Aggregation of cateslytin beta-sheets on negatively charged lipids promotes rigid membrane domains. A new mode of action for antimicrobial peptides?

Cateslytin, a positively charged (5+) arginine-rich antimicrobial peptide (bCgA, RSMRLSFRARGYGFR), was chemically synthesized and studied against membranes that mimic bacterial or mammalian systems. Circular dichroism, polarized attenuated total reflection infrared spectroscopy, (1)H high-resolution MAS NMR, and (2)H and (31)P solid state NMR were used to follow the interaction from peptide and membrane points of view. Cateslytin, which is unstructured in solution, is converted into antiparallel beta-sheets that aggregate mainly flat at the surface of negatively charged bacterial mimetic membranes. Arginine residues are involved in the binding to negatively charged lipids. Following the interaction of the cateslytin peptide, rigid and thicker membrane domains enriched in negatively charged lipids are found. Much less interaction is detected with neutral mammalian model membranes, as reflected by only minor percentages of beta-sheets or helices in the peptide secondary structure. No membrane destruction was detected for both bacterial and mammalian model membranes. A molecular model is proposed in which zones of different rigidity and thickness bring about phase boundary defects that ultimately lead to permeability induction and peptide crossing through bacterial membranes.     

Membrane binding and pore formation of the antibacterial peptide PGLa: thermodynamic and mechanistic aspects

The antibacterial peptide PGLa exerts its activity by permeabilizing bacterial membranes whereas eukaryotic membranes are not affected. To provide insight into the selectivity and the permeabilization mechanism, the binding of PGLa to neutral and negatively charged model membranes was studied with high-sensitivity isothermal titration calorimetry (ITC), circular dichroism (CD), and solid-state deuterium nuclear magnetic resonance ((2)H NMR). The binding of PGLa to negatively charged phosphatidylcholine (PC)/phosphatidylglycerol (PG) (3:1) vesicles was by a factor of approximately 50 larger than that to neutral PC vesicles. The negatively charged membrane accumulates the cationic peptide at the lipid-water interface, thus facilitating the binding to the membrane. However, if bulk concentrations are replaced by surface concentrations, very similar binding constants are obtained for neutral and charged membranes (K approximately 800-1500 M(-)(1)). Membrane selectivity is thus caused almost exclusively by electrostatic attraction to the membrane surface and not by hydrophobic insertion. Membrane insertion is driven by an exothermic enthalpy (DeltaH approximately -11 to -15 kcal/mol) but opposed by entropy. An important contribution to the binding process is the membrane-induced random coil --> alpha-helix transition of PGLa. The peptide is random coil in solution but adopts an approximately 80% alpha-helical conformation when bound to the membrane. Helix formation is an exothermic process, contributing approximately 70% to the binding enthalpy and approximately 30% to the free energy of binding. The (2)H NMR measurements with selectively deuterated lipids revealed small structural changes in the lipid headgroups and in the hydrocarbon interior upon peptide binding which were continuous over the whole concentration range. In contrast, isothermal titration calorimetry of PGLa solutions with PC/PG(3:1) vesicles gave rise to two processes: (i) an exothermic binding of PGLa to the membrane followed by (ii) a slower endothermic process. The latter is only detected at peptide-to-lipid ratios >17 mmol/mol and is paralleled by the induction of membrane leakiness. Dye efflux measurements are consistent with the critical limit derived from ITC measurements. The endothermic process is assigned to peptide pore formation and/or lipid perturbation. The enthalpy of pore formation is 9.7 kcal/mol of peptide. If the same excess enthalpy is assigned to the lipid phase, the lipid perturbation enthalpy is 180 cal/mol of lipid. The functional synergism between PGLa and magainin 2 amide could also be followed by ITC and dye release experiments and is traced back to an enhanced pore formation activity of a peptide mixture.     

Preferential association of apocytochrome c with negatively charged phospholipids in mixed model membranes

The mitochondrial precursor protein, apocytochrome c, binds to model membranes containing negatively charged phospholipids (Rietveld, A., Sijens, R., Verkleij, A.J. and Kruijff, B. (1983) EMBO J. 2, 907-913). In the present paper the effect of apocytochrome c on the lipid distribution in model membranes, consisting of neutral and acidic phospholipids, is examined. Both ESR and fluorescence energy transfer experiments show that the protein preferentially interacts with the negatively charged phospholipid in the mixed model membranes. Semi-quantitative analysis of the fluorescence energy transfer from the single tryptophan in apocytochrome c to the parinaric acid in phosphatidylserine or phosphatidylcholine in mixed bovine brain phosphatidylserine/egg phosphatidylcholine vesicles reveals and average donor-acceptor distance of 22-26 A and 26-30 A for phosphatidylserine and phosphatidylcholine, respectively. In addition, these experiments demonstrate that this preferential interaction does not induce the separation of large domains enriched in complexes of apocytochrome c with negatively charged phospholipids and domains enriched in neutral lipids.     

The interaction of neuropeptide Y with negatively charged and zwitterionic phospholipid membranes

The interaction of the 36 amino acid neuropeptide Y (NPY) with liposomes was studied using the intrinsic tyrosine fluorescence of NPY and an NPY fragment comprising amino acids 18–36. The vesicular membranes were composed of phosphatidylcholine and phosphatidylserine at varying mixing ratios. From the experimentally measured binding curves, the standard Gibbs free energy for the peptide transfer from aqueous solution to the lipid membrane was calculated to be around ?30 kJ/mol for membrane mixtures containing physiological amounts of acidic lipids at pH 5. The effective charge of the peptide depends on the pH of the buffer and is about half of its theoretical net charge. The results were confirmed using the fluorescence of the NPY analogue [Trp³²]-NPY. Further, the position of NPY’s α-helix in the membrane was estimated from the intrinsic tyrosine fluorescence of NPY, from quenching experiments with spin-labelled phospholipids using [Trp³²]-NPY, and from ¹H magic-angle spinning NMR relaxation measurements using spin-labelled [Ala³¹, TOAC³²]-NPY. The results suggest that the immersion depth of NPY into the membrane is triggered by the membrane composition. The α-helix of NPY is located in the upper chain region of zwitterionic membranes but its position is shifted to the glycerol region in negatively charged membranes. For membranes composed of phosphatidylcholine and phosphatidylserine, an intermediate position of the α-helix is observed.     

Membrane destabilization induced by the human immunodeficiency virus type-1 fusion peptide

Summary The human immunodeficiency virus type-1 (HIV-1) fusion peptide, corresponding to a sequence of 23 amino acid residues at the N-terminus of the spike transmembrane subunit gp41, has the capacity to destabilize negatively charged and neutral large unilamellar vesicles, representing, respectively, the acidic and the neutral fraction of the plasma membrane lipids of viral target cells. As revealed by infrared spectroscopy, the peptide associated with the vesicles may exist in different conformations. In negatively charged membranes the structure is mainly an α-helix, while in Ca²⁺-neutralized negatively charged membranes the conformation switches to a predominantly extended conformation. In membranes composed of zwitterionic phospholipids and cholesterol, the peptide also adopts a predominant extended structure. The α-helical structure permeabilizes negatively charged vesicles but does not induce membrane fusion. The peptide in β-type conformation, on the other hand, permeabilizes neutral membranes and triggers fusion. As seen by³¹P NMR, the latter structure also exhibits the capacity to alter the lamellar organization of the membrane.     

A study of the regional effects of alpha-synuclein on the organization and stability of phospholipid bilayers

Associations between the protein alpha-synuclein (alpha-syn) and presynaptic vesicles have been implicated in synaptic plasticity and neurotransmitter release and may also affect how the protein aggregates into fibrils found in Lewy bodies, the cellular inclusions associated with neurodegenerative diseases. This work investigated how alpha-syn interacts with model phospholipid membranes and examined what effect protein binding has upon the physical properties of lipid bilayers. Wide line 2H and 31P NMR spectra of phospholipid vesicles revealed that alpha-syn associates with membranes containing lipids with anionic headgroups and can disrupt the integrity of the lipid bilayer, but the protein has little effect on membranes of zwitterionic phosphatidylcholine. A peptide, alpha-syn(10-48), which corresponds to the lysine-rich N-terminal region of alpha-syn, was found to associate with lipid headgroups with a preference for a negative membrane surface charge. Another peptide, alpha-syn(120-140), which corresponds to the glutamate-rich C-terminal region, also associates weakly with lipid headgroups but with a slightly higher affinity for membranes with no net surface charge than for negatively charged membrane surfaces. Binding of alpha-syn(10-48) and alpha-syn(120-140) to the lipid vesicles did not disrupt the lamellar structure of the membranes, but both peptides appeared to induce the lateral segregation of the lipids into clusters of acidic lipid-enriched and acidic lipid-deficient domains. From these findings, it is speculated that the N-terminal and C-terminal domains of full-length alpha-syn might act in concert to organize the membrane components during normal protein function and perhaps play a role in presynaptic vesicle synthesis, maintenance, and fusion.     

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.     

Fusogenic Alzheimer's peptide fragment Abeta (29-42) in interaction with lipid bilayers: secondary structure, dynamics, and specific interaction with phosphatidyl ethanolamine polar heads as revealed by solid-state NMR

The interaction of the native Alzheimer's peptide C-terminal fragment Abeta (29-42), and two mutants (G33A and G37A) with neutral lipid bilayers made of POPC and POPE in a 9:1 molar ratio was investigated by solid-state NMR. This fragment and the lipid composition were selected because they represent the minimum requirement for the fusogenic activity of the Alzheimer's peptide. The chemical shifts of alanine methyl isotropic carbon were determined by MAS NMR, and they clearly demonstrated that the major form of the peptide equilibrated in membrane is not in a helical conformation. (2)H NMR, performed with acyl chain deuterated POPC, demonstrated that there is no perturbation of the acyl chain's dynamics and of the lipid phase transition temperature. (2)H NMR, performed with alanine methyl-deuterated peptide demonstrated that the peptide itself has a limited mobility below and above the lipid phase transition temperature (molecular order parameter equal to 0.94). MAS (31)P NMR revealed a specific interaction with POPE polar head as seen by the enhancement of POPE phosphorus nuclei T(2) relaxation. All these results are in favor of a beta-sheet oligomeric association of the peptide at the bilayer interface, preferentially recruiting phosphatidyl ethanolamine polar heads.     

How Lipid Headgroups Sense the Membrane Environment: An Application of 14N NMR

The orientation of lipid headgroups may serve as a powerful sensor of electrostatic interactions in membranes. As shown previously by ²H NMR measurements, the headgroup of phosphatidylcholine (PC) behaves like an electrometer and varies its orientation according to the membrane surface charge. Here, we explored the use of solid-state ¹⁴N NMR as a relatively simple and label-free method to study the orientation of the PC headgroup in model membrane systems of varying composition. We found that ¹⁴N NMR is sufficiently sensitive to detect small changes in headgroup orientation upon introduction of positively and negatively charged lipids and we developed an approach to directly convert the ¹⁴N quadrupolar splittings into an average orientation of the PC polar headgroup. Our results show that inclusion of cholesterol or mixing of lipids with different length acyl chains does not significantly affect the orientation of the PC headgroup. In contrast, measurements with cationic (KALP), neutral (Ac-KALP), and pH-sensitive (HALP) transmembrane peptides show very systematic changes in headgroup orientation, depending on the amount of charge in the peptide side chains and on their precise localization at the interface, as modulated by varying the extent of hydrophobic peptide/lipid mismatch. Finally, our measurements suggest an unexpectedly strong preferential enrichment of the anionic lipid phosphatidylglycerol around the cationic KALP peptide in ternary mixtures with PC. We believe that these results are important for understanding protein/lipid interactions and that they may help parametrization of membrane properties in computational studies.     

Lipid modulation of nicotinic acetylcholine receptor function: the role of neutral and negatively charged lipids.

The effects of negatively charged and neutral lipids on the function of the reconstituted nicotinic acetylcholine receptor from Torpedo californica were determined with two assays using acetylcholine receptor-containing vesicles: the ion flux response and the affinity-state transition. The receptor was reconstituted into three different lipid environments, with and without neutral lipids: (1) phosphatidylcholine/phosphatidylserine; (2) phosphatidylcholine/phosphatidic acid; and (3) phosphatidylcholine/cardiolipin. Analysis of the ion flux responses showed that: (1) all three negatively charged lipid environments gave fully functional acetylcholine receptor ion channels, provided neutral lipids were added; (2) in each lipid environment, the neutral lipids tested were functionally equivalent to cholesterol; and (3) the rate of receptor desensitization depends upon the type of neutral lipid and negatively charged phospholipid reconstituted with the receptor. The functional effects of neutral and negatively charged lipids on the acetylcholine receptor are discussed in terms of protein-lipid interactions and stabilization of protein structure by lipids.     

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).     

Concentration-dependent behavior of nisin interaction with supported bilayer lipid membrane

Nisin is a positively charged antibacterial peptide that binds to the negatively charged membranes of gram-positive bacteria. The initial interaction of the peptide with the model membrane of negatively charged DPPG (dipalmitoylphosphatidylglycerol) was studied by cyclic voltammetry and a.c. impedance spectroscopy. Nisin could induce pores in the supported bilayer lipid membrane, thus, it led to the marker ions Fe(CN)(6)(3-/4-) crossing the lipid membrane and giving the redox reaction on the glassy carbon electrode (GCE). Experimental results suggested that the pore formation on supported bilayer lipid membrane was dependent on the concentration of nisin and it included three main concentration stages: low, middling, high concentration.     

Peptide:lipid ratio and membrane surface charge determine the mechanism of action of the antimicrobial peptide BP100. Conformational and functional studies

The cecropin-melittin hybrid antimicrobial peptide BP100 (H-KKLFKKILKYL-NH2) is selective for Gram-negative bacteria, negatively charged membranes, and weakly hemolytic. We studied BP100 conformational and functional properties upon interaction with large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs, containing variable proportions of phosphatidylcholine (PC) and negatively charged phosphatidylglycerol (PG). CD and NMR spectra showed that upon binding to PG-containing LUVs BP100 acquires α-helical conformation, the helix spanning residues 3–11. Theoretical analyses indicated that the helix is amphipathic and surface-seeking. CD and dynamic light scattering data evinced peptide and/or vesicle aggregation, modulated by peptide:lipid ratio and PG content. BP100 decreased the absolute value of the zeta potential (ζ) of LUVs with low PG contents; for higher PG, binding was analyzed as an ion-exchange process. At high salt, BP100-induced LUVS leakage requires higher peptide concentration, indicating that both electrostatic and hydrophobic interactions contribute to peptide binding. While a gradual release took place at low peptide:lipid ratios, instantaneous loss occurred at high ratios, suggesting vesicle disruption. Optical microscopy of GUVs confirmed BP100-promoted disruption of negatively charged membranes. The mechanism of action of BP100 is determined by both peptide:lipid ratio and negatively charged lipid content. While gradual release results from membrane perturbation by a small number of peptide molecules giving rise to changes in acyl chain packing, lipid clustering (leading to membrane defects), and/or membrane thinning, membrane disruption results from a sequence of events – large-scale peptide and lipid clustering, giving rise to peptide-lipid patches that eventually would leave the membrane in a carpet-like mechanism.     

Non-electrostatic binding and self-association of amyloid beta-peptide on the surface of tightly packed phosphatidylcholine membranes

Self-association of amyloid β-peptide (Aβ) is considered to be an initial step in the development of Alzheimer’s disease and is known to be promoted by negatively charged lipid membranes. We have examined the possibility of non-electrostatic Aβ-membrane interaction by using neutral phosphatidylcholine lipids. Fluorescence and circular dichroism spectra have clearly shown that Aβ binds to the phosphatidylcholine membrane in the lamellar gel phase but not in the ripple gel or liquid crystalline phase, indicating the importance of the tight lipid packing characteristic of the lamellar gel phase. The Aβ-membrane binding occurs at both low and high salt concentrations, ensuring the non-electrostatic nature of the interaction. The membrane-bound Aβ molecule takes a monomeric α-helical or self-associated β-sheet structure depending on the temperature, peptide/lipid ratio, and salt concentration. The flat surface of tightly packed phosphatidylcholine membranes appears to serve as a platform for non-electrostatic binding and self-association of Aβ.     

Solid-state nuclear magnetic resonance relaxation studies of the interaction mechanism of antimicrobial peptides with phospholipid bilayer membranes

An 18-residue peptide, KWGAKIKIGAKIKIGAKI-NH(2) was designed to form amphiphilic beta-sheet structures when bound to lipid bilayers. The peptide possesses high antimicrobial activity when compared to naturally occurring linear antimicrobial peptides, most of which adopt an amphipathic alpha-helical conformation upon binding to the lipids. The perturbation of the bilayer by the peptide was studied by static (31)P and (2)H solid-state NMR spectroscopy using POPC and POPG/POPC (3/1) bilayer membranes with sn-1 chain perdeuterated POPC and POPG as the isotopic labels. (31)P NMR powder spectra exhibited two components for POPG/POPC bilayers upon addition of the peptide but only a slight change in the line shape for POPC bilayers, indicating that the peptide selectively disrupted the membrane structure consisting of POPG lipids. (2)H NMR powder spectra indicated a reduction in the lipid chain order for POPC bilayers and no significant change in the ordering for POPG/POPC bilayers upon association of the peptide with the bilayers, suggesting that the peptide acts as a surface peptide in POPG/POPC bilayers. Relaxation rates are more sensitive to the motions of the membranes over a large range of time scales. Longer (31)P longitudinal relaxation times for both POPG and POPC in the presence of the peptide indicated a direct interaction between the peptide and the POPG/POPC bilayer membranes. (31)P longitudinal relaxation studies also suggested that the peptide prefers to interact with the POPG phospholipids. However, inversion-recovery (2)H NMR spectroscopic experiments demonstrated a change in the relaxation rate of the lipid acyl chains for both the POPC membranes and the POPG/POPC membranes upon interaction with the peptide. Transverse relaxation studies indicated an increase in the spectral density of the collective membrane motion caused by the interaction between the peptide and the POPG/POPC membrane. The experimental results demonstrate significant dynamic changes in the membrane in the presence of the antimicrobial peptide and support a carpet mechanism for the disruption of the membranes by the antimicrobial peptide.     

A spectroscopic study of the membrane interaction of the antimicrobial peptide Pleurocidin

The cationic amphipathic alpha-helical antibiotic peptide, pleurocidin, from the winter flounder Pleuronectes americanus associates strongly with anionic membranes where it is able to translocate across the membrane, cause dye leakage from vesicles and induce pore like channel conductance. To investigate the mechanism of pleurocidin antibiotic activity in more detail we have applied a variety of spectroscopic techniques to study the interaction of pleurocidin with model membranes. At neutral pH the peptide inserts into membranes containing anionic lipids and, as shown by proton-decoupled 15N solid-state NMR spectroscopy of macroscopically oriented samples, is aligned parallel to the membrane surface. 2H solid-state NMR spectroscopy of chain deuterated phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) lipids in mixed membranes shows that pleurocidin interacts with both the zwitterionic PE and anionic PG but disrupts the lipid acyl chain order of the anionic PG lipids more effectively. At acidic pH the three histidine residues of pleurocidin become protonated and positively charged which does not alter the membrane disrupting effect nor the location of the peptide in the membrane. The results are interpreted in terms of a structural model for pleurocidin inserted into anionic lipid membranes and the implications of our data are discussed in terms of a general mechanism for the antibiotic activity.     

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.     

2H and31P nuclear magnetic resonance studies of membranes containing bovine rhodopsin

Summary Purified, delipidated rhodopsin is recombined with phospholipid using octyl-glucoside (OG) and preformed vesicles. Normal egg phosphatidylcholine, phosphatidylcholine in which the N-methyl groups are fully deuterated, and dioleoyl phosphatidylcholine labeled with deuterium at carbons 9 and 10 were used.³¹P nuclear magnetic resonance (NMR) and²H NMR measurements were obtained of the pure phospholipids and of the recombined membranes containing rhodopsin.³¹P NMR of the recombined membrane (containing the deuterated phospholipid) showed two overlapping resonances. One resembled a normal phospholipid bilayer, and the other was much broader, representing a motionally restricted phospholipid headgroup environment. The population of phospholipids in the motionally restricted environment can be modulated by conditions in the media.²H NMR spectra of the same recombined membranes showed only one component. These experimental results agree with a theoretical analysis that predicts an insensitivity of²H NMR to lipids bound to membrane proteins. A model containing at least three different phospholipid environments in the presence of the membrane protein rhodopsin is described.Deceased.