Solid-state NMR study of antimicrobial peptides from Australian frogs in phospholipid membranes

Antimicrobial peptides, isolated from the dorsal glands of Australian tree frogs, possess a wide spectrum of biological activity and some are specific to certain pathogens. These peptides have the capability of disrupting bacterial membranes and lysing lipid bilayers. This study focused on the following amphibian peptides: (1) aurein 1.2, a 13-residue peptide; (2) citropin 1.1, with 16 residues; and (3) maculatin 1.1, with 21 residues. The antibiotic activity and structure of these peptides have been studied and compared and possible mechanisms by which the peptides lyse bacterial membrane cells have been proposed. The peptides adopt amphipathic -helical structures in the presence of lipid micelles and vesicles. Specifically ¹⁵N-labelled peptides were studied using solid-state NMR to determine their structure and orientation in model lipid bilayers. The effect of these peptides on phospholipid membranes was determined by ²H and ³¹P solid-state NMR techniques in order to understand the mechanisms by which they exert their biological effects that lead to the disruption of the bacterial cell membrane. Aurein 1.2 and citropin 1.1 are too short to span the membrane bilayer while the longer maculatin 1.1, which may be flexible due to the central proline, would be able to span the bilayer as a transmembrane -helix. All three peptides had a peripheral interaction with phosphatidylcholine bilayers and appear to be located in the aqueous region of the membrane bilayer. It is proposed that these antimicrobial peptides have a "detergent"-like mechanism of membrane lysis.This paper was submitted as a record of the 2002 Australian Biophysical Society     

Cholesterol attenuates the interaction of the antimicrobial peptide gramicidin S with phospholipid bilayer membranes

We have investigated the effect of the presence of 25 mol percent cholesterol on the interactions of the antimicrobial peptide gramicidin S (GS) with phosphatidylcholine and phosphatidylethanolamine model membrane systems using a variety of methods. Our circular dichroism spectroscopic measurements indicate that the incorporation of cholesterol into egg phosphatidylcholine vesicles has no significant effect on the conformation of the GS molecule but that this peptide resides in a range of intermediate polarity as compared to aqueous solution or an organic solvent. Our Fourier transform infrared spectroscopic measurements confirm these findings and demonstrate that in both cholesterol-containing and cholesterol-free dimyristoylphosphatidylcholine liquid-crystalline bilayers, GS is located in a region of intermediate polarity at the polar--nonpolar interfacial region of the lipid bilayer. However, GS appears to be located in a more polar environment nearer the bilayer surface when cholesterol is present. Our (31)P-nuclear magnetic resonance studies demonstrate that the presence of cholesterol markedly reduces the tendency of GS to induce the formation of inverted nonlamellar phases in model membranes composed of an unsaturated phosphatidylethanolamine. Finally, fluorescence dye leakage experiments indicate that cholesterol inhibits the GS-induced permeabilization of phosphatidylcholine vesicles. Thus in all respects the presence of cholesterol attenuates but does not abolish the interactions of GS with, and the characteristic effects of GS on, phospholipid bilayers. These findings may explain why it is more potent at disrupting cholesterol-free bacterial than cholesterol-containing eukaryotic membranes while nevertheless disrupting the integrity of the latter at higher peptide concentrations. This additional example of the lipid specificity of GS may aid in the rational design of GS analogs with increased antibacterial but reduced hemolytic activities.     

Proton magnetic resonance spectroscopic studies of the interaction of ubiquinone-10 with phospholipid model membranes

Proton magnetic resonance spectra of ubiquinone-10 and ubiquinone-10 dispersed with dipalmitoylglycerophosphocholine or egg phosphatidylcholine in aqueous medium have been obtained. The dispersions are in the form of multilamellar liposomes as judged by 31P-NMR spectra and the thermal history of the samples have ensured that ubiquinone not incorporated into the phospholipid structure only gives rise to a broad-line NMR proton spectrum. A high-resolution proton spectrum of ubiquinone is observed with upfield shifts of the O-methyl protons of the benzoquinone rings, indicating close proximity of the molecules but with an arrangement different from the pure liquid ubiquinone. Spectra obtained in the presence of the lanthanide shift reagents, dysprosium fluorooctanedionate and Dy(NO3)3, which have a preferred location in the hydrophobic and hydrophilic domains, respectively, of ubiquinone/phospholipid codispersions, are consistent with the partitioning of ubiquinone into a hydrophobic phospholipid environment remote from the aqueous phase. The type of arrangements of ubiquinone that could be accommodated within bilayers of phospholipid are discussed.     

Proton magnetic resonance studies of lipid bilayer membranes Experimental determination of inter- and intramolecular nuclear relaxation rates in sonicated phosphatidylcholine bilayer vesicles

We have determined the relative magnitudes of the intra- and intermolecular contributions to the nuclear magnetic relaxation rates of the methylene protons of the hydrocarbon chains in phosphatidylcholine bilayer vesicles over a range of temperatures and at two NMR frequencies (100 and 220 MHz). These measurements have been made by the isotopic dilution method using deuterated phosphatidylcholines containing fully deuterated hydrocarbon chains. The results showed that both the methylene linewidths and the spin-lattice relaxation rates are dominated by intramolecular dipolar interactions. Both the intra- and intermolecular contributions to the spin-lattice relaxation rate were found to decrease with increasing temperature and to exhibit a frequency dependence, the rates being higher at the lower NMR frequency in both cases. These observations indicate that both intra- and intermolecular dipolar interactions are modulated by anisotropic motions. In the case of the intermolecular dipolar fields, it is proposed that they are modulated both by the rapid rotational isomerization of the hydrocarbon chains as well as by lateral diffusion of the lipid molecules. That the hydrocarbon chain motion must be fairly effective in effecting efficient spin-lattice relaxation is evident from the negligible intramolecular interchain contribution to the relaxation found in the present work.     

The interaction of lanthanide and calcium salts with phospholipid bilayer vesicles: The validity of the nuclear magnetic resonance method for determination of vesicle bilayer phospholipid surface ratios

Contrary to a recent report (B. Sears et al., Biochemistry 15 (1976) 1635), it has been determined that the ratio of the number of phospholipids on the inner and outer surfaces of phospholipid bilayer vesicles can be accurately determined by NMR paramagnetic ion shift reagent studies of vesicles. It is concluded that the metal interacts with all of the phospholipid on the exposed bilayer surface. A ratio of outer phospholipid to inner surface phospholipid of 2.1 ± 0.1 is obtained regardless of the nucleus studies, position of the nucleus relative to the metal ion binding site, molar ratio of metal to phospholipid over three orders of magnitude, or location of the metal ion on the inside or outside of the vesicle. Additionally, P-31 NMR studies using LaCl₃ and CaCl₂ indicate that Ca²⁺ weakly interacts with egg PC vesicles and than the lanthanides are adequate substitutes for Ca²⁺ since neither metal is found to perturb measurably the average polar head group conformation.     

Spin-label electron spin resonance studies on the interactions of lysine peptides with phospholipid membranes.

The interactions of lysine oligopeptides with dimyristoyl phosphatidylglycerol (DMPG) bilayer membranes were studied using spin-labeled lipids and electron spin resonance spectroscopy. Tetralysine and pentalysine were chosen as models for the basic amino acid clusters found in a variety of cytoplasmic membrane-associating proteins, and polylysine was chosen as representative of highly basic peripherally bound proteins. A greater motional restriction of the lipid chains was found with increasing length of the peptide, while the saturation ratio of lipids per peptide was lower for the shorter peptides. In DMPG and dimyristoylphosphatidylserine host membranes, the perturbation of the lipid chain mobility by polylysine was greater for negatively charged spin-labeled lipids than for zwitterionic lipids, but for the shorter lysine peptides these differences were smaller. In mixed bilayers composed of DMPG and dimyristoylphosphatidylcholine, little difference was found in selectivity between spin-labeled phospholipid species on binding pentalysine. Surface binding of the basic lysine peptides strongly reduced the interfacial pK of spin-labeled fatty acid incorporated into the DMPG bilayers, to a greater extent for polylysine than for tetralysine or pentalysine at saturation. The results are consistent with a predominantly electrostatic interaction with the shorter lysine peptides, but with a closer surface association with the longer polylysine peptide.     

2H nuclear magnetic resonance of the gramicidin A backbone in a phospholipid bilayer.

Solid-state 2H NMR spectroscopy has been employed to study the channel conformation of gramicidin A (GA) in unoriented 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC) multilayers. Quadrupolar echo spectra were obtained at 44 degrees C and 53 degrees C, from gramicidin A labels in which the proton attached to the alpha carbon of residue 3, 4, 5, 10, 12, or 14 was replaced with deuterium. Because of the nearly axially symmetric electric field gradient tensor, the quadrupolar splittings obtained from an unoriented multilamellar dispersion of DMPC and singly labeled GA directly yield unambiguous orientational constraints on the C-2H bonds. The average of the ratios of the quadrupolar splittings of the left-handed amino acids to those of the right-handed amino acids, (delta vQL/delta vQD), is expected to be 0.97 +/- 0.04 for a relaxed right-handed beta 6.3LD helix, while a ratio of 0.904 +/- 0.003 is expected for a left-handed beta LD6.3 helix. Since we have experimentally determined this ratio to be 1.01 +/- 0.04, we conclude that that the helix sense of the channel conformation of GA is right-handed. Assuming that the dominant motions are fast axial diffusion of the gramicidin molecule and reorientation of the diffusion axis with respect to the local bilayer normal, then the theoretical splittings may all be scaled down by a constant motional narrowing factor. In this case, a relaxed right-handed beta LD6.3 helix, whose axis of motional averaging is roughly along the presumed helix axis, gave the best fit to experimental results. The reasonably uniform correspondence between the splittings predicted by the relaxed right-handed beta LD6.3 helix and the observed splittings, for labels from both the inner and outer turn of GA, did not reflect a peptide backbone flexibility gradient, since an outer turn (i.e., the turn of the helix closest to the interface with water) with greater flexibility would show additional motional narrowing for labels located there.     

Study of F-actin interaction with planar and liposomal bilayer phospholipid membranes

Interaction of the cytoskeletal protein F-actin with planar bilayer lipid membrane (BLM) induced formation of single ionic channels in both NaCl and KCl bathing solutions. We also recorded noiselike high-currentjumps with a mean conductivity of approximately 160 pS, which might represent the simultaneous opening and closing of several channels of lower conductivity. The ratio of cation to anion permeabilities (Pc/Pa) of the BLM with many channels in KCl was 26 +/- 2. Freeze-fracture electron microscopy revealed fibrillar-like structures on the hydrophobic surfaces of liposomal membranes. We also observed some structural features giving evidence for the penetration of F-actin fibers through an artificial phospholipid membrane. We suggest that the F-actin/lipids complexes can transmit electric signals in synaptic and other intercellular contacts.     

Nuclear magnetic resonance studies of cation transport across vesicle bilayer membranes.

We analyze an increasingly popular NMR method analogous to the black lipid membrane (BLM) isotopic tracer experiment for the study of mediated cation transport but involving the preparation of vesicles with an environment asymmetric in that paramagnetic metal ions are present only outside the vesicles. This asymmetry is manifest in the NMR spectrum as two distinct resonances for magnetic nuclei in outside and inside lipid headgroups. As mediated transport begins and for the paramagnetic metal ions enter the vesicles, the inner headgroup resonance line shifts and changes shape with a time course containing much information on the actual ion transport mechanism. Processes by which the ions enter the vesicles one or a few at a time (such as via a diffusive carrier) are easily distinguishable from those by which the ions enter in large bursts (such as by pore activation). The limiting case where intervesicular mediator exchange is slow relative to cation transport (the situation for integral membrane proteins) is treated analytically. Computer simulated curves indicate conditions necessary for certain changes in the line shape which are analogous to the "current jumps" observed in BLM conductance studies. The theory derived allows estimates of the average number of ions entering the first few bursts, how often the bursts occur, and how they depend on the concentration of the mediating species in the vesicular membrane. Preliminary experimental spectra illustrating some of the various possible line shape behaviors are presented.     

The interaction of pseudohalides with the phospholipid head-group: a nuclear magnetic resonance study.

Liposomes have been studied by means of high-field magnetic resonance techniques. The choline N+(CH3)3 group showed two proton resonances for phosphatidylcholine whereas the addition of a charged species to the phospholipid resulted in a single N+(CH3)3 resonance. Upon the addition of either of two linear pseudohalide anions, the two resonances for phosphatidylcholine were further split whereas for the mixture of lipids containing a charged species, the single head-group resonance was now split. The presence of a negative charge on the liposome does not prevent the anion-liposome interaction observed for neutral liposomes. Incorporation of cholesterol into the negatively charged liposomes results in a clear initial splitting of the head-group proton signal in a manner very similar to that for neutral liposomes; this head-group signal is then further split upon anion addition. The small splitting involved suggests a weak pseudohalide-liposome interaction whose magnitude depends on the position of the anion in the lyotropic series. The phosphorous NMR signal from the head-group is unaffected by the pseudohalide interaction whereas the carbon signals from the N+(CH3)3 groups are affected, indicating that the initial anion interaction is localized to the region of the choline groups of the liposome. After the initial exposure of the liposome to the anion, however, the splitting decreases with time, indicating that the anions have entered the liposome and interact with both inside and outside head-groups.     

Surface plasmon resonance and nuclear magnetic resonance studies of ABAD-Abeta interaction

Abeta binding alcohol dehydrogenase (ABAD) is an NAD-dependent mitochondrial dehydrogenase. The binding between ABAD and Abeta is likely a direct link between Abeta and mitochondrial toxicity in Alzheimer's disease. In this study, surface plasmon resonance (SPR) was employed to determine the temperature dependence of the affinity of the ABAD-Abeta interaction. A van't Hoff analysis revealed that the ABAD-Abeta association is driven by a favorable entropic change (DeltaS = 300 +/- 30 J mol-1 K-1) which overcomes an unfavorable enthalpy change (DeltaH = 49 +/- 7 kJ/mol). Therefore, hydrophobic interactions and changes in protein dynamics are the dominant driving forces of the ABAD-Abeta interaction. This is the first dissection of the entropic and enthalpic contribution to the energetics of a protein-protein interaction involving Abeta. SPR confirmed the conformational changes in the ABAD-Abeta complex after Abeta binding, consistent with differences seen in the crystal structures of free ABAD and the ABAD-Abeta complex. Saturation transfer difference (STD) NMR experiments directly and unambiguously demonstrated the inhibitory effect of Abeta on the ABAD-NAD interaction. Conversely, NAD inhibits the Abeta-ABAD interaction. Binding of Abeta and binding of NAD to ABAD are likely mutually exclusive. Thus, Abeta binding induces conformational and subsequently functional changes in ABAD, which may have a role in the mechanism of Abeta toxicity in Alzheimer's disease.     

Isothermal titration calorimetry studies of the binding of the antimicrobial peptide gramicidin S to phospholipid bilayer membranes

The binding of the amphiphilic, positively charged, cyclic beta-sheet antimicrobial decapeptide gramicidin S (GS) to various lipid bilayer model membrane systems was studied by isothermal titration calorimetry. Large unilamellar vesicles composed of the zwitterionic phospholipid 1-palmitoyl-2-oleoylphosphatidylcholine or the anionic phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol, or a binary mixture of the two, with or without cholesterol, were used to mimic the lipid compositions of the outer monolayers of the lipid bilayers of mammalian and bacterial membranes, respectively. Dynamic light scattering results suggest the absence of major alterations in vesicle size or appreciable vesicle fusion upon the binding of GS to the lipid vesicles under our experimental conditions. The binding isotherms can be reasonably well described by a one-site binding model. GS is found to bind with higher affinity to anionic phosphatidylglycerol than to zwitterionic phosphatidylcholine vesicles, indicating that electrostatic interactions in the former system facilitate peptide binding. However, the presence of cholesterol reduced binding only slightly, indicating that the binding of GS is not highly sensitive to the order of the phospholipid bilayer system. Similarly, the measured positive endothermic binding enthalpy (DeltaH) varies only modestly (2.6 to 4.4 kcal/mol), and the negative free energy of binding (DeltaG) also remains relatively constant (-10.9 to -12.1 kcal/mol). The relatively large but invariant positive binding entropy, reflected in relatively large TDeltaS values (13.4 to 16.4 kcal/mol), indicates that GS binding to phospholipid bilayers is primarily entropy driven. Finally, the relative binding affinities of GS for various phospholipid vesicles correlate relatively well with the relative lipid specificity for GS interactions with bacterial and erythrocyte membranes observed in vivo.     

Deuteron nuclear magnetic resonance study of the dynamic organization of phospholipid/cholesterol bilayer membranes: molecular properties and viscoelastic behavior.

The influence of cholesterol on the dynamic organization of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers was studied by deuteron nuclear magnetic resonance (2H NMR) using unoriented and macroscopically aligned samples. Analysis of the various temperature- and orientation-dependent experiments were performed using a comprehensive NMR model based on the stochastic Liouville equation. Computer simulations of the relaxation data obtained from phospholipids deuterated at the 6-, 13- and 14-position of the sn-2 chain and cholesterol labeled at the 3 alpha-position of the rigid steroid ring system allowed the unambiguous assignment of the various motional modes and types of molecular order present in the system. Above the phospholipid gel-to-liquid-crystalline phase transition, TM, 40 mol % cholesterol was found to significantly increase the orientational and conformational order of the phospholipid with substantially increased trans populations even at the terminal sn-2 acyl chain segments. Lowering the temperature continuously increases both inter- and intramolecular ordering, yet indicates less ordered chains than found for the pure phospholipid in its paracrystalline gel phase. Trans-gauche isomerization rates on all phospholipid alkyl chain segments are slowed down by incorporated cholesterol to values characteristic of gel-state lipid. However, intermolecular dynamics remain fast on the NMR time scale up to 30 K below TM, with rotational correlation times tau R parallel for DMPC ranging from 10 to 100 ns and an activation energy of ER = 35 kJ/mol. Below 273 K a continuous noncooperative condensation of both phospholipid and cholesterol is observed in the mixed membranes, and at about 253 K only a motionally restricted component is left, exhibiting slow fluctuations with correlation times of tau R perpendicular greater than 1 microsecond. In the high-temperature region (T greater than TM), order director fluctuations are found to constitute the dominant transverse relaxation process. Analysis of these collective lipid motions provides the viscoelastic parameters of the membranes. The results (T = 318 K) show that cholesterol significantly reduces the density of the cooperative motions by increasing the average elastic constant of the membrane from K = 1 x 10(-11) N for the pure phospholipid bilayers to K = 3.5 x 10(-11) N for the mixed system.     

Anisotropic 2H-nuclear magnetic resonance spin-lattice relaxation in cerebroside- and phospholipid-cholesterol bilayer membranes.

The axially symmetric powder pattern 2H-nuclear magnetic resonance (NMR) lineshapes observed in the liquid crystalline phase of pure lipid or lipid/cholesterol bilayers are essentially invariant to temperature, or, equivalently, to variations in the correlation times characterizing C-2H bond reorientations. In either of these melted phases, where correlation times for C-2H bond motions are shorter than 10(-7) s, information on the molecular dynamics of the saturated hydrocarbon chain would be difficult to obtain using lineshape analyses alone, and one must resort to other methods, such as the measurement of 2H spin-lattice relaxation rates, in order to obtain dynamic information. In pure lipid bilayers, the full power of the spin-lattice relaxation technique has yet to be realized, since an important piece of information, namely the orientation dependence of the 2H spin-lattice relaxation rates is usually lost due to orientational averaging of T1 by rapid lateral diffusion. Under more favorable circumstances, such as those encountered in the lipid/cholesterol mixtures of this study, the effects of orientational averaging by lateral diffusion are nullified, due to either a marked reduction (by at least an order of magnitude) in the diffusion rate, or a marked increase in the radii of curvature of the liposomes. In either case, the angular dependence of 2H spin-lattice relaxation is accessible to experimental study, and can be used to test models of molecular dynamics in these systems. Simulations of the partially recovered lineshapes indicate that the observed T1 anisotropies are consistent with large amplitude molecular reorientation of the C-2H bond among a finite number of sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of small molecules with phospholipid bilayer membranes: permeability studies

The passage of a phospholipid through the gel to liquid crystal phase transition is associated with an increase in the motional freedom of its fatty acyl chains as measured by spectroscopic techniques and an essentially isothermal absorption of heat as measured by differential scanning calorimetry (DSC). In addition, bilayers formed from that phospholipid display a permeability maximum for both non-electrolytes and electrolytes in the temperature region of the phase transition. In this study the sodium (and in some cases glucose) permeabilities of liposomes composed of either dimyristoyl or dipalmitoyl phosphatidylcholine plus dicetylphosphate were measured in the presence of a group of benzene and adamantane derivatives known to increase fatty acyl chain motion below the lipid transition temperature (T_c) and in the case of the adamantanes to also lower the T_c as measured by DSC. None of these compounds change the temperature at which the permeability maximum occurs despite their lowering of the phospholipid T_c. That is, in the presence of these additives there is observed an apparent dissociation between the phase transition and the permeability maximum. It is proposed that the permeability maximum normally observed in the temperature region of the T_c is associated with the completion of the ‘melting’ process. Hence a compound could cause early ‘melting’ of the bilayer but not change its permeability properties if the temperature at which the ‘melting’ process neared completion was not changed.     

Charge-pulse relaxation studies with lipid bilayer membranes modified by alamethicin

Charge-pulse relaxation studies with the alamethicin-lipid membrane system reveal a triphasic decay of membrane voltage. At short times (resolution time 2 microseconds), where a voltage decay due to the orientation of alamethicin dipoles from the interface into the membranes interior ("gating current") could possibly be expected, only a slow decrease with a time constant determined by the bare membrane conductance occurs. After approximately 1 ms (depending on the experimental conditions) the formation of alamethicin pores starts, leading to an increase in the voltage decay rate. When the characteristic voltage Vcpc is approached, pores close and after passing Vcpc the voltage decreases slowly again according to the bare membrane conductance. Vcpc is determined as a function of the initially applied voltage Vo, alamethicin and KCl concentration. Since the membrane voltage decreases continuously, the system does not reach the equilibrium states obtained at constant voltages. Taking the presented experimental results into account the estimate of the electrical potential at the functional membrane of photosynthesis induced by a saturating single turnover flash of deltaphio approximately 105-135 mV (Zickler, Witt and Boheim (1976) FEBS Lett. 66, 142-148) is changed to deltaphio approximately 200 mV.