Membrane interactions in small fast-tumbling bicelles as studied by 31P NMR

Small fast-tumbling bicelles are ideal for studies of membrane interactions at molecular level; they allow analysis of lipid properties using solution-state NMR. In the present study we used ³¹P NMR relaxation to obtain detailed information on lipid head-group dynamics. We explored the effect of two topologically different membrane-interacting peptides on bicelles containing either dimyristoylphosphocholine (DMPC), or a mixture of DMPC and dimyristoylphosphoglycerol (DMPG), and dihexanoylphosphocholine (DHPC). KALP21 is a model transmembrane peptide, designed to span a DMPC bilayer and dynorphin B is a membrane surface active neuropeptide. KALP21 causes significant increase in bicelle size, as evidenced by both dynamic light scattering and ³¹P T₂ relaxation measurements. The effect of dynorphin B on bicelle size is more modest, although significant effects on T₂ relaxation are observed at higher temperatures. A comparison of ³¹P T₁ values for the lipids with and without the peptides showed that dynorphin B has a greater effect on lipid head-group dynamics than KALP21, especially at elevated temperatures. From the field-dependence of T₁ relaxation data, a correlation time describing the overall lipid motion was derived. Results indicate that the positively charged dynorphin B decreases the mobility of the lipid molecules  – in particular for the negatively charged DMPG – while KALP21 has a more modest influence. Our results demonstrate that while a transmembrane peptide has severe effects on overall bilayer properties, the surface bound peptide has a more dramatic effect in reducing lipid head-group mobility. These observations may be of general importance for understanding peptide–membrane interactions.     

Insights on the interactions of synthetic amphipathic peptides with model membranes as revealed by 31P and 2H solid-state NMR and infrared spectroscopies

We studied the interaction between synthetic amphipathic peptides and model membranes by solid-state NMR and infrared spectroscopies. Peptides with 14 and 21 amino acids composed of leucines and phenylalanines modified by the addition of crown ethers were synthesized. The 14-mer and 21-mer peptides both possess a helical amphipathic structure. To shed light on their membrane interaction, (31)P and (2)H solid-state NMR experiments were performed on both peptides in interaction with dimyristoylphosphatidylcholine vesicles in the absence and presence of cholesterol, dimyristoylphosphatidylglycerol vesicles, and oriented bicelles. (31)P NMR experiments on multilamellar vesicles reveal that the dynamics and/or orientation of the polar headgroups are weakly yet markedly affected by the presence of the peptides, whereas (31)P NMR experiments on bicelles indicate no significant changes in the morphology and orientation of the bicelles. On the other hand, (2)H NMR experiments on vesicles reveal that the acyl chain order is affected differently depending on the membrane lipidic composition and on the peptide hydrophobic length. Finally, infrared spectroscopy was used to study the interfacial region of the bilayer. Based on these studies, mechanisms of membrane perturbation are proposed for the 14-mer and 21-mer peptides in interaction with model membranes depending on the bilayer composition and peptide length.     

Membrane topology of a 14-mer model amphipathic peptide: a solid-state NMR spectroscopy study

We have investigated the interaction between a synthetic amphipathic 14-mer peptide and model membranes by solid-state NMR. The 14-mer peptide is composed of leucines and phenylalanines modified by the addition of crown ethers and forms a helical amphipathic structure in solution and bound to lipid membranes. To shed light on its membrane topology, 31P, 2H, 15N solid-state NMR experiments have been performed on the 14-mer peptide in interaction with mechanically oriented bilayers of dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), and dipalmitoylphosphatidylcholine (DPPC). The 31P, 2H, and 15N NMR results indicate that the 14-mer peptide remains at the surface of the DLPC, DMPC, and DPPC bilayers stacked between glass plates and perturbs the lipid orientation relative to the magnetic field direction. Its membrane topology is similar in DLPC and DMPC bilayers, whereas the peptide seems to be more deeply inserted in DPPC bilayers, as revealed by the greater orientational and motional disorder of the DPPC lipid headgroup and acyl chains. 15N{31P} rotational echo double resonance experiments have also been used to measure the intermolecular dipole-dipole interaction between the 14-mer peptide and the phospholipid headgroup of DMPC multilamellar vesicles, and the results indicate that the 14-mer peptide is in contact with the polar region of the DMPC lipids. On the basis of these studies, the mechanism of membrane perturbation of the 14-mer peptide is associated to the induction of a positive curvature strain induced by the peptide lying on the bilayer surface and seems to be independent of the bilayer hydrophobic thickness.     

Dynamics of transportan in bicelles is surface charge dependent

In this study we investigated the dynamic behavior of the chimeric cell-penetrating peptide transportan in membrane-like environments using NMR. Backbone amide ¹⁵N spin relaxation was used to investigate the dynamics in two bicelles: neutral DMPC bicelles and partly negatively charged DMPG-containing bicelles. The structure of the peptide as judged from CD and chemical shifts is similar in the two cases. Both the overall motion as well as the local dynamics is, however, different in the two types of bicelles. The overall dynamics of the peptide is significantly slower in the partly negatively charged bicelle environment, as evidenced by longer global correlation times for all measured sites. The local motion, as judged from generalized order parameters, is for all sites in the peptide more restricted when bound to negatively charged bicelles than when bound to neutral bicelles (increase in S ² is on average 0.11 ± 0.07). The slower dynamics of transportan in charged membrane model systems cause significant line broadening in the proton NMR spectrum, which in certain cases limits the observation of ¹H signals for transportan when bound to the membrane. The effect of transportan on DMPC and DHPC motion in zwitterionic bicelles was also investigated, and the motion of both components in the bicelle was found to be affected.Electronic Supplementary Material Supplementary material is available for this article at http://dx.doi.org/10.1007/s10858-006-9008-y and is accessible for authorized users.     

Opsin stability and folding: modulation by phospholipid bicelles

Integral membrane proteins do not fare well when extracted from biological membranes and are unstable or lose activity in detergents commonly used for structure and function investigations. We show that phospholipid bicelles provide a valuable means of preserving alpha-helical membrane proteins in vitro by supplying a soluble lipid bilayer fragment. Both 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/3-[(cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate (Chaps) and DMPC/l-α-1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) bicelles dramatically increase the stability of the mammalian vision receptor rhodopsin as well as its apoprotein, opsin. Opsin is particularly unstable in detergent solution but can be directly purified into DMPC/Chaps. We show that opsin can also be directly purified in DMPC/DHPC bicelles to give correctly folded functional opsin, as shown by the ability to regenerate rhodopsin to  70% yield. These well-characterised DMPC/DHPC bicelles enable us to probe the influence of bicelle properties on opsin stability. These bicelles are thought to provide DMPC bilayer fragments with most DHPC capping the bilayer edge, giving a soluble bilayer disc. Opsin stability is shown to be modulated by the q value, the ratio of DMPC to DHPC, which reflects changes in the bicelle size and, thus, proportion of DMPC bilayer present. The observed changes in stability also correlate with loss of opsin secondary structure as determined by synchrotron far-UV circular dichroism spectroscopy; the most stable bicelle results in the least helix loss. The inclusion of Chaps rather than DHPC in the DMPC/Chaps bicelles, however, imparts the greatest stability. This suggests that it is not just the DMPC bilayer fragment in the bicelles that stabilises the protein, but that Chaps provides additional stability either through direct interaction with the protein or by altering the DMPC/Chaps bilayer properties within the bicelle. The significant stability enhancements and preservation of secondary structure reported here in bicelles are pertinent to other membrane proteins, notably G-protein-coupled receptors, which are unstable in detergent solution.     

Biophysical studies of the interactions between 14-mer and 21-mer model amphipathic peptides and membranes: Insights on their modes of action

We have investigated the interactions between synthetic amphipathic peptides and zwitterionic model membranes. Peptides with 14 and 21 amino acids composed of leucines and phenylalanines modified by the addition of crown ethers have been synthesized. The 14-mer and 21-mer peptides both possess a helical amphipathic structure as revealed by circular dichroism. To shed light on their mechanism of membrane interaction, different complementary biophysical techniques have been used such as circular dichroism, fluorescence, membrane conductivity measurement and NMR spectroscopy. Results obtained by these different techniques show that the 14-mer peptide is a membrane perturbator that facilitate the leakage of species such as calcein and Na ions, while the 21-mer peptide acts as an ion channel. ³¹P solid-state NMR experiments on multilamellar vesicles reveal that the dynamics and/or orientation of the polar headgroups are greatly affected by the presence of the peptides. Similar results have also been obtained in mechanically oriented DLPC and DMPC bilayers where different acyl chain lengths seem to play a role in the interaction. On the other hand, ²H NMR experiments on multilamellar vesicles demonstrate that the acyl chain order is affected differently by the two peptides. Based on these studies, mechanisms of action are proposed for the 14-mer and 21-mer peptides with zwitterionic membranes.     

Antimicrobial and Membrane Disrupting Activities of a Peptide Derived from the Human Cathelicidin Antimicrobial Peptide LL37

A 21-residue peptide segment, LL7-27 (RKSKEKIGKEFKRIVQRIKDF), corresponding to residues 7-27 of the only human cathelicidin antimicrobial peptide, LL37, is shown to exhibit potent activity against microbes (particularly Gram-positive bacteria) but not against erythrocytes. The structure, membrane orientation, and target membrane selectivity of LL7-27 are characterized by differential scanning calorimetry, fluorescence, circular dichroism, and NMR experiments. An anilinonaphthalene-8-sulfonic acid uptake assay reveals two distinct modes of Escherichia coli outer membrane perturbation elicited by LL37 and LL7-27. The circular dichroism results show that conformational transitions are mediated by lipid-specific interactions in the case of LL7-27, unlike LL37. It folds into an α-helical conformation upon binding to anionic (but not zwitterionic) vesicles, and also does not induce dye leakage from zwitterionic lipid vesicles. Differential scanning calorimetry thermograms show that LL7-27 is completely integrated with DMPC/DMPG (3:1) liposomes, but induces peptide-rich and peptide-poor domains in DMPC liposomes. ¹⁵N NMR experiments on mechanically aligned lipid bilayers suggest that, like the full-length peptide LL37, the peptide LL7-27 is oriented close to the bilayer surface, indicating a carpet-type mechanism of action for the peptide. ³¹P NMR spectra obtained from POPC/POPG (3:1) bilayers containing LL7-27 show substantial disruption of the lipid bilayer structure and agree with the peptide's ability to induce dye leakage from POPC/POPG (3:1) vesicles. Cholesterol is shown to suppress peptide-induced disorder in the lipid bilayer structure. These results explain the susceptibility of bacteria and the resistance of erythrocytes to LL7-27, and may have implications for the design of membrane-selective therapeutic agents.     

Biophysical studies of the membrane location of the voltage-gated sensors in the HsapBK and KvAP K channels

The membrane location of two fragments in two different K⁺-channels, the KvAP (from Aeropyrum pernix) and the HsapBK (human) corresponding to the putative “paddle” domains, has been investigated by CD, fluorescence and NMR spectroscopy. Both domains interact with q = 0.5 phospholipid bicelles, DHPC micelles and with POPC vesicles. CD spectra demonstrate that both peptides become largely helical in the presence of phospholipid bicelles. Fluorescence quenching studies using soluble acrylamide or lipid-attached doxyl-groups show that the arginine-rich domains are located within the bilayered region in phospholipid bicelles. Nuclear magnetic relaxation parameters, T₁ and ¹³C-¹H NOE, for DMPC in DMPC/DHPC bicelles and for DHPC in micelles showed that the lipid acyl chains in the bicelles become less flexible in the presence of either of the fragments. An even more pronounced effect is seen on the glycerol carbons. ²H NMR spectra of magnetically aligned bicelles showed that the peptide derived from KvAP had no or little effect on bilayer order, while the peptide derived from HsapBK had the effect of lowering the order of the bilayer. The present study demonstrates that the fragments derived from the full-length proteins interact with the bilayered interior of model membranes, and that they affect both the local mobility and lipid order of model membrane systems.     

Morphology of fast-tumbling bicelles: a small angle neutron scattering and NMR study

Bilayered micelles, or bicelles, which consist of a mixture of long- and short-chain phospholipids, are a popular model membrane system. Depending on composition, concentration, and temperature, bicelle mixtures may adopt an isotropic phase or form an aligned phase in magnetic fields. Well-resolved (1)H NMR spectra are observed in the isotropic or so-called fast-tumbling bicelle phase, over the range of temperatures investigated (10-40 degrees C), for molar ratios of long-chain lipid to short-chain lipid between 0.20 and 1.0. Small angle neutron scattering data of this phase are consistent with the model in which bicelles were proposed to be disk-shaped. The experimentally determined dimensions are roughly consistent with the predictions of R.R. Vold and R.S. Prosser (J. Magn. Reson. B 113 (1996)). Differential paramagnetic shifts of head group resonances of dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC), induced by the addition of Eu(3+), are also consistent with the bicelle model in which DHPC is believed to be primarily sequestered to bicelle rims. Selective irradiation of the DHPC aliphatic methyl resonances results in no detectable magnetization transfer to the corresponding DMPC methyl resonances (and vice versa) in bicelles, which also suggests that DHPC and DMPC are largely sequestered in the bicelle. Finally, (1)H spectra of the antibacterial peptide indolicidin (ILPWKWPWWPWRR-NH(2)) are compared, in a DPC micellar phase and the above fast-tumbling bicellar phases for a variety of compositions. The spectra exhibit adequate resolution and improved dispersion of amide and aromatic resonances in certain bicelle mixtures.     

Magnetically oriented dodecylphosphocholine bicelles for solid-state NMR structure analysis

A mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) with the short-chain detergent n-dodecylphosphocholine (DPC) is introduced here as a new membrane-mimetic bicelle system for solid-state NMR structure analysis of membrane proteins in oriented samples. Magnetically aligned DMPC/DPC bicelles are stable over a range of concentrations, with an optimum lipid ratio of q=3:1, and they can be flipped with lanthanide ions. The advantage of DMPC/DPC over established bicelle systems lies in the possibility to use one and the same detergent for purification and NMR analysis of the membrane protein, without any need for detergent exchange. Furthermore, the same batch of protein can be studied in both micelles and bicelles, using liquid-state and solid-state NMR, respectively. The applicability of the DMPC/DPC bicelles is demonstrated here with the (15)N-labeled transmembrane protein TatA.     

Interaction of the neuropeptide met-enkephalin with zwitterionic and negatively charged bicelles as viewed by 31P and 2H solid-state NMR

The interaction of the neuropeptide methionine-enkephalin (Menk) with bicelles was investigated by solid-state NMR. Bicelles composed of dimyristoylphosphatidylcholine (DMPC) and dicaproylphosphatidylcholine (DCPC) were modified to investigate the effect of the lipid headgroup and electrostatic charges on the association with Menk. A total of 10 mol % of DMPC was replaced by zwitterionic phosphatidylethanolamine (DMPE), anionic phosphatidylglycerol (DMPG), or phosphatidylserine (DMPS). The preparation of DMPE-doped bicelles (Bic/PE) is reported for the first time. The (31)P and (2)H NMR results revealed changes in the lipid dynamics when Menk interacts with the bicellar systems. (2)H NMR experiments showed a disordering effect of Menk on the lipid chains in all the bicelles except Bic/PG, whereas the study of the choline headgroups indicated a decreased order of the lipids only in Bic/PE and Bic/PG. Our results suggest that the insertion depth of Menk into bicelles is modulated by their composition, more specifically by the balance between hydrophobic and electrostatic interactions. Menk would be buried at the lipid polar/apolar interface, the depth of penetration into the hydrophobic membrane core following the scaling Bic > Bic/PE > Bic/PS at the slightly acidic pH used in this study. The peptide would not insert into the bilayer core of Bic/PG and would rather remain at the surface.     

Structural evaluation of phospholipid bicelles for solution-state studies of membrane-associated biomolecules

Several complementary physical techniques have been used to characterize the aggregate structures formed in solutions containing dimyristoylphosphatidylcholine (DMPC)/dihexanoylphosphatidylcholine (DHPC) at ratios of < or =0.5 and to establish their morphology and lipid organization as that of bicelles. (31)P NMR studies showed that the DMPC and DHPC components were highly segregated over a wide range of DMPC/DHPC ratios (q = 0.05-0.5) and temperatures (15 degrees C and 37 degrees C). Only at phospholipid concentrations below 130 mM did the bicelles appear to undergo a change in morphology. These results were corroborated by fluorescence data, which demonstrated the inverse dependence of bicelle size on phospholipid concentration as well as a distinctive change in phospholipid arrangement at low concentrations. In addition, dynamic light scattering and electron microscopy studies supported the hypothesis that the bicellar phospholipid aggregates are disk-shaped. The radius of the planar domain of the disk was found to be directly proportional to the ratio of DMPC/DHPC and inversely proportional to the total phospholipid concentration when the DMPC/DHPC ratio was held constant at 0.5. Taken together, these results suggest that bicelles with low q retain the morphology and bilayer organization typical of their liquid-crystalline counterparts, making them useful membrane mimetics.     

The membrane-induced structure of melittin is correlated with the fluidity of the lipids

The effect of the bee toxin melittin on DMPC dynamics in fast-tumbling bicelles has been investigated. The ¹³C R₁ and ¹³C-¹H NOE relaxation parameters for DMPC were used to monitor the effect of melittin and cholesterol on lipid dynamics. It was found that melittin has the largest effect on the DMPC mobility in DMPC/DHPC bicelles, while less effect was observed in cholesterol-doped bicelles, or in bicelles made with CHAPS, indicating that the rigidity of the membrane affects the melittin-membrane interaction. CD spectra were analysed in terms of cooperativity of the α-helix to random coil transition in melittin, and these results also indicated similar differences between the bicelles. The study shows that bicelles can be used to investigate lipid dynamics by spin relaxation, and in particular of peptide-induced changes in membrane fluidity.     

Position of residues in transmembrane peptides with respect to the lipid bilayer: A combined lipid NOEs and water chemical exchange approach in phospholipid bicelles

The model transmembrane peptide P16 (Ac-KKGLLLALLLLALLLALLLKKA-NH₂) was incorporated into small unaligned phospholipid bicelles, which provide a `native-like' lipid bilayer compatible with high-resolution solution NMR techniques. Using amide-water chemical exchange and amide-lipid cross-relaxation measurements, the interactions between P16 and bicelles were investigated. Distinctive intermolecular NOE patterns observed in band-selective 2D-NOESY spectra of bicellar solutions with several lipid deuteration schemes indicated that P16 is preferentially interacting with the `bilayered' region of the bicelle rather than with the rim. Furthermore, when amide-lipid NOEs were combined with amide-water chemical exchange cross-peaks of selectively ¹⁵N-labeled P16 peptides, valuable information was obtained about the position of selected residues relative to the membrane-water interface. Specifically, three main classes were identified. Class I residues lie outside the bilayer and show amide-water exchange cross-peaks but no amide-lipid NOEs. Class II residues reside in the bilayer-water interface and show both amide-water exchange cross-peaks and amide-lipid NOEs. Class III residues are embedded within the hydrophobic core of the membrane and show no amide-water exchange cross-peaks but strong amide-lipid NOEs.     

Determining the Mode of Action Involved in the Antimicrobial Activity of?Synthetic Peptides: A Solid-State NMR and FTIR Study

We have previously shown that leucine to lysine substitution(s) in neutral synthetic crown ether containing 14-mer peptide affect the peptide structure and its ability to permeabilize bilayers. Depending on the substitution position, the peptides adopt mainly either a α-helical structure able to permeabilize dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) vesicles (nonselective peptides) or an intermolecular β-sheet structure only able to permeabilize DMPG vesicles (selective peptides). In this study, we have used a combination of solid-state NMR and Fourier transform infrared spectroscopy to investigate the effects of nonselective α-helical and selective intermolecular β-sheet peptides on both types of bilayers. ³¹P NMR results indicate that both types of peptides interact with the headgroups of DMPC and DMPG bilayers. ²H NMR and Fourier transform infrared results reveal an ordering of the hydrophobic core of bilayers when leakage is noted, i.e., for DMPG vesicles in the presence of both types of peptides and DMPC vesicles in the presence of nonselective peptides. However, selective peptides have no significant effect on the ordering of DMPC acyl chains. The ability of these 14-mer peptides to permeabilize lipid vesicles therefore appears to be related to their ability to increase the order of the bilayer hydrophobic core.     

A 2H solid-state NMR spectroscopic investigation of biomimetic bicelles containing cholesterol and polyunsaturated phosphatidylcholine

Deuterium solid-state NMR spectroscopy was used to qualitatively study the effects of both 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLiPC) and cholesterol on magnetically aligned phospholipid bilayers (bicelles) as a function of temperature utilizing the chain-perdeuterated probe 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC-d54) in DMPC/dihexanoylPC (DHPC) phospholipid bilayers. The results demonstrate that polyunsaturated PC and cholesterol were successfully incorporated into DMPC/DHPC phospholipid bilayers, leading to a bicelle that will be useful for investigations of eukaryotic membrane protein-lipid interactions. The data indicate that polyunsaturated PC increases membrane fluidity and decreases the minimum magnetic alignment temperature for DMPC/DHPC bicelles. Conversely, the introduction of cholesterol into aligned DMPC/DHPC bilayers decreases fluidity in the membrane and increases the minimum temperature necessary to magnetically align the phospholipid bilayers. Finally, the addition of Tm3+ to magnetically aligned DMPC/DMPC-d54/PLiPC/DHPC bilayers doubles the quadrupolar splittings, indicating that this unique bicelle system can be aligned with the bilayer normal parallel to the static magnetic field.     

NMR-based conformational analysis of sphingomyelin in bicelles

Sphingomyelin (SM) is a common sphingolipid in mammalian membranes and is known to be substantially involved in cellular events such as the formation of lipid rafts. Despite its biological significance, conformation of SM in a membrane environment remains unclear because the noncrystalline property and anisotropic environment of lipid bilayers hampers the application of X-ray crystallography and NMR measurements. In this study, to elucidate the conformation of SM in membranes, we utilized bicelles as a substitute for a lipid bilayer membrane. First, we demonstrated through (31)P NMR, (2)H NMR, and dynamic light scattering experiments that SM forms both oriented and isotropic bicelles by changing the ratio of SM/dihexanoyl phosphatidylcholine. Then, we determined the conformation of SM in isotropic bicelles on the basis of coupling constants and NOE correlations in (1)H NMR and found that the C2-C6 and amide groups of SM take a relatively rigid conformation in bicelles.     

Interaction of antimicrobial peptides from Australian amphibians with lipid membranes

Solid-state NMR and CD spectroscopy were used to study the effect of antimicrobial peptides (aurein 1.2, citropin 1.1, maculatin 1.1 and caerin 1.1) from Australian tree frogs on phospholipid membranes. 31P NMR results revealed some effect on the phospholipid headgroups when the peptides interact with DMPC/DHPC (dimyristoylphosphatidylcholine/dihexanoylphosphatidylcholine) bicelles and aligned DMPC multilayers. 2H NMR showed a small effect of the peptides on the acyl chains of DMPC in bicelles or aligned multilayers, suggesting interaction with the membrane surface for the shorter peptides and partial insertion for the longer peptides. 15N NMR of selectively labelled peptides in aligned membranes and oriented CD spectra indicated an alpha-helical conformation with helix long axis approximately 50 degrees to the bilayer surface at high peptide concentrations. The peptides did not appear to insert deeply into PC membranes, which may explain why these positively charged peptides preferentially lyse bacterial rather than eucaryotic cells.     

Antimicrobial activity and membrane selective interactions of a synthetic lipopeptide MSI-843

Lipopeptide MSI-843 consisting of the nonstandard amino acid ornithine (Oct-OOLLOOLOOL-NH₂) was designed with an objective towards generating non-lytic short antimicrobial peptides, which can have significant pharmaceutical applications. Octanoic acid was coupled to the N-terminus of the peptide to increase the overall hydrophobicity of the peptide. MSI-843 shows activity against bacteria and fungi at micromolar concentrations. It permeabilizes the outer membrane of Gram-negative bacterium and a model membrane mimicking bacterial inner membrane. Circular dichroism investigations demonstrate that the peptide adopts α-helical conformation upon binding to lipid membranes. Isothermal titration calorimetry studies suggest that the peptide binding to membranes results in exothermic heat of reaction, which arises from helix formation and membrane insertion of the peptide. ²H NMR of deuterated-POPC multilamellar vesicles shows the peptide-induced disorder in the hydrophobic core of bilayers. ³¹P NMR data indicate changes in the lipid head group orientation of POPC, POPG and Escherichia colitotal lipid bilayers upon peptide binding. Results from ³¹P NMR and dye leakage experiments suggest that the peptide selectively interacts with anionic bilayers at low concentrations (up to 5 mol%). Differential scanning calorimetry experiments on DiPOPE bilayers and ³¹P NMR data from E.coli total lipid multilamellar vesicles indicate that MSI-843 increases the fluid lamellar to inverted hexagonal phase transition temperature of bilayers by inducing positive curvature strain. Combination of all these data suggests the formation of a lipid-peptide complex resulting in a transient pore as a plausible mechanism for the membrane permeabilization and antimicrobial activity of the lipopeptide MSI-843.     

Biophysical studies of the interactions between 14-mer and 21-mer model amphipathic peptides and membranes: insights on their modes of action

We have investigated the interactions between synthetic amphipathic peptides and zwitterionic model membranes. Peptides with 14 and 21 amino acids composed of leucines and phenylalanines modified by the addition of crown ethers have been synthesized. The 14-mer and 21-mer peptides both possess a helical amphipathic structure as revealed by circular dichroism. To shed light on their mechanism of membrane interaction, different complementary biophysical techniques have been used such as circular dichroism, fluorescence, membrane conductivity measurement and NMR spectroscopy. Results obtained by these different techniques show that the 14-mer peptide is a membrane perturbator that facilitate the leakage of species such as calcein and Na ions, while the 21-mer peptide acts as an ion channel. (31)P solid-state NMR experiments on multilamellar vesicles reveal that the dynamics and/or orientation of the polar headgroups are greatly affected by the presence of the peptides. Similar results have also been obtained in mechanically oriented DLPC and DMPC bilayers where different acyl chain lengths seem to play a role in the interaction. On the other hand, (2)H NMR experiments on multilamellar vesicles demonstrate that the acyl chain order is affected differently by the two peptides. Based on these studies, mechanisms of action are proposed for the 14-mer and 21-mer peptides with zwitterionic membranes.