPG headgroups. The depth of insertion and the change in lipid orientation are concentration-dependent and argue for an electroporation-like mechanism for membrane penetration.     

The gene expression and deficiency phenotypes of Cockayne syndrome B protein in Caenorhabditis elegans

Maculatin 1.1 is an antimicrobial peptide isolated from the Australian tree frog Litoria genimaculata that adopts an amphipathic, alpha-helical structure in solution. Its orientation and conformation when incorporated to pre-formed DMPG (1,2-dimyristoyl-sn-glycero-3-phosphoglycerol) and DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) vesicles was determined using polarised Fourier transform infrared-attenuated total reflection infrared and deuterium exchange experiments. For DMPG membranes, our results show insertion of 70% of the maculatin 1.1 molecules, with an angle of insertion of approximately 35 degrees to the membrane normal and with a predominant alpha-helical structure. These results suggest that maculatin 1.1 acts through a pore-forming mechanism to lyse bacterial membranes. A similar degree of insertion in DMPG (65%) and alpha-helical structure was observed for a biologically inactive, less amphipathic maculatin 1.1 analogue, P15A, although the helix tilt was found to be greater (46 degrees) than for maculatin 1.1. Similar experiments performed using DMPC liposomes showed poor insertion, less than 5%, for both maculatin 1.1 and its analogue. In addition, the shape of the amide I band in these samples is consistent with alpha-helix, beta-structure and disordered structures being present in similar proportion. These results clearly show that maculatin 1.1 inserts preferentially in negatively charged membranes (DMPG) which mimic the negatively charged membrane of Gram-positive bacteria. We attribute the high percentage of insertion of the biologically inactive analogue in DMPG to the fact that its concentration on the membrane surface in our experiments is likely to be much higher than that found in physiological conditions.     

Characterization of the structure and membrane interaction of the antimicrobial peptides aurein 2.2 and 2.3 from Australian southern bell frogs

The structure and membrane interaction of the antimicrobial peptide aurein 2.2 (GLFDIVKKVVGALGSL-CONH(2)), aurein 2.3 (GLFDIVKKVVGAIGSL-CONH(2)), both from Litoria aurea, and a carboxy C-terminal analog of aurein 2.3 (GLFDIVKKVVGAIGSL-COOH) were studied to determine which features of this class of peptides are key to activity. Circular dichroism and solution-state NMR data indicate that all three peptides adopt an alpha-helical structure in the presence of trifluoroethanol or lipids such as 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and a 1:1 mixture of DMPC and 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG). Oriented circular dichroism was used to determine the orientation of the peptides in lipid bilayers over a range of concentrations (peptide/lipid molar ratios (P/L) = 1:15-1:120) in DMPC and 1:1 DMPC/DMPG, in the liquid crystalline state. The results demonstrate that in DMPC all three peptides are surface adsorbed over a range of low peptide concentrations but insert into the bilayers at high peptide concentrations. This finding is corroborated by (31)P-solid-state NMR data of the three peptides in DMPC, which shows that at high peptide concentrations the peptides perturb the membrane. Oriented circular dichroism data of the aurein peptides in 1:1 DMPC/DMPG, on the other hand, show that the peptides with amidated C-termini readily insert into the membrane bilayers over the concentration range studied (P/L = 1:15-1:120), whereas the aurein 2.3 peptide with a carboxy C-terminus inserts at a threshold concentration of P/L* between 1:80 and 1:120. Overall, the data presented here suggest that all three peptides studied interact with phosphatidylcholine membranes in a manner which is similar to aurein 1.2 and citropin 1.1, as reported in the literature, with no correlation to the reported activity. On the other hand, both aurein 2.2 and aurein 2.3 behave similarly in phosphatidylcholine/phosphatidylglycerol (PC/PG) membranes, whereas aurein 2.3-COOH inserts less readily. As this does not correlate with reported activities, minimal inhibitory concentrations of the three peptides against Staphylococcus aureus (strain C622, ATCC 25923) and Staphylococcus epidermidis (strain C621--clinical isolate) were determined. The correlation between structure, membrane interaction, and activity are discussed in light of these results.     

Serum albumin-lipid membrane interaction influencing the uptake of porphyrins

It is frequently observed in pharmaceutical practice that entrapped substances are lost rapidly when liposomes are used as carriers to introduce substances into cells. The reason for the loss is the interaction of serum components with liposomes. To elucidate the mechanism of this phenomenon the partition of mesoporphyrin (MP) was systematically studied in model systems composed of various lipids and human serum albumin (HSA). As surface charge is an important factor in the interaction, neutral (1, 2-dimyristoyl-sn-glycero-3-phosphatidylcoline, DMPC) and negatively charged (1,2-dimyristoyl-sn-glycero-3-phosphatidylcoline/1, 2-dimyristoyl-sn-glycero-3-phosphatidylglycerol, DMPC/DMPG = 19/1 w/w) lipids were compared. The liposome/apomyoglobin system was the negative control. The size distribution of sonicated samples was carefully analyzed by dynamic light scattering. Constants of association of MP to the proteins and to the liposomes were determined: K(p,1) = (2.5 +/- 0.7) x 10(7) M(-1), K(p,2) = (1.0 +/- 0.7) x 10(8) M(-1), K(L,1) = (1.3 +/- 0.3) x 10(5) M(-1), and K(L,2) = (3.2 +/- 0.6) x 10(4) M(-1) for HSA, apomyoglobin, DMPC, and DMPC/DMPG liposomes, respectively. These data were used to evaluate the partition experiments. The transfer of MP from the liposomes to the proteins was followed by fluorescence spectroscopy. In the case of apomyoglobin, the experimental points could be interpreted by ruling out the protein-liposome interaction. In the case of HSA, the efflux of MP from the liposomes was strongly inhibited above a critical HSA concentration range for negatively charged vesicles. This effect was interpreted as the result of HSA coat formation on the liposome surface. This direct interaction is significant for small liposomes. The interpretation is fully supported by differential scanning calorimetry experiments.     

Real-time quantitative analysis of lipid disordering by aurein 1.2 during membrane adsorption, destabilisation and lysis

Effective antimicrobial peptides (AMPs) distinguish between the host and microbial cells, show selective antimicrobial activity and exhibit a fast killing mechanism. Although understanding the structure-function characteristics of AMPs is important, the impact of the peptides on the architecture of membranes with different lipid compositions is also critical in understanding the molecular mechanism and specificity of membrane destabilisation. In this study, the destabilisation of supported lipid bilayers (SLBs) by the AMP aurein 1.2 was quantitatively analysed by dual polarisation interferometry. The lipid bilayers were formed on a planar silicon oxynitride chip, and composed of mixed synthetic lipids, or Escherichiacoli lipid extract. The molecular events leading sequentially from peptide adsorption to membrane lysis were examined in real time by changes in bilayer birefringence (lipid molecular ordering) as a function of membrane-bound peptide mass. Aurein 1.2 bound weakly without any change in membrane ordering at low peptide concentration (5 μM), indicating a surface-associated state without significant perturbation in membrane structure. At 10 μM peptide, marked reversible changes in molecular ordering were observed for all membranes except DMPE/DMPG. However, at 20 μM aurein 1.2, removal of lipid molecules, as determined by mass loss with a concomitant decrease in birefringence during the association phase, was observed for DMPC and DMPC/DMPG SLBs, which indicates membrane lysis by aurein. The membrane destabilisation induced by aurein 1.2 showed cooperativity at a particular peptide/lipid ratio with a critical mass/molecular ordering value. Furthermore, the extent of membrane lysis for DMPC/DMPG was nearly double that for DMPC. However, no lysis was observed for DMPC/DMPG/cholesterol, DMPE/DMPG and E. coli SLBs. The extent of birefringence changes with peptide mass suggested that aurein 1.2 binds to the membrane without inserting through the bilayer and membrane lysis occurs through detergent-like micellisation above a critical P/L ratio. Real-time quantitative analysis of the structural properties of membrane organisation has allowed the membrane destabilisation process to be resolved into multiple steps and provides comprehensive information to determine the molecular mechanism of aurein 1.2 action.     

Interactions of a synthetic Leu–Lys-rich antimicrobial peptide with phospholipid bilayers

The interaction of the synthetic antimicrobial peptide P5 (KWKKLLKKPLLKKLLKKL-NH₂) with model phospholipid membranes was studied using solid-state NMR and circular dichroism (CD) spectroscopy. P5 peptide had little secondary structure in buffer, but addition of large unilamellar vesicles (LUV) composed of dimyristoylphosphatidylcholine (DMPC) increased the β-sheet content to ~20%. Addition of negatively charged LUV, DMPC–dimyristoylphosphatidylglycerol (DMPG) 2:1, led to a substantial (~40%) increase of the α-helical conformation. The peptide structure did not change significantly above and below the phospholipid phase transition temperature. P5 peptide interacted differently with DMPC bilayers with deuterated acyl chains (d₅₄-DMPC) and mixed d₅₄-DMPC–DMPG bilayers, used to mimic eukaryotic and prokaryotic membranes, respectively. In DMPC vesicles, P5 peptide had no significant interaction apart from slightly perturbing the upper region of the lipid acyl chain with minimum effect at the terminal methyl groups. By contrast, in the DMPC–DMPG vesicles the peptide increased disorder throughout the entire acyl chain of DMPC in the mixed bilayer. P5 promoted disordering of the headgroup of neutral membranes, observed by ³¹P NMR. However, no perturbations in the T ₁ relaxation nor the T ₂- values were observed at 30°C, although a slight change in the dynamics of the headgroup at 20°C was noticeable compared with peptide-free vesicles. However, the P5 peptide caused similar perturbations of the headgroup of negatively charged vesicles at both temperatures. These data correlate with the non-haemolytic activity of the P5 peptide against red blood cells (neutral membranes) while inhibiting bacterial growth (negatively charged membranes).     

Derivative spectrophotometry as a tool for the determination of drug partition coefficients in water/dimyristoyl-L-alpha-phosphatidylglycerol (DMPG) liposomes.

The partition coefficients (K(p)) between lipid bilayers of dimyristoyl-L-alpha-phosphatidylglycerol (DMPG) unilamellar liposomes and water were determined using derivative spectrophotometry for chlordiazepoxide (benzodiazepine), isoniazid and rifampicin (tuberculostatic drugs) and dibucaine (local anaesthetic). A comparison of the K(p) values in water/DMPG with those in water/DMPC (dimyristoyl-L-alpha-phosphatidylcholine) revealed that for chlordiazepoxide and isoniazid, neutral drugs at physiological pH, the partition coefficients are similar in anionic (DMPG) and zwitterionic (DMPC) liposomes. However, for ionised drugs at physiological pH, the electrostatic interactions are different with DMPG and DMPC, with the cationic dibucaine having a stronger interaction with DMPG, and the anionic rifampicin having a much larger K(p) in zwitterionic DMPC. These results show that liposomes are a better model membrane than an isotropic two-phase solvent system, such as water-octanol, to predict drug-membrane partition coefficients, as they mimic better the hydrophobic part and the outer polar charged surface of the phospholipids of natural membranes.     

Lipid-protein interactions with cardiac phospholamban studied by spin-label electron spin resonance

Phospholamban is a cardiac regulatory protein that, in its monomeric form, inhibits the Ca(2+)-ATPase. Lipid-protein interactions with a synthetic variant of phospholamban, in which all cysteine residues are replaced with alanine, have been studied by spin-label electron spin resonance (ESR) in different lipid host membranes. Both the stoichiometry and selectivity of lipid interactions were determined from the two-component ESR spectra of phospholipid species spin-labeled on the 14 C atom of the sn-2 chain. The lipid stoichiometry is determined by the oligomeric state of the protein and the selectivity by the membrane disposition of the positively charged residues in the N-terminal section of the protein. In dimyristoylphosphatidylcholine (DMPC) membranes, the stoichiometry (N(b)) is 7 lipids/monomer for the full-length protein and 4 for the transmembrane section (residues 26-52). These stoichiometries correspond to the dimeric and pentameric forms, respectively. In palmitoyloleoylphosphatidylcholine, N(b) = 4 for both the whole protein and the transmembrane peptide. In negatively charged membranes of dimyristoylphosphatidylglycerol (DMPG), the lipid stoichiometry is N(b) = 10-11 per monomer for both the full-length protein and the transmembrane peptide. This stoichiometry corresponds to monomeric dispersion of the protein in the negatively charged lipid. The sequence of lipid selectivity is as follows: stearic acid > phosphatidic acid > phosphatidylserine = phosphatidylglycerol = phosphatidylcholine > phosphatidylethanolamine for both the full-length protein and the transmembrane peptide in DMPC. Absolute selectivities are, however, lower for the transmembrane peptide. A similar pattern of lipid selectivity is obtained in DMPG, but the absolute selectivities are reduced considerably. The results are discussed in terms of the integration of the regulatory species in the lipid membrane.     

Orientation and lipid-peptide interactions of gramicidin A in lipid membranes: polarized attenuated total reflection infrared spectroscopy and spin-label electron spin resonance

Gramicidin A was incorporated at a peptide/lipid ratio of 1:10 mol/mol in aligned bilayers of dimyristoyl phosphatidylcholine (DMPC), phosphatidylserine (DMPS), phosphatidylglycerol (DMPG), and phosphatidylethanolamine (DMPE), from trifluoroethanol. Orientations of the peptide and lipid chains were determined by polarized attenuated total reflection infrared spectroscopy. Lipid-peptide interactions with gramicidin A in DMPC bilayers were studied with different spin-labeled lipid species by using electron spin resonance spectroscopy. In DMPC membranes, the orientation of the lipid chains is comparable to that in the absence of peptide, in both gel and fluid phases. In gel-phase DMPC, the effective tilt of the peptide exceeds that of the lipid chains, but in the fluid phase both are similar. For gramicidin A in DMPS, DMPG, and DMPE, the degree of orientation of the peptide and lipid chains is less than in DMPC. In the fluid phase of DMPS, DMPG, and DMPE, gramicidin A is also less well oriented than are the lipid chains. In DMPE especially, gramicidin A is largely disordered. In DMPC membranes, three to four lipids per monomer experience direct motional restriction on interaction with gramicidin A. This is approximately half the number of lipids expected to contact the intramembranous perimeter of the gramicidin A monomer. A selectivity for certain negatively charged lipids is found in the interaction with gramicidin A in DMPC. These results are discussed in terms of the integration of gramicidin A channels in lipid bilayers, and of the interactions of lipids with integral membrane proteins.     

Mechanism of membrane binding by the bovine seminal plasma protein,PDC-109: a surface plasmon resonance study

PDC-109, the major protein of bovine seminal plasma, binds to sperm plasma membranes upon ejaculation and plays a crucial role in the subsequent events leading to fertilization. The binding process is mediated primarily by the specific interaction of PDC-109 with choline-containing phospholipids. In the present study the kinetics and mechanism of the interaction of PDC-109 with phospholipid membranes were investigated by the surface plasmon resonance technique. Binding of PDC-109 to different phospholipid membranes containing 20% cholesterol (wt/wt) indicated that binding occurs by a single-step mechanism. The association rate constant (k(1)) for the binding of PDC-109 to dimyristoylphosphatidylcholine (DMPC) membranes containing cholesterol was estimated to be 5.7 x 10(5) M(-1) s(-1) at 20 degrees C, while the values of k(1) estimated at the same temperature for the binding to membranes of negatively charged phospholipids such as dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidic acid (DMPA) containing 20% cholesterol (wt/wt) were at least three orders of magnitude lower. The dissociation rate constant (k(-1)) for the DMPC/PDC-109 system was found to be 2.7 x 10(-2) s(-1) whereas the k(-1) values obtained with DMPG and DMPA was about three to four times higher. From the kinetic data, the association constant for the binding of PDC-109 to DMPC was estimated as 2.1 x 10(7) M(-1). The association constants for different phospholipids investigated decrease in the order: DMPC > DMPG > DMPA > DMPE. Thus the higher affinity of PDC-109 for choline phospholipids is reflected in a faster association rate constant and a slower dissociation rate constant for DMPC as compared to the other phospholipids. Binding of PDC-109 to dimyristoylphosphatidylethanolamine and dipalmitoylphosphatidylethanolamine, which are also zwitterionic, was found to be very weak, clearly indicating that the charge on the lipid headgroup is not the determining factor for the binding. Analysis of the activation parameters indicates that the interaction of PDC-109 with DMPC membranes is favored by a strong entropic contribution, whereas negative entropic contribution is primarily responsible for the rather weak interaction of this protein with DMPA and DMPG.     

Interaction of purified human proteinase 3 (PR3) with reconstituted lipid bilayers.

Proteinase 3 (PR3), the major target autoantigen in Wegener's granulomatosis is a serine proteinase that is normally stored intracellularly in the primary granules of quiescent neutrophils and monocytes. Upon cell activation, a significant portion of this antigen is detected on the cell surface membrane. The nature of the association of PR3 with the membrane and its functional significance are unknown. We investigated the interaction of purified human PR3 with mixtures of zwitterionic (dimyristoyl-L-alpha-phosphatidylcholine, DMPC) and anionic (dimyristoyl-L-alpha-phosphatidylglycerol, DMPG) phospholipids in reconstituted lipid bilayers using differential scanning calorimetry and lipid photolabeling, and measured the affinity of this interaction using spectrophotometry. Two other primary granule constituents, human neutrophil elastase (HNE) and myeloperoxidase (MPO) were investigated for comparison. In calorimetric assays, using lipid vesicles of mixed DMPC/DMPG, increasing PR3 concentrations (protein/lipid molar ratio from 0 to 1 : 110) induced a significant decrease of the main chain transition enthalpy and a shift in chain melting temperatures which is indicative of partial insertion of PR3 into the hydrophobic region of the lipid membranes. This was confirmed by hydrophobic photolabeling using liposomes containing trace amounts of the photoactivable [125I]-labeled phosphatidylcholine analog TID-PC/16. The molar affinity of PR3, HNE, and MPO to lipid vesicles of different DMPC/DMPG ratios was then determined by spectrophotometry. At a DMPC/DMPG ratio of 1 : 1, molar affinities of PR3, Kd = 4.5 +/- 0.3 microm; HNE, 14.5 +/- 1.2 microm; and MPO, 50 +/- 5 microm (n = 3) were estimated. The lipid-associated PR3 exhibited two-fold lower Vmax and Km values, and its enzyme activity was slightly more inhibited (Ki) by the natural alpha1-proteinase inhibitor (alpha1-PI) or an autoantibody to PR3.     

Structural effects of the antimicrobial peptide maculatin 1.1 on supported lipid bilayers

The interactions of the antimicrobial peptide maculatin 1.1 (GLFGVLAKVAAHVVPAIAEHF-NH₂) with model phospholipid membranes were studied by use of dual polarisation interferometry and neutron reflectometry and dimyristoylphosphatidylcholine (DMPC) and mixed DMPC–dimyristoylphosphatidylglycerol (DMPG)-supported lipid bilayers chosen to mimic eukaryotic and prokaryotic membranes, respectively. In DMPC bilayers concentration-dependent binding and increasing perturbation of bilayer order by maculatin were observed. By contrast, in mixed DMPC–DMPG bilayers, maculatin interacted more strongly and in a concentration-dependent manner with retention of bilayer lipid order and structure, consistent with pore formation. These results emphasise the importance of membrane charge in mediating antimicrobial peptide activity and emphasise the importance of using complementary methods of analysis in probing the mode of action of antimicrobial peptides.     

Effect of acyl chain structure and bilayer phase state on binding and penetration of a supported lipid bilayer by HPA3

The effect of acyl chain structure and bilayer phase state on binding and penetration by the peptide HPA3 was studied using dual polarisation interferometry. This peptide is an analogue of Hp(2-20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RpL1) which has been shown to have antimicrobial and cell-penetrating properties. The binding of HPA3 to zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1-palmitolyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and negatively charged membranes composed of DMPC and 1,2-dimyristoyl-sn-glycero-3-(phosphor-rac-(1-glycerol)) (DMPG) or POPC and 1-palmitolyl-2-oleyl-sn-glycero-3-(phosphor-rac-(1-glycerol)) (POPG) was determined using dual polarisation interferometry (DPI). Mass and birefringence were measured in real time, enabling the creation of birefringence–mass plots for detailed analysis of the changes in lipid bilayer order during the peptide-binding process. HPA3 bound to all four lipids and the binding progressed as a single phase for the saturated gel phase bilayers DMPC and DMPC–DMPG. However, the binding process involved two or more phases, with penetration of the unsaturated fluid phase POPC and POPC–POPG bilayers. Structural changes in the saturated bilayer were partially reversible whereas binding to the unsaturated bilayer resulted in irreversible changes in membrane structure. These results demonstrate that more disordered unsaturated bilayers are more susceptible to further disorganisation and have a lower capacity to recover from peptide-induced structural changes than saturated ordered bilayers. In addition, this study further establishes DPI as powerful tool for analysis of multiphase peptide-insertion processes associated with complex structural changes in the liquid-crystalline membrane.     

Interactions of the Australian tree frog antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with lipid model membranes: Differential scanning calorimetric and Fourier transform infrared spectroscopic studies

The interactions of the antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidylethanolamine (DMPE) were studied by differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy. The effects of these peptides on the thermotropic phase behavior of DMPC and DMPG are qualitatively similar and manifested by the suppression of the pretransition, and by peptide concentration-dependent decreases in the temperature, cooperativity and enthalpy of the gel/liquid-crystalline phase transition. However, at all peptide concentrations, anionic DMPG bilayers are more strongly perturbed than zwitterionic DMPC bilayers, consistent with membrane surface charge being an important aspect of the interactions of these peptides with phospholipids. However, at all peptide concentrations, the perturbation of the thermotropic phase behavior of zwitterionic DMPE bilayers is weak and discernable only when samples are exposed to high temperatures. FTIR spectroscopy indicates that these peptides are unstructured in aqueous solution and that they fold into α-helices when incorporated into lipid membranes. All three peptides undergo rapid and extensive H-D exchange when incorporated into D₂O-hydrated phospholipid bilayers, suggesting that they are located in solvent-accessible environments, most probably in the polar/apolar interfacial regions of phospholipid bilayers. The perturbation of model lipid membranes by these peptides decreases in magnitude in the order maculatin 1.1 > aurein 1.2 > citropin 1.1, whereas the capacity to inhibit Acholeplasma laidlawii B growth decreases in the order maculatin 1.1 > aurein 1.2 ≅ citropin 1.1. The higher efficacy of maculatin 1.1 in disrupting model and biological membranes can be rationalized by its larger size and higher net charge. However, despite its smaller size and lower net charge, aurein 1.2 is more disruptive of model lipid membranes than citropin 1.1 and exhibits comparable antimicrobial activity, probably because aurein 1.2 has a higher propensity for partitioning into phospholipid membranes.