Molecular dynamics simulations of local anesthetic articaine in a lipid bilayer

In order to investigate structural and dynamical properties of local anesthetic articaine in a model lipid bilayer, a series of molecular dynamics simulations have been performed. Simulations were carried out for neutral and charged (protonated) forms of articaine inserted in fully hydrated dimyristoylphosphatidylcholine (DMPC) lipid bilayer. For comparison purpose, a fully hydrated DMPC bilayer without articaine was also simulated. The length of each simulation was 200 ns. Various properties of the lipid bilayer systems in the presence of both charged and uncharged forms of articaine taken at two different concentrations have been examined: membrane area per lipid, mass density distributions, order parameters, radial distribution functions, head group tilt, diffusion coefficients, electrostatic potential, etc, and compared with results of previous simulations of DMPC bilayer in the presence of lidocaine. It was shown that addition of both charged and neutral forms of articaine causes increase of the dipole electrostatic potential in the membrane interior.     

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.     

Mechanism of structural transformations induced by antimicrobial peptides in lipid membranes

It has long been suggested that pore formation is responsible for the increase in membrane permeability by antimicrobial peptides (AMPs). To better understand the mechanism of AMP activity, the disruption of model membrane by protegrin-1 (PG-1), a cationic antimicrobial peptide, was studied using atomic force microscopy. We present here the direct visualization of the full range of structural transformations in supported lipid bilayer patches induced by PG-1 on zwitterionic 1,2-dimyristoyl-snglycero-phospho-choline (DMPC) membranes. When PG-1 is added to DMPC, the peptide first induces edge instability at low concentrations, then pore-like surface defects at intermediate concentrations, and finally wormlike structures with a specific length scale at high concentrations. The formation of these structures can be understood using a mesophase framework of a binary mixture of lipids and peptides, where PG-1 acts as a line-active agent. Atomistic molecular dynamics simulations on lipid bilayer ribbons with PG-1 molecules placed at the edge or interior positions are carried out to calculate the effect of PG-1 in reducing line tension. Further investigation of the placement of PG-1 and its association with defects in the bilayer is carried out using unbiased assembly of a PG-1 containing bilayer from a random mixture of PG-1, DMPC, and water. A generalized model of AMP induced structural transformations is also presented in this work. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Effects of temperature and pressure on the lateral organization of model membranes with functionally reconstituted multidrug transporter LmrA

To contribute to the understanding of membrane protein function upon application of pressure, we investigated the influence of hydrostatic pressure on the conformational order and phase behavior of the multidrug transporter LmrA in biomembrane systems. To this end, the membrane protein was reconstituted into various lipid bilayer systems of different chain length, conformation, phase state and heterogeneity, including raft model mixtures as well as some natural lipid extracts. In the first step, we determined the temperature stability of the protein itself and verified its reconstitution into the lipid bilayer systems using CD spectroscopic and AFM measurements, respectively. Then, to yield information on the temperature and pressure dependent conformation and phase state of the lipid bilayer systems, generalized polarization values by the Laurdan fluorescence technique were determined, which report on the conformation and phase state of the lipid bilayer system. The temperature-dependent measurements were carried out in the temperature range 5-70 °C, and the pressure dependent measurements were performed in the range 1-200 MPa. The data show that the effect of the LmrA reconstitution on the conformation and phase state of the lipid matrix depends on the fluidity and hydrophobic matching conditions of the lipid system. The effect is most pronounced for fluid DMPC and DMPC with low cholesterol levels, but minor for longer-chain fluid phospholipids such as DOPC and model raft mixtures such as DOPC/DPPC/cholesterol. The latter have the additional advantage of using lipid sorting to avoid substantial hydrophobic mismatch. Notably, the most drastic effect was observed for the neutral/glycolipid natural lipid mixture. In this case, the impact of LmrA incorporation on the increase of the conformational order of the lipid membrane was most pronounced. As a consequence, the membrane reaches a mechanical stability which makes it very insensitive to application of pressures as high as 200 MPa. The results are correlated with the functional properties of LmrA in these various lipid environments and upon application of high hydrostatic pressure and are discussed in the context of other work on pressure effects on membrane protein systems.     

Structural divergence among cannabinoids influences membrane dynamics: a 2H solid-state NMR analysis

Cannabinoids are compounds that can modulate neuronal functions and immune responses via their activity at the CB(1) receptor. We used (2)H NMR order parameters and relaxation rate determination to delineate the behavior of magnetically aligned phospholipid bilayers in the presence of several structurally distinct cannabinoid ligands. THC (Delta(9)-Tetrahydrocannabinol) and WIN-55,212-2 were found to lower the phase transition temperature of the DMPC and to destabilize their acyl chains leading to a lower average S(CD) ( approximately 0.13), while methanandamide and CP-55,940 exhibited unusual properties within the lipid bilayer resulting in a greater average S(CD) ( approximately 0.14) at the top of the phospholipid upper chain. The CB(1) antagonist AM281 had average S(CD) values that were higher than the pure DMPC lipids, indicating a stabilization of the lipid bilayer. R(1Z) versus |S(CD)|(2) plots indicated that the membrane fluidity is increased in the presence of THC and WIN-55,212-2. The interaction of CP-55,940 with a variety of zwitterionic and charged membranes was also assessed. The unusual effect of CP-55,940 was present only in bicelles composed of DMPC. These studies strongly suggest that cannabinoid action on the membrane depends upon membrane composition as well as the structure of the cannabinoid ligands.     

Cholesterol effects on the phospholipid condensation and packing in the bilayer: a molecular simulation study

A 15-ns molecular dynamics simulation of the fully hydrated liquid-crystalline dimyristoylphosphatidylcholine-cholesterol (DMPC-Chol) bilayer containing approximately 22 mol% Chol was carried out. The generated trajectory was analysed to investigate the mechanism of the Chol condensing effect on DMPC hydrocarbon chains and the influence of Chol on the chain packing in the membrane. Chol was found to induce stronger van der Waals interactions among the chains, whereas its interactions with the chains were weak. In the DMPC-Chol bilayer, as in the DMPC bilayer, DMPC chains were regularly packed around a chosen chain but around a Chol molecule they were not. DMPC gamma chains made closer contacts with Chol than the beta chains.     

Membrane interactions and dynamics of a 21-mer cytotoxic peptide: A solid-state NMR study

We have investigated the membrane interactions and dynamics of a 21-mer cytotoxic model peptide that acts as an ion channel by solid-state NMR spectroscopy. To shed light on its mechanism of membrane perturbation, ³¹P and ²H NMR experiments were performed on 21-mer peptide-containing bicelles. ³¹P NMR results indicate that the 21-mer peptide stabilizes the bicelle structure and orientation in the magnetic field and perturbs the lipid polar head group conformation. On the other hand, ²H NMR spectra reveal that the 21-mer peptide orders the lipid acyl chains upon binding. ¹⁵N NMR experiments performed in DMPC bilayers stacked between glass plates also reveal that the 21-mer peptide remains at the bilayer surface. ¹⁵N NMR experiments in perpendicular DMPC bicelles indicate that the 21-mer peptide does not show a circular orientational distribution in the bicelle planar region. Finally, ¹³C NMR experiments were used to study the 21-mer peptide dynamics in DMPC multilamellar vesicles. By analyzing the ¹³CO spinning sidebands, the results show that the 21-mer peptide is immobilized upon membrane binding. In light of these results, we propose a model of membrane interaction for the 21-mer peptide where it lies at the bilayer surface and perturbs the lipid head group conformation.     

Hydrophobic matching controls the tilt and stability of the dimeric platelet-derived growth factor receptor (PDGFR) β transmembrane segment

The platelet-derived growth factor receptor β is a member of the cell surface receptor tyrosine kinase family and dimerizes upon activation. We determined the structure of the transmembrane segment in dodecylphosphocholine micelles by liquid-state NMR and found that it forms a stable left-handed helical dimer. Solid-state NMR and oriented circular dichroism were used to measure the tilt angle of the helical segments in macroscopically aligned model membranes with different acyl chain lengths. Both methods showed that decreasing bilayer thickness (DEPC-POPC-DMPC) led to an increase in the helix tilt angle from 10° to 30° with respect to the bilayer normal. At the same time, reconstitution of the comparatively long hydrophobic segment became less effective, eventually resulting in complete protein aggregation in the short-chain lipid DLPC. Unrestrained molecular dynamics simulations of the dimer were carried out in explicit lipid bilayers (DEPC, POPC, DMPC, sphingomyelin), confirming the observed dependence of the helix tilt angle on bilayer thickness. Notably, molecular dynamics revealed that the left-handed dimer gets tilted en bloc, whereas conformational transitions to alternative (e.g. right-handed dimeric) states were not supported. The experimental data along with the simulation results demonstrate a pronounced interplay between the platelet-directed growth factor receptor β transmembrane segment and the bilayer thickness. The effect of hydrophobic mismatch might play a key role in the redistribution and activation of the receptor within different lipid microdomains of the plasma membrane in vivo.     

Identifying interactions between transmembrane helices from the adenosine A2A receptor

The human adenosine A(2A) receptor (A(2A)R) is an integral membrane protein and a member of the G-protein-coupled receptor (GPCR) superfamily, characterized by seven transmembrane (TM) helices. Although helix-helix association in the lipid bilayer is known to be an essential step in the folding of GPCRs, the determinants of their structures, folding, and assembly in the cell membrane are poorly understood. Previous studies in our group showed that while peptides corresponding to all seven TM domains of A(2A)R form stable helical structures in detergent micelles and lipid vesicles, they display significant variability in their helical propensity. This finding suggested to us that some TM domains might need to interact with other domains to properly insert and fold in hydrophobic environments. In this study, we assessed the ability of TM peptides to interact in pairwise combinations. We analyzed peptide interactions in hydrophobic milieus using circular dichroism spectroscopy and F?rster resonance energy transfer. We find that specific interactions between TM helices occur, leading to additional helical content, especially in weakly helical TM domains, suggesting that some TM domains need a partner for proper folding in the membrane. The approach developed in this study will enable complete analysis of the TM domain interactions and the modeling of a folding pathway for A(2A)R.     

Effects of phospholipid unsaturation on the membrane/water interface: a molecular simulation study.

Molecular dynamics (MD) simulations of fully hydrated bilayers in the liquid-crystalline state made of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) or 1-palmitoyl-2-elaidoyl-phosphatidylcholine (PEPC) were carried out to investigate the effect of the incorporation of a double bond in the phosphatidylcholine (PC) beta-chain (cis or trans) on the membrane/water interface. The bilayers reached thermal equilibrium after 3 and 1 ns of MD simulations, respectively, and productive runs were carried out for 3 ns for each bilayer. As reference systems, the 1,2-dimyristoyl-phosphatidylcholine (DMPC) bilayer (M. Pasenkiewicz-Gierula, Y. Takaoka, H. Miyagawa, K. Kitamura, and A. Kusumi, 1999, Biophys. J. 76:1228-1240) and DMPC-cholesterol (Chol) bilayer containing 22 mol % Chol (M. Pasenkiewicz-Gierula, T. Róg, K. Kitamura, A. and Kusumi, 2000, Biophys. J. 78:1376-1389) were used. The study shows that at the interface of POPC, PEPC, and DMPC-Chol bilayers, average numbers of PC-water and PC-PC interactions are similar and, respectively, greater and smaller than in the DMPC bilayer. The average area/PC in mono-unsaturated bilayers is approximately 4 A(2) larger than in the DMPC bilayer; nevertheless, a strong correlation was found between a single molecular area (SMA) of a PC and the number of interactions this PC makes; i.e., PCs (either saturated or unsaturated) with the same SMA form similar numbers of intermolecular links. The numbers and corresponding SMAs are distributed about averages pertinent to each bilayer. No significant difference between cis and trans bonds was found.     

Membrane Curvature Revisited—the Archetype of Rhodopsin Studied by Time-Resolved Electronic Spectroscopy

G-protein-coupled receptors (GPCRs) comprise the largest and most pharmacologically targeted membrane protein family. Here, we used the visual receptor rhodopsin as an archetype for understanding membrane lipid influences on conformational changes involved in GPCR activation. Visual rhodopsin was recombined with lipids varying in their degree of acyl chain unsaturation and polar headgroup size using 1-palmitoyl-2-oleoyl-sn-glycero- and 1,2-dioleoyl-sn-glycerophospholipids with phosphocholine (PC) or phosphoethanolamine (PE) substituents. The receptor activation profile after light excitation was measured using time-resolved ultraviolet-visible spectroscopy. We discovered that more saturated POPC lipids back shifted the equilibrium to the inactive state, whereas the small-headgroup, highly unsaturated DOPE lipids favored the active state. Increasing unsaturation and decreasing headgroup size have similar effects that combine to yield control of rhodopsin activation, and necessitate factors beyond proteolipid solvation energy and bilayer surface electrostatics. Hence, we consider a balance of curvature free energy with hydrophobic matching and demonstrate how our data support a flexible surface model (FSM) for the coupling between proteins and lipids. The FSM is based on the Helfrich formulation of membrane bending energy as we previously first applied to lipid-protein interactions. Membrane elasticity and curvature strain are induced by lateral pressure imbalances between the constituent lipids and drive key physiological processes at the membrane level. Spontaneous negative monolayer curvature toward water is mediated by unsaturated, small-headgroup lipids and couples directly to GPCR activation upon light absorption by rhodopsin. For the first time to our knowledge, we demonstrate this modulation in both the equilibrium and pre-equilibrium evolving states using a time-resolved approach.     

Molecular aspects of the interaction between amphotericin B and a phospholipid bilayer: molecular dynamics studies

Amphotericin B (AmB) is a polyene macrolide antibiotic used to treat systemic fungal infections. The molecular mechanism of AmB action is still only partly characterized. AmB interacts with cell-membrane components and forms membrane channels that eventually lead to cell death. The interaction between AmB and the membrane surface can be regarded as the first (presumably crucial) step on the way to channel formation. In this study molecular dynamics simulations were performed for an AmB–lipid bilayer model in order to characterize the molecular aspects of AmB–membrane interactions. The system studied contained a box of 200 dimyristoylphosphatidylcholine (DMPC) molecules, a single AmB molecule placed on the surface of the lipid bilayer and 8,065 water molecules. Two molecular dynamics simulations (NVT ensemble), each lasting 1 ns, were performed for the model studied. Two different programs, CHARMM and NAMD2, were used in order to test simulation conditions. The analysis of MD trajectories brought interesting information concerning interactions between polar groups of AmB and both DMPC and water molecules. Our studies show that AmB preferentially took a vertical position, perpendicular to the membrane surface, with no propensity to enter the membrane. Our finding may suggest that a single AmB molecule entering the membrane is very unlikely.Figure The figure presents the whole structure of the system simulated—starting point. AmB is presented as a space-filling model, DMPC molecules—green sticks, water molecules—red sticks     

Molecular Dynamics Simulation of Conformational Flexibility of Alamethicin Fragments in Aqueous and Membranous Environment

Abstract

We present here results on molecular dynamics (MD) simulation on two fragments of channel forming antibiotic peptide Alamethicin, containing isoamino butyric acid (Aib). Simulations are carried out in aqueous and membranous environment in a bilayer of 39 molecules of Dimyristoyl phosphatidyl choline (DMPC). The peptides Boc—;Pro-Aib-Ala-Aib- OBzl (Alam 1) and Boc-Leu-Aib-Pro-OBzl (Alam 2) were simulated from their crystallography coordinates. The bilayers were built from two different conformations (A and B) of DMPC reported in crystal data. The P-N dipoles were arranged hexagonally with surface area per lipid molecule 66.5 A°² and P-P separation across the bilayer 34 A°. They were hydrated by 28.6 and 25.5 water molecules per DMPC molecule. Simulations are done using AMBER 4.0 package in constant number volume temperature (NVT) condition for 100 pico seconds (ps) in aqueous environment and 250 ps of equilibrated bilayer. Geometric parameters of lipids as: bilayer thickness, order parameter of the chains, transfraction of chain torsional angles were monitored. We also monitored geometric parameters of the peptides as backbone torsional angles, distances amongst Ca atoms, angles between Cα atoms, movement of center of gravity (CG) along and perpendicular to bilayer normal. We find that membrane bilayer is slightly disturbed due to the presence of peptides. In case of alam 2 in water angles ψ1 and ψ3 showed larger variation in water compared to same in the bilayer. The peptide conformation is more stable in DMPC bilayer. However the peptides showed movement along and perpendicular to bilayer normal. This we believe is due to hydrophobic nature of these peptides.     

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.     

Kinetic and conformational properties of a novel T-cell antigen receptor transmembrane peptide in model membranes

Core peptide (CP; GLRILLLKV) is a 9-amino acid peptide derived from the transmembrane sequence of the T-cell antigen receptor (TCR) alpha-subunit. CP inhibits T-cell activation both in vitro and in vivo by disruption of the TCR at the membrane level. To elucidate CP interactions with lipids, surface plasmon resonance (SPR) and circular dichroism (CD) were used to examine CP binding and secondary structure in the presence of either the anionic dimyristoyl-L-alpha-phosphatidyl-DL-glycerol (DMPG), or the zwitterionic dimyristoyl-L-alpha-phoshatidyl choline (DMPC).Using lipid monolayers and bilayers, SPR experiments demonstrated that irreversible peptide-lipid binding required the hydrophobic interior provided by a membrane bilayer. The importance of electrostatic interactions between CP and phospholipids was highlighted on lipid monolayers as CP bound reversibly to anionic DMPG monolayers, with no detectable binding observed on neutral DMPC monolayers.CD revealed a dose-dependent conformational change of CP from a dominantly random coil structure to that of beta-structure as the concentration of lipid increased relative to CP. This occurred only in the presence of the anionic DMPG at a lipid : peptide molar ratio of 1.6:1 as no conformational change was observed when the zwitterionic DMPC was tested up to a lipid : peptide ratio of 8.4 : 1.     

Transmembrane helix-helix interactions: comparative simulations of the glycophorin a dimer

The glycophorin helix dimer is a paradigm for the exploration of helix-helix interactions in integral membrane proteins. Two NMR structures of the dimer are known, one in a detergent micelle and one in a lipid bilayer. Multiple (4 x 50 ns) molecular dynamics simulations starting from each of the two NMR structures, with each structure in either a dodecyl phosphocholine (DPC) micelle or a dimyristoyl phosphatidylcholine (DMPC) bilayer, have been used to explore the conformational dynamics of the helix dimer. Analysis of the helix-helix interaction, mediated by the GxxxG sequence motif, suggests convergence of the simulations to a common model. This is closer to the NMR structure determined in a bilayer than to micelle structure. The stable dimer interface in the final simulation model is characterized by (i) Gly/Gly packing and (ii) Thr/Thr interhelix H-bonds. These results demonstrate the ability of extended molecular dynamics simulations in a lipid bilayer environment to refine membrane protein structures or models derived from experimental data obtained in protein/detergent micelles.     

Cholesterol effects on the phosphatidylcholine bilayer polar region: a molecular simulation study

A molecular dynamics (MD) simulation of a fully hydrated, liquid-crystalline dimyristoylphosphatidylcholine (DMPC)-Chol bilayer membrane containing approximately 22 mol% Chol was carried out for 4.3 ns. The bilayer reached thermal equilibrium after 2.3 ns of MD simulation. A 2.0-ns trajectory generated during 2.3-4.3 ns of MD simulation was used for analyses to determine the effects of Chol on the membrane/water interfacial region. In this region, 70% of Chol molecules are linked to DMPC molecules via short-distance interactions, where the Chol hydroxyl group (OH-Chol) is 1) charge paired to methyl groups of the DMPC choline moiety ( approximately 34%), via the hydroxyl oxygen atom (Och); 2) water bridged to carbonyl ( approximately 19%) and nonester phosphate ( approximately 14%) oxygen atoms, via both Och and the hydroxyl hydrogen atom (Hch); and 3) directly hydrogen (H) bonded to carbonyl ( approximately 11%) and nonester phosphate ( approximately 5%) oxygen atoms, via Hch ( approximately 17% of DMPC-Chol links are multiple). DMPC's gamma-chain carbonyl oxygen atom is involved in 44% of water bridges and 51% of direct H bonds formed between DMPC and Chol. On average, a Chol molecule forms 0.9 links with DMPC molecules, while a DMPC molecule forms 2.2 and 0.3 links with DMPC and Chol molecules, respectively. OH-Chol makes hydrogen bonds with 1.1 water molecules, preferentially via Hch. The average number of water molecules H bonded to the DMPC headgroup is increased by 7% in the presence of Chol. These results indicate that inclusion of Chol decreases interlipid links and increases hydration in the polar region of the membrane.     

MD Simulations of a 5-HT2A Receptor Model in DOPC Membranes

Abstract

This work presents MD calculations on a model 5-HT_2A G protein-coupled receptor embedded in DOPC membrane bilayers at different lipid:protein ratios. The primary purpose is to evaluate physical properties of the system to probe membrane dynamics and the solvation interactions. This showed several kinds of apparent cooperativity phenomena in distributions, lipid dynamics, and hydrogen bond interactions. The integral protein appears to cause a disordered condensation of the local lipid structure which does not extend far beyond the surface layer of lipids. This, and changes in the lipid-protein interaction profiles give a start toward understanding membrane-protein selectivity. The presence of a partially immobilized surface layer of lipids may generally slow down kinetics of solute-solute interactions in similar liquid-crystal membranes. A hydration algorithm was also applied to the GPCR model producing a detailed water structure in some of the internal cavities, including in the areas proposed for ligand binding. Other properties examined included the distributions of lipid groups, the membrane electrostatic potential, and some of the short-time protein dynamics.     

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.