Binding of pediocin PA-1 with anionic lipid induces model membrane destabilization

To obtain molecular insights into the action mode of antimicrobial activity of pediocin PA-1, the interactions between this bacteriocin and dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylglycerol (DMPG) model membranes have been investigated in D(2)O at pD 6 by Fourier transform infrared spectroscopy. The interactions were monitored with respect to alteration of the secondary structure of pediocin, as registered by the amide I' band, and phospholipid conformation, as revealed by the methylene nu(s)(CH(2)) and carbonyl nu(C;O) stretching vibrations. The results show that no interaction between pediocin and DMPC occurs. By contrast, pediocin undergoes a structural reorganization in the presence of DMPG. Upon heating, pediocin self-aggregates, which is not observed for this pD in aqueous solution. The gel-to-crystalline phase transition of DMPG shifts to higher temperatures with a concomitant dehydration of the interfacial region. Our results indicate that pediocin is an extrinsic peptide and that its action mechanism may lie in a destabilization of the cell membrane.     

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.     

Interaction study between maltose-modified PPI dendrimers and lipidic model membranes

The influence of maltose-modified poly(propylene imine) (PPI) dendrimers on dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) (3%) liposomes was studied. Fourth generation (G4) PPI dendrimers with primary amino surface groups were partially (open shell glycodendrimers — OS) or completely (dense shell glycodendrimers — DS) modified with maltose residues. As a model membrane, two types of 100 nm diameter liposomes were used to observe differences in the interactions between neutral DMPC and negatively charged DMPC/DMPG bilayers. Interactions were studied using fluorescence spectroscopy to evaluate the membrane fluidity of both the hydrophobic and hydrophilic parts of the lipid bilayer and using differential scanning calorimetry to investigate thermodynamic parameter changes. Pulsed-filed gradient NMR experiments were carried out to evaluate common diffusion coefficient of DMPG and DS PPI in D₂O when using below critical micelle concentration of DMPG. Both OS and DS PPI G4 dendrimers show interactions with liposomes. Neutral DS dendrimers exhibit stronger changes in membrane fluidity compared to OS dendrimers. The bilayer structure seems more rigid in the case of anionic DMPC/DMPG liposomes in comparison to pure and neutral DMPC liposomes. Generally, interactions of dendrimers with anionic DMPC/DMPG and neutral DMPC liposomes were at the same level. Higher concentrations of positively charged OS dendrimers induced the aggregation process with negatively charged liposomes. For all types of experiments, the presence of NaCl decreased the strength of the interactions between glycodendrimers and liposomes. Based on NMR diffusion experiments we suggest that apart from electrostatic interactions for OS PPI hydrogen bonds play a major role in maltose-modified PPI dendrimer interactions with anionic and neutral model membranes where a contact surface is needed for undergoing multiple H-bond interactions between maltose shell of glycodendrimers and surface membrane of liposome.     

Structural effects of the antimicrobial peptide maculatin 1.1 on supported lipid bilayers

The interactions of the antimicrobial peptide maculatin 1.1 (GLFGVLAKVAAHVVPAIAEHF-NH₂) with model phospholipid membranes were studied by use of dual polarisation interferometry and neutron reflectometry and dimyristoylphosphatidylcholine (DMPC) and mixed DMPC–dimyristoylphosphatidylglycerol (DMPG)-supported lipid bilayers chosen to mimic eukaryotic and prokaryotic membranes, respectively. In DMPC bilayers concentration-dependent binding and increasing perturbation of bilayer order by maculatin were observed. By contrast, in mixed DMPC–DMPG bilayers, maculatin interacted more strongly and in a concentration-dependent manner with retention of bilayer lipid order and structure, consistent with pore formation. These results emphasise the importance of membrane charge in mediating antimicrobial peptide activity and emphasise the importance of using complementary methods of analysis in probing the mode of action of antimicrobial peptides.     

Comparative study of the interaction of synthetic methionine-enkephalin and its amidated derivate with monolayers of zwitterionic and negatively charged phospholipids

Using Langmuir’s monolayer technique, the surface behavior and the interaction of the synthetic neuropeptide methionine-enkephalin (Met-enk) and its amidated derivate (Met-enk-NH₂) with monolayers of the zwitterionic dimyristoylphosphatidylcholine (DMPC) and the negatively charged dimyristoylphosphatidylglycerol (DMPG) were studied. The surface tension (γ, mN/m) of DMPG and DMPC monolayers as a function of time (after injection of the peptide under the interface) was detected. The decrease in γ values showed that there was a strong penetration effect of both types of Met-enk molecules into the monolayers, being significantly stronger for the amidated derivate, Met-enk-NH₂. We suggest that the interaction between the neuropeptides and DMPC was predominantly determined by peptides amphiphilicity, while the electrostatic forces play significant role for the insertion of the cationic Met-enk-NH₂ in DMPG monolayers, especially at high packing densities. Our results demonstrate the potential of lipid monolayers formed in Langmuir’s trough to be successfully used as an elegant and simple membrane models to study lipid–peptide interactions at the air/water interface.     

Resonance Raman Studies of Co—O2 and O—O Stretching Vibrations in Oxy-Cobalt Hemes

Strong evidence suggests that the stretching vibration of the bound oxygen can be perturbed by an accidentally degenerate porphyrin ring mode, resulting in two split frequencies. In the Co(II)(TpivPP) (pyridine) ¹⁸O₂ complex, we demonstrate that the ν(¹⁸O—¹⁸O) mode, after being shifted from its ν(¹⁶O—¹⁶O) value at 1,156 cm^-1, undergoes a resonance interaction with the 1,080 cm^-1 porphyrin mode, giving rise to two lines at 1,067 and 1,089 cm^-1. In the O₂ complex of Co(II) mesoporphyrin IX-substituted sperm whale myoglobin, we observed a dramatic intensity increase at 1,132 cm^-1 upon ¹⁶O₂ → ¹⁸O₂ substitution, which is due to the reappearance of the 1,132-cm^-1 porphyrin mode after the removal of resonance conditions. A decrease in O₂ binding affinity, caused by the proximal base tension, corresponds to an increase in the Co—O₂ stretching frequency. The ν(Co—O₂) at 527 cm^-1 for the low affinity Co(II)(TpivPP)(1,2-Me₂Im) O₂ complex is 11 cm^-1 higher than the 516-cm^-1 value for the high affinity complex (with N-MeIm replacing 1,2-Me₂Im). However, in the corresponding iron complexes the reverse behavior is observed, i.e., the ν(Fe—O₂) decreases for the (1,2-Me₂Im) complex. There is a 24-cm^-1 difference in the Co—O₂ stretching frequencies between Co(II)(TpivPP)(N-MeIm)O₂ (at 516 cm^-1) and oxy meso CoMb (at 540 cm^-1), suggesting a protein induced distortion of the Co—O—O linkage. However, the values for ν(Fe—O₂) are nearly identical between Fe(II)(TpivPP)(N-MeIm)O₂ (at 571 cm^-1) and oxy Mb (at 573 cm^-1), indicating that O₂ binds to myoglobin in the same manner as in the sterically unhindered “picket fence” complex. Evidence is presented that suggests the presence of two dioxygen stretching frequencies due to two different conformers in each of the N-MeIm and 1,2-Me₂Im complex of oxy Co(II)(TpivPP).     

Calorimetric and spectroscopic studies of the phase behavior and organization of lipid bilayer model membranes composed of binary mixtures of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol

The thermotropic phase behavior of hydrated bilayers derived from binary mixtures of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) was investigated by differential scanning calorimetry, Fourier-transform infrared spectroscopy and ³¹P-nuclear magnetic resonance spectroscopy. Binary mixtures of DMPC and DMPG that have not been annealed at low temperatures exhibit broad, weakly energetic pretransitions (∼11-15 °C) and highly cooperative, strongly energetic gel/liquid-crystalline phase transitions (∼23-25 °C). After low temperature incubation, these mixtures also exhibit a thermotropic transition form a lamellar-crystalline to a lamellar gel phase at temperatures below the onset of the gel/liquid-crystalline phase transition. The midpoint temperatures of the pretransitions and gel/liquid-crystalline phase transitions of these lipid mixtures are both maximal in mixtures containing ∼30 mol% DMPG but the widths and enthalpies of the same thermotropic events exhibit no discernable composition dependence. In contrast, thermotropic transitions involving the L_c phase exhibit a very strong composition dependence, and the midpoint temperatures and transition enthalpies are both maximal with mixtures containing equimolar amounts of the two lipids. Our spectroscopic studies indicate that the L_c phases formed are structurally similar as regards their modes of hydrocarbon chain packing, interfacial hydration and hydrogen-bonding interactions, as well as the range and amplitudes of the reorientational motions of their phosphate headgroups. Our results indicate that although DMPC and DMPG are highly miscible, their mixtures do not exhibit ideal mixing. We attribute the non-ideality in their mixing behavior to the formation of preferential PC/PG contacts in the L_c phase due to the combined effects of steric crowding of the DMPC headgroups and charge repulsion between the negatively charged DMPG molecules.     

Elastic-Mathematical Theory of Cells and Mitochondria in Swelling Process: II. Effect of Temperature upon Modulus of Elasticity of Membranous Material of Egg Cells of Sea Urchin, Strongylocentrotus purpuratus, and of Oyster, Crassostrea virginica

The elastic behavior of the cell wall as a function of the temperature has been studied with particular attention being given to the swelling of egg cells of Strongylocentrotus purpuratus and Crassostrea virginica in different sea water concentrations at different temperatures. It was found that the modulus of elasticity is a nonlinear function of temperature. At about 12-13°C the modulus of elasticity (E) is constant, independent of the stress (σ) and strain (ε_ν) which exist at the cell wall; the membranous material follows Hooke's law, and E ≈ 3 × 10⁷ dyn/cm² for S. purpuratus and C. virginica. When the temperature is higher or lower than 12-13°C, the modulus of elasticity increases, and the membranous material does not follow Hooke's law, but is almost directly proportional to the stresses existing at the cell wall. On increasing the stress, the function E_σ = E(σ) approaches saturation. The corresponding stress-strain diagrams, σ = σ(ε_ν), and the graphs, E_σ = E(σ) and E_σ = E(t) are given. The cyto-elastic phenomena at the membrane are discussed.     

Faster Synthesis of Beta-Diketonate Ternary Europium Complexes: Elapsed Times & Reaction Yields

β-diketonates are customary bidentate ligands in highly luminescent ternary europium complexes, such as Eu(β-diketonate)₃(L)₂, where L stands for a nonionic ligand. Usually, the syntheses of these complexes start by adding, to an europium salt such as EuCl₃(H₂O)₆, three equivalents of β-diketonate ligands to form the complexes Eu(β-diketonate)₃(H₂O)₂. The nonionic ligands are subsequently added to form the target complexes Eu(β-diketonate)₃(L)₂. However, the Eu(β-diketonate)₃(H₂O)₂ intermediates are frequently both difficult and slow to purify by recrystallization, a step which usually takes a long time, varying from days to several weeks, depending on the chosen β-diketonate. In this article, we advance a novel synthetic technique which does not use Eu(β-diketonate)₃(H₂O)₂ as an intermediate. Instead, we start by adding 4 equivalents of a monodentate nonionic ligand L straight to EuCl₃(H₂O)₆ to form a new intermediate: EuCl₃(L)₄(H₂O)_n, with n being either 3 or 4. The advantage is that these intermediates can now be easily, quickly, and efficiently purified. The β-diketonates are then carefully added to this intermediate to form the target complexes Eu(β-diketonate)₃(L)₂. For the cases studied, the 20-day average elapsed time reduced to 10 days for the faster synthesis, together with an improvement in the overall yield from 42% to 69%.     

The Structure of a Melittin-Stabilized Pore

Melittin has been reported to form toroidal pores under certain conditions, but the atomic-resolution structure of these pores is unknown. A 9-μs all-atom molecular-dynamics simulation starting from a closely packed transmembrane melittin tetramer in DMPC shows formation of a toroidal pore after 1 μs. The pore remains stable with a roughly constant radius for the rest of the simulation. Surprisingly, one or two melittin monomers frequently transition between transmembrane and surface states. All four peptides are largely helical. A simulation in a DMPC/DMPG membrane did not lead to a stable pore, consistent with the experimentally observed lower activity of melittin on anionic membranes. The picture that emerges from this work is rather close to the classical toroidal pore, but more dynamic with respect to the configuration of the peptides.     

Calcium Enhances Binding of Aβ Monomer to DMPC Lipid Bilayer

Using isobaric-isothermal replica-exchange molecular dynamics and the all-atom explicit-solvent model, we studied the equilibrium binding of Aβ monomers to a zwitterionic dimyristoylphosphatidylcholine (DMPC) bilayer coincubated with calcium ions. Using our previous replica-exchange molecular dynamics calcium-free simulations as a control, we reached three conclusions. First, calcium ions change the tertiary structure of the bound Aβ monomer by destabilizing several long-range intrapeptide interactions, particularly the salt bridge Asp²³-Lys²⁸. Second, calcium strengthens Aβ peptide binding to the DMPC bilayer by enhancing electrostatic interactions between charged amino acids and lipid polar headgroups. As a result, Aβ monomer penetrates deeper into the bilayer, making disorder in proximal lipids and bilayer thinning more pronounced. Third, because calcium ions demonstrate strong affinity to negatively charged amino acids, a considerable influx of calcium into the area proximal to the bound Aβ monomer is observed. Consequently, the localizations of negatively charged amino acids and calcium ions in the Aβ binding footprint overlap. Based on our data, we propose a mechanism by which calcium ions strengthen Aβ-bilayer interactions. This mechanism involves two factors: 1) calcium ions make the DMPC bilayer partially cationic and thus attractive to the anionic Aβ peptide; and 2) destabilization of the Asp²³-Lys²⁸ salt bridge makes Lys²⁸ available for interactions with the bilayer. Finally, we conclude that a single Aβ monomer does not promote permeation of calcium ions through the zwitterionic bilayer.     

Studies on the mechanism of the protective action of 16,16-dimethyl PGE2 in carbon tetrachloride induced acute hepatic injury in the rat

We have previously demonstrated the partial protection of the rat liver by 16,16-dmPGE₂ (DMPG) against a number of hepatotoxins including carbon tetrachloride (CCl₄). However, it has not been determined whether hepatoprotection by DMPG represents a true “cytoprotective” action or if merely accomplished through inhibition of CCl₄ metabolism to reactive, toxic trichoromethyl (CCl₃·) free radicals. This report details a series of experiments in which the effects of DMPG on CCl₄ metabolism was evaluated in the rat.These data indicate that pretreatment with DMPG may reduce the hepatic concentration of the toxic CCl₃· free radicals in CCl₄ poisoned rats. Evidence is presented which suggests that this reduction in binding may have been due to a decrease in the rate of CCl₄ metabolism. However, DMPG did not affect the hepatic concentration of total microsomal cytochrome P450, the necessary enzyme in this metabolic process. On the other hand, free radical spin trapping experiments indicate that the rate of free radical formation from CCl₄ was slowed by treatment. Also, indirect evidence suggests that the metabolism of another cytochrome P450 substrate, phenobarbital, was slowed in DMPG treated rats. We conclude that the rate of CCl₄ metabolism may be reduced by pretreatment with DMPG. Furthermore, some measure of hepatic protection might be expected to occur as a result of the reduction in the rate of CCl₄ metabolism. However, we are unable to determine if this action was solely responsible for the observed hepatic protection.     

Interactions of the Australian tree frog antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with lipid model membranes: Differential scanning calorimetric and Fourier transform infrared spectroscopic studies

The interactions of the antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidylethanolamine (DMPE) were studied by differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy. The effects of these peptides on the thermotropic phase behavior of DMPC and DMPG are qualitatively similar and manifested by the suppression of the pretransition, and by peptide concentration-dependent decreases in the temperature, cooperativity and enthalpy of the gel/liquid-crystalline phase transition. However, at all peptide concentrations, anionic DMPG bilayers are more strongly perturbed than zwitterionic DMPC bilayers, consistent with membrane surface charge being an important aspect of the interactions of these peptides with phospholipids. However, at all peptide concentrations, the perturbation of the thermotropic phase behavior of zwitterionic DMPE bilayers is weak and discernable only when samples are exposed to high temperatures. FTIR spectroscopy indicates that these peptides are unstructured in aqueous solution and that they fold into α-helices when incorporated into lipid membranes. All three peptides undergo rapid and extensive H-D exchange when incorporated into D₂O-hydrated phospholipid bilayers, suggesting that they are located in solvent-accessible environments, most probably in the polar/apolar interfacial regions of phospholipid bilayers. The perturbation of model lipid membranes by these peptides decreases in magnitude in the order maculatin 1.1 > aurein 1.2 > citropin 1.1, whereas the capacity to inhibit Acholeplasma laidlawii B growth decreases in the order maculatin 1.1 > aurein 1.2 ≅ citropin 1.1. The higher efficacy of maculatin 1.1 in disrupting model and biological membranes can be rationalized by its larger size and higher net charge. However, despite its smaller size and lower net charge, aurein 1.2 is more disruptive of model lipid membranes than citropin 1.1 and exhibits comparable antimicrobial activity, probably because aurein 1.2 has a higher propensity for partitioning into phospholipid membranes.     

Pore Structure and Synergy in Antimicrobial Peptides of the Magainin Family

Magainin 2 and PGLa are among the best-studied cationic antimicrobial peptides. They bind preferentially to negatively charged membranes and apparently cause their disruption by the formation of transmembrane pores, whose detailed structure is still unclear. Here we report the results of 5–9 μs all-atom molecular dynamics simulations starting from tetrameric transmembrane helical bundles of these two peptides, as well as their stoichiometric mixture, and the analog MG-H2 in DMPC or 3:1 DMPC/DMPG membranes. The simulations produce pore structures that appear converged, although some effect of the starting peptide arrangement (parallel vs. antiparallel) is still observed on this timescale. The peptides remain mostly helical and adopt tilted orientations. The calculated tilt angles for PGLa are in excellent agreement with recent solid state NMR experiments. The antiparallel dimer structure in the magainin 2 simulations resembles previously determined NMR and crystal structures. More transmembrane orientations and a larger and more ordered pore are seen in the 1:1 heterotetramer with an antiparallel helix arrangement. Insights into the mechanism of synergy between these two peptides are obtained via implicit solvent modeling of homo- and heterodimers and analysis of interactions in the atomistic simulations. This analysis suggests stronger pairwise interactions in the heterodimer than in the two homodimers.     

A recurrent magnesium-binding motif provides a framework for the ribosomal peptidyl transferase center

The ribosome is an ancient macromolecular machine responsible for the synthesis of all proteins in all living organisms. Here we demonstrate that the ribosomal peptidyl transferase center (PTC) is supported by a framework of magnesium microclusters (Mg²⁺-μc's). Common features of Mg²⁺-μc's include two paired Mg²⁺ ions that are chelated by a common bridging phosphate group in the form Mg_(a)²⁺–(O1P-P-O2P)–Mg_(b)²⁺. This bridging phosphate is part of a 10-membered chelation ring in the form Mg_(a)²⁺–(OP-P-O5′-C5′-C4′-C3′-O3′-P-OP)–Mg_(a)²⁺. The two phosphate groups of this 10-membered ring are contributed by adjacent residues along the RNA backbone. Both Mg²⁺ ions are octahedrally coordinated, but are substantially dehydrated by interactions with additional RNA phosphate groups. The Mg²⁺-μc's in the LSU (large subunit) appear to be highly conserved over evolution, since they are unchanged in bacteria (Thermus thermophilus, PDB entry 2J01) and archaea (Haloarcula marismortui, PDB entry 1JJ2). The 2D elements of the 23S rRNA that are linked by Mg²⁺-μc's are conserved between the rRNAs of bacteria, archaea and eukarya and in mitochondrial rRNA, and in a proposed minimal 23S-rRNA. We observe Mg²⁺-μc's in other rRNAs including the bacterial 16S rRNA, and the P4–P6 domain of the tetrahymena Group I intron ribozyme. It appears that Mg²⁺-μc's are a primeval motif, with pivotal roles in RNA folding, function and evolution.     

How Many Processed Pseudogenes Are Accumulated in a Gene Family?

A simple kinetic model is developed that describes the accumulation of processed pseudogenes in a functional gene family. Insertion of new pseudogenes occurs at rate ν per gene and is countered by spontaneous deletion (at rate δ per DNA segment) of segments containing processed pseudogenes. If there are k functional genes in a gene family, the equilibrium number of processed pseudogenes is k(ν/δ), and the percentage of functional genes in the gene family at equilibrium is 1/[1 + (ν/δ)]. ν/δ values estimated for five gene families ranged from 1.7 to 15. This fairly narrow range suggests that the rates of formation and deletion of processed pseudogenes may be positively correlated for these families. If δ is sufficiently large relative to the per nucleotide mutation rate µ (δ > 20µ), processed pseudogenes will show high homology with each other, even in the absence of gene conversion between pseudogenes. We argue that formation of processed pseudogenes may share common pathways with transposable elements and retroviruses, creating the potential for correlated responses in the evolution of processed pseudogenes due to direct selection for control of transposable elements and/or retroviruses. Finally, we discuss the nature of the selective forces that may act directly or indirectly to influence the evolution of processed pseudogenes.

Anything produced by evolution is bound to be a bit of a mess—S. Brenner

     

Raman dispersion spectroscopy probes heme distortions in deoxyHb-trout IV involved in its T-state Bohr effect

The depolarization ratios of heme protein Raman lines arising from vibrations of the heme group exhibit significant dependence on the excitation wavelength. From the analysis of this depolarization ratio dispersion, one obtains information about symmetry-lowering distortions δQ^Γ of the heme group that can be classified in terms of the symmetry races Γ = A_1g, B_1g, B_2g, and A_2g in D_4h symmetry. The heme-protein interaction can be changed by the protonation of distinct amino acid side chains (i.e., for instance the Bohr groups in hemoglobin derivates), which gives rise to specific static heme distortions for each protonation state. From the Raman dispersion data, it is possible to obtain parameters by fitting to a theoretical expression of the Raman tensor, which provide information on these static distortions and also about the pK values of the involved titrable side chains. We have applied this method to the ν₄ (1,355 cm^-1) and ν₁₀ (1,620 cm^-1) lines of deoxygenated hemoglobin of the fourth component of trout and have measured their depolarization ratio dispersion as a function of pH between 6 and 9. From the pH dependence of the thus derived parameters, we obtain pK values identical to those of the Bohr groups, which were earlier derived from the corresponding O₂-binding isotherms. These are pK_α1 = pK_α2 = 8.5 for the α and pK_β1 = 7.5, pK_β2 = 7.4 for the β chains. We also obtain the specific distortion parameters for each protonation state. As shown in earlier studies, the ν₄ mode mainly probes distortions from interactions between the proximal histidine and atoms of the heme core (i.e., the nitrogens and the C_α atoms of the pyrroles). Group theoretical argumentation allows us to relate specific changes of the imidazole geometry as determined by its tilt and azimuthal angle and the iron-out-of-plane displacement to distinct variations of the normal distortions δQ^Γ derived from the Raman dispersion data. Thus, we found that the pH dependence of the heme distortions δQ^A1g (totally symmetric) and δQ^B1g (asymmetric) is caused by variations of the azimuthal rather than the tilt angle of the Fe-His (F8) bond. In contrast to this, the ν₁₀ line mainly monitors changes resulting from the interaction between peripheral substituents of the porphyrin macrocycle (vinyl). From the pH dependence of the parameters, it is possible to separately identify distortions δQ^Γ affecting the hemes in the α and β chains, respectively. From this, we find that in the α subunit structural changes induced on protonation of the corresponding Bohr groups are mainly transferred via the Fe—N_ε bond and give rise to changes in the azimuthal angle. In the β subunit, however, in addition, structural changes of the heme pocket arise, which most probably result from protonation of the imidazole of the COOH-terminal His (HC3 β). This rearranges the net of H bonds between His HC3 β, Ser (F9 β), and Glu (F7 β).     

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.     

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).