protegrin-1 and 1-palmitoyl-2-oleoylphosphatidylcholine

Protegrin-1 (PG-1) belongs to the family of antimicrobial peptides. It interacts specifically with the membrane of a pathogen and kills the pathogen by releasing its cellular contents. To fully understand the energetics governing the orientation of PG-1 in different membrane environments and its effects on the physicochemical properties of the peptide and membrane bilayers, we have performed the potential of mean force (PMF) calculations as a function of its tilt angle at four distinct rotation angles in explicit membranes composed of either DLPC (1,2-dilauroylphosphatidylcholine) or POPC (1-palmitoyl-2-oleoylphosphatidylcholine) lipid molecules. The resulting PMFs in explicit lipid bilayers were then used to search for the optimal hydrophobic thickness of the EEF1/IMM1 implicit membrane model in which a two-dimensional PMF in the tilt and rotation space was calculated. The PMFs in explicit membrane systems clearly reveal that the energetically favorable tilt angle is affected by both the membrane hydrophobic thickness and the PG-1 rotation angle. Local thinning of the membrane around PG-1 is observed upon PG-1 tilting. The thinning is caused by both hydrophobic mismatch and arginine-lipid head group interactions. The two-dimensional PMF in the implicit membrane is in good accordance with those from the explicit membrane simulations. The ensemble-averaged Val16 (15)N and (13)CO chemical shifts weighted by the two-dimensional PMF agree fairly well with the experimental values, suggesting the importance of peptide dynamics in calculating such ensemble properties for direct comparison with experimental observables.

Topics: Antimicrobial Cationic Peptides; Chemical Phenomena; Lipid Bilayers; Magnetic Resonance Spectroscopy; Phosphatidylcholines; Reference Standards

Antimicrobial peptides interact specifically with the membrane of a pathogen and kill the pathogen by releasing its cellular contents. Protegrin-1 (PG-1), a beta-hairpin antimicrobial peptide, is known to exist as a transmembrane monomer in a 1,2-dilauroylphosphatidylcholine (DLPC) bilayer and shows concentration-dependent oligomerization in a 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayer. To examine its structure, dynamics, orientation, and interaction in membranes, we performed comparative molecular dynamics simulations of PG-1 monomer and dimer in DLPC and POPC bilayers for a total of 840 ns. The PG-1 monomer exhibits larger tilting in DLPC than in POPC due to a hydrophobic mismatch. PG-1 tilting is dependent on its rotation angle. The specific orientation of PG-1 in membranes is governed by the interactions of its aromatic residues with lipid headgroups. The calculated (15)N and (13)CO chemical shifts of Val(16) in DLPC reveal that there are different sets of tilt and rotation angles that satisfy the experimental values reasonably, suggesting that more experiments are needed to determine its orientation. The dimer simulations show that the dimer interface is better preserved in POPC than in DLPC because POPC's greater hydrophobic thickness causes reduced flexibility of the C-terminal strands. Both monomer and dimer simulations show membrane thinning around PG-1, largely due to arginine-lipid interactions.

Topics: Antimicrobial Cationic Peptides; Computer Simulation; Guanidine; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Models, Chemical; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Phosphatidylcholines; Protein Multimerization; Protein Stability; Protein Structure, Secondary; Rotation; Water

Membrane pores that are induced in oriented membranes by an antimicrobial peptide (AMP), protegrin-1 (PG-1), are investigated by (31)P and (2)H solid state NMR spectroscopy. We incorporated a well-studied peptide, protegrin-1 (PG-1), a beta-sheet AMP, to investigate AMP-induced dynamic supramolecular lipid assemblies at different peptide concentrations and membrane compositions. Anisotropic NMR line shapes specifying toroidal pores and thinned membranes, which are formed in membrane bilayers by the binding of AMPs, have been analyzed for the first time. Theoretical NMR line shapes of lipids distributed on the surface of toroidal pores and thinned membranes reproduce reasonably well the line shape characteristics of our experimentally measured (31)P and (2)H solid-state NMR spectra of oriented lipids binding with PG-1. The lateral diffusions of lipids are also analyzed from the motionally averaged one- and two-dimensional (31)P and (2)H solid-state NMR spectra of oriented lipids that are binding with AMPs.

Topics: Antimicrobial Cationic Peptides; Deuterium; Diffusion; Lipid Bilayers; Magnetic Resonance Spectroscopy; Phosphatidylcholines; Phosphorus Isotopes; Porosity

Disulfide-bonded beta-hairpin structures are common among antimicrobial peptides. Disulfide bonds are known to be important for antimicrobial activity, but the underlying structural reason is not well understood. We have investigated the membrane-bound structure of a disulfide-deleted analogue of the antimicrobial peptide protegrin-1, in which the four Cys residues were replaced by Ala. The secondary structure, dynamics, and topology of this Ala-PG1 peptide in the membrane were determined by using magic-angle-spinning NMR spectroscopy. Conformation-dependent (13)C isotropic chemical shifts of multiple (13)C-labeled residues were obtained from 1D cross-polarization and direct-polarization spectra, and from 2D J-coupling-mediated (13)C-(13)C correlation spectra. Most labeled residues exhibited two conformations: a random coil and a beta-sheet structure. The dual-conformation property was present in both anionic lipid bilayers, which mimic the bacterial membrane, and zwitterionic cholesterol-containing bilayers, which mimic the eukaryotic cell membrane. The mobility of the peptide was measured by using a 2D C-H dipolar-shift correlation experiment. The random-coil fraction was highly mobile whereas the beta-sheet component was rigid. (1)H spin diffusion from the lipid chains to the peptide indicates that the beta-sheet component was well inserted into the anionic membrane, but surface bound in the cholesterol-containing neutral membrane. Thus, the removal of disulfide bonds changed some PG-1 molecules to highly mobile random coils that were poorly associated with the lipid membrane, but other molecules retained a beta-sheet conformation and had a similar membrane-binding topology to the parent peptide. Thus, the reduced antimicrobial activity of Ala-PG1 was largely due to the reduced number of insertion-competent beta-sheet molecules, rather than uniformly weakened activity of identically structured peptides.

Topics: Alanine; Amino Acid Sequence; Anti-Infective Agents; Antimicrobial Cationic Peptides; Carbon Radioisotopes; Cell Membrane; Cholesterol; Cysteine; Disulfides; Lipid Bilayers; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Conformation; Proteins

The intermolecular packing of a beta-hairpin antimicrobial peptide, PG-1, in lipid bilayers is determined using solid-state NMR distance measurements. Previous spin counting experiments showed that PG-1 associates as dimers in POPC bilayers; however, the detailed dimer structure was unknown. We have now measured several intermolecular 13C-19F, 1H-13C, and 15N-13C distances in site-specifically labeled PG-1 to constrain the structure of the intermolecular interface. The distances are measured using the rotational-echo double-resonance (REDOR) technique under magic-angle spinning. The results indicate that two PG-1 molecules align in a parallel fashion with the C-terminal strand of the hairpin forming the dimer interface. Six hydrogen bonds stabilize this interface, and the Phe12 side chain adopts the g- conformation in the membrane as in solution. The parallel packing of the peptide in the lipid bilayer differs from the antiparallel dimer found in DPC micelles and may be stabilized by its strong amphipathic character, which should facilitate its insertion into the amphipathic lipid bilayer. This study demonstrates the utility of the REDOR NMR technique for the elucidation of the oligomeric structure of membrane proteins.

Topics: Antimicrobial Cationic Peptides; Dimerization; Hydrogen Bonding; Lipid Bilayers; Magnetic Resonance Spectroscopy; Membrane Proteins; Phosphatidylcholines; Protein Structure, Secondary; Proteins

Protegrins (PG) are important in defending host tissues, preventing infection via an attack on the membrane surface of invading microorganisms. Protegrins have powerful antibiotic abilities, but the molecular-level mechanisms underlying the interactions of their beta-sheet motifs with the membrane are not known. Protegrin-1 (PG-1) is composed of 18 amino acids with a high content of basic residues and two disulfide bonds. Here we focused on the stability of PG-1 at the amphipathic interface in lipid bilayers and on the details of the peptide-membrane interactions. We simulated all-atom models of the PG-1 monomer with explicit water and lipid bilayers composed of both homogeneous POPC (palmitoyl-oleyl-phosphatidylcholine) lipids and a mixture of POPC/POPG (palmitoyl-oleyl-phosphatidylglycerol) (4:1) lipids. We observed that local thinning of the lipid bilayers mediated by the peptide is enhanced in the lipid bilayer containing POPG, consistent with experimental results of selective membrane targeting. The beta-hairpin motif of PG-1 is conserved in both lipid settings, whereas it is highly bent in aqueous solution. The conformational dynamics of PG-1, especially the highly charged beta-hairpin turn region, are found to be mostly responsible for disturbing the membrane. Even though the eventual membrane disruption requires PG-1 oligomers, our simulations clearly show the first step of the monomeric effects. The thinning effects in the bilayer should relate to pore/channel formation in the lipid bilayer and thus be responsible for further defects in the membrane caused by oligomer.

Topics: Antimicrobial Cationic Peptides; Computer Simulation; Lipid Bilayers; Models, Molecular; Peptides; Phosphatidylcholines; Phosphatidylglycerols; Protein Structure, Secondary; Proteins; Water

We used solid-state NMR spectroscopy to investigate the oligomeric structure and insertion of protegrin-1 (PG-1), a beta-hairpin antimicrobial peptide, in lipid bilayers that mimic either the bacterial inner membrane [palmitoyloleoylphosphatidyl ethanolamine and palmitoyloleoylphosphatidylglycerol (POPE/POPG) bilayers] or the red blood cell membrane [neutral palmitoyloleoylphosphatidylcholine (POPC)/cholesterol bilayers]. (1)H spin diffusion from lipids to the peptide indicates that PG-1 contacts both the lipid acyl chains and the headgroups in the anionic membrane but resides far from the lipid chains in the POPC/cholesterol bilayer. (19)F spin diffusion data indicates that 75% of the beta-hairpins have homodimerized N strands and C strands in the anionic membrane. The resulting (NCCN)(n) multimer suggests a membrane-inserted beta-barrel enclosing a water pore. The lipids surrounding the beta-barrel have high orientational disorder and chain upturns, thus they may act as fillers for the pore. These results revise several features of the toroidal pore model, first proposed for magainin and subsequently applied to PG-1. In the POPC/cholesterol membrane, the N and C strands of PG-1 cluster into tetramers, suggesting the formation of beta-sheets on the membrane surface. Thus, the membrane composition plays a decisive role in defining the assembly and insertion of PG-1. The different oligomeric structures of PG-1 help to explain its greater toxicity for bacteria than for eukaryotic cells.

Topics: Anions; Anti-Infective Agents; Antimicrobial Cationic Peptides; Cholesterol; Lipid Bilayers; Magnetic Resonance Spectroscopy; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Peptides; Phosphatidylcholines; Phosphatidylglycerols; Protein Conformation; Proteins

Aggregation or oligomerization is important for the function of many membrane peptides such as ion channels and antimicrobial peptides. However, direct proof of aggregation and the determination of the number of molecules in the aggregate have been difficult due to the lack of suitable high-resolution methods for membrane peptides. We propose a 19F spin diffusion magic-angle-spinning NMR technique to determine the oligomeric state of peptides bound to the lipid bilayer. Magnetization transfer between chemically equivalent but orientationally different 19F spins on different molecules reduces the 19F magnetization in an exchange experiment. At long mixing times, the equilibrium 19F magnetization is 1/M, where M is the number of orientationally different molecules in the aggregate. The use of the 19F spin increases the homonuclear dipolar coupling and thus the distance reach. We demonstrate this technique on crystalline model compounds with known numbers of molecules in the asymmetric unit cell, and show that 19F spin diffusion is more efficient than that of 13C by a factor of approximately 500. Application to a beta-hairpin antimicrobial peptide, protegrin-1, shows that the peptide is almost completely dimerized in POPC bilayers at a concentration of 7.4 mol %. Decreasing the peptide concentration reduced the dimer fraction. Using a monomer-dimer equilibrium model, we estimate the DeltaG for dimer formation to be -10.2 +/- 2.3 kJ/mol. This is in good agreement with the previously measured free energy reduction for partitioning and aggregating beta-sheet peptides into phospholipid membranes. This 19F spin diffusion technique opens the possibility of determining the oligomeric structures of membrane peptides.

Topics: Anti-Infective Agents; Antimicrobial Cationic Peptides; Dimerization; Fluorine; Glycine; Lipid Bilayers; Membrane Proteins; Nuclear Magnetic Resonance, Biomolecular; Peptides; Phenylalanine; Phosphatidylcholines; Proteins; Thermodynamics

The influence of an antimicrobial peptide, protegrin-1 (PG-1), on the curvature and lateral diffusion coefficient (D(L)) of phosphocholine bilayers is investigated using one- (1D) and two-dimensional (2D) (31)P exchange NMR. The experiments utilize the fact that lipid lateral diffusion over the curved surface of vesicles changes the molecular orientation and thus the (31)P chemical shift anisotropy. This reorientation is manifested in 2D spectra as off-diagonal intensities and in 1D stimulated-echo experiments as reduced echo heights. The 2D spectra give information on the reorientation-angle distribution while the decay of the stimulated-echo intensity, which closely tracks the second-order correlation function in our experiments, yields the correlation times of the reorientation. The relationships among the 2D exchange spectra, stimulated-echo intensities, the correlation function, and reorientation-angle distributions are analyzed in detail. In the absence of PG-1, both dilaurylphosphotidylcholine (DLPC) and palmitoyloleoylphosphatidylcholine (POPC) vesicles show biexponential decays of the stimulated-echo intensities to equilibrium values of 0.20-0.25, suggesting that the curvature of the lipid vesicles has a bimodal distribution. The addition of PG-1 to DLPC vesicles increased the decay time constants, indicating that D(L) decreases due to peptide binding. In contrast, the addition of PG-1 to POPC vesicles decreased the decay constants by three to fivefold, indicating that the POPC vesicles are fragmented into smaller vesicles. On the basis of the changes in D(L) and the decay constants, we estimate that the radius of the POPC vesicles decreases by threefold due to PG-1 binding. Simulations of the 2D exchange spectra yielded quantitative reorientation-angle distributions that are consistent with the bimodal distributions of the vesicle curvature and the effects of the peptide on the two types of lipid bilayers. Thus, (31)P exchange NMR provides useful insights into the membrane morphological changes induced by this antimicrobial peptide.

Topics: Antimicrobial Cationic Peptides; Magnetic Resonance Spectroscopy; Membranes, Artificial; Phosphatidylcholines; Phosphorus Isotopes; Proteins; Sensitivity and Specificity; Time Factors

The interaction of a beta-hairpin antimicrobial peptide, protegrin-1 (PG-1), with various lipid membranes is investigated by (31)P, (2)H, and (13)C solid-state NMR. Mixed lipid bilayers containing anionic lipids and cholesterol are used to mimic the bacterial and mammalian cell membranes, respectively. (31)P and (2)H spectra of macroscopically oriented samples show that PG-1 induces the formation of an isotropic phase in anionic bilayers containing phosphatidylglycerol. Two-dimensional (31)P exchange experiments indicate that these isotropic lipids are significantly separate from the residual oriented lamellar bilayers, ruling out toroidal pores as the cause for the isotropic signal. (1)H spin diffusion experiments show that PG-1 is not exclusively bound to the isotropic phase but is also present in the residual oriented lamellar bilayers. This dynamic and morphological heterogeneity of the anionic membranes induced by PG-1 is supported by the fact that (13)C T(2) relaxation times measured under cross polarization and direct polarization conditions differ significantly. In contrast to the anionic membrane, the zwitterionic phosphatidylcholine (PC) membrane does not form an isotropic phase in the presence of PG-1 but shows significant orientational disorder. The addition of cholesterol to the PC bilayer significantly reduces this orientational disorder. The (13)C T(2) relaxation times of the PC lipids in the presence of both cholesterol and PG-1 suggest that the peptide may decrease the dynamic heterogeneity of the cholesterol-containing membrane. The observed selective interaction of PG-1 with different lipid membranes is consistent with its biological function and may be caused by its strong cationic and amphipathic structure.

Topics: Animals; Anions; Antimicrobial Cationic Peptides; Carbon Isotopes; Cholesterol; Deuterium; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Nuclear Magnetic Resonance, Biomolecular; Phosphatidylcholines; Phosphatidylglycerols; Phosphorus Isotopes; Proteins; Protons; Swine; Thermodynamics