Maturation of McjA precursor peptide into active microcin MccJ25

Microcin J25 is a ribosomally synthesised 21-residue antimicrobial peptide produced by certain strains of enterobacteria, that adopts an extraordinary 'threaded lasso' structure. To date, the biosynthesis of this peptide is little understood. Here we report the in vitro maturation of the microcin precursor peptide into active microcin J25 for the first time. Furthermore, we show that the enzymes required for the posttranslational modification of this precursor peptide are associated with the bacterial inner membrane.     

Penetration of surfactin into phospholipid monolayers: nanoscale interfacial organization

Atomic force microscopy (AFM) combined with surface pressure-area isotherms were used to probe the interfacial behavior of phospholipid monolayers following penetration of surfactin, a cyclic lipopeptide produced by Bacillus subtilis strains. Prior to penetration experiments, interfacial behavior of different surfactin molecules (cyclic surfactins with three different aliphatic chain lengths--S13, S14, and S15--and a linear surfactin obtained by chemical cleavage of the cycle of the surfactin S15) has been investigated. A more hydrophobic aliphatic chain induces greater surface-active properties of the lipopeptide. The opening of the peptide ring reduces the surface activity. The effect of phospholipid acyl chain length (dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine- (DPPC), and distearoylphosphatidylcholine) and phospholipid polar head (DPPC, dipalmitoylphosphatidylethanolamine and dipalmitoylphosphatidylserine) on monolayer penetration properties of the surfactin S15 has been explored. Results showed that while the lipid monolayer thickness and the presence of electrostatic repulsions from the interfacial film do not significantly influence surfactin insertion, these parameters strongly modulate the ability of the surfactin to alter the nanoscale organization of the lipid films. We also probed the effect of surfactin structure (influence of the aliphatic chain length and of the cyclic structure of the peptide ring) on the behavior of DPPC monolayers. AFM images and isotherms showed that surfactin penetration is promoted by longer lipopeptide chain length and a cyclic polar head. This indicates that hydrophobic interactions are of main importance for the penetration power of surfactin molecules.     

Structure of microcin J25, a peptide inhibitor of bacterial RNA polymerase, is a lassoed tail

Microcin J25 (MccJ25) is a 21-amino acid peptide inhibitor active against the DNA-dependent RNA polymerase of Gram negative bacteria. Previously, the structure of MccJ25 was reported to be a head-to-tail circle, cyclo(-G(1)GAGHVPEYF(10)VGIGTPISFY(20)G-). On the basis of biochemical studies, mass spectrometry, and NMR, we show that this structure is incorrect, and that the peptide has an extraordinary structural fold. MccJ25 contains an internal lactam linkage between the alpha-amino group of Gly1 and the gamma-carboxyl of Glu8. The tail (Tyr9-Gly21) passes through the ring (Gly1-Glu8), with Phe19 and Tyr20 straddling each side of the ring, sterically trapping the tail in a noncovalent interaction we call a lassoed tail.     

Penetration of beta-lactoglobulin into monoglyceride monolayers. dynamics, interactions, and topography of mixed films

In this work we have analyzed the penetration of betalactoglobulin into a monoglyceride monolayer (monopalmitin or monoolein) spread at the air-water interface and its effects on the structural, dilatational, and topographical characteristics of mixed films. Dynamic tensiometry, surface film balance, Brewster angle microscopy (BAM), and surface dilatational rheology have been used, maintaining the temperature constant at 20 degrees C and the pH and ionic strength at 7 and 0.05 M, respectively. The initial surface pressure (mN/m) of the spread monoglyceride monolayer (pii(MONOGLYCERIDE)) at 10, 20, and the collapse point is the variable studied. Beta-lactoglobulin can penetrate into a spread monoglyceride monolayer at every surface pressure. The penetration of beta-lactoglobulin into the monoglyceride monolayer with a more condensed structure, at the collapse point of the monoglyceride, requires monoglyceride molecular loss by collapse and/or desorption. However, the structural, topographical, and dilatational characteristics of monoglyceride penetrated by beta-lactoglobulin mixed monolayers are essentially dominated by the presence of monoglyceride (either monopalmitin or monoolein) in the mixed film. In fact, monoglyceride molecules have the capacity to re-enter the monolayer after expansion and recompression of the mixed monolayer. Thus, monoglyceride molecular loss by collapse and/or desorption is reversible. The topography of the monolayer under dynamic conditions corroborates these conclusions.     

Penetration of a GPI-anchored protein into phospholipid monolayers spread at the air/water interface

Mammalian alkaline phosphatases (AP) belong to glycosylphosphatidyl inositol (GPI) anchored proteins family, which are localised and clustered on the outer layer of the plasma membranes forming microdomains. Using Langmuir film and polarisation modulation infrared reflection absorption spectroscopy (PMIRRAS) techniques, the penetration process of the protein into a phospholipid monolayer have been studied at the air–buffer interface. The penetration of AP-GPI in distearoylphosphatidylcholine monolayers (DSPC) induces a more important surface pressure increase than in dioleoylphosphatidylcholine (DOPC) monolayer. However, the exclusion surface pressure rather similar for both lipids, 20.5 and 22 mN m⁻¹ for, respectively, DSPC and DOPC, indicates that the AP-GPI cannot, in similar conditions, insert by itself into bilayer membranes of either biological or mimetic origin. PMIRRAS suggests that the pure acyl chains perdeuterated DSPC (d₇₀-DSPC) interact with Mg²⁺ present into the buffer. AP-GPI inserts progressively into the d₇₀-DSPC monolayer changing the environment of phospholipid molecules. Amide I band exhibits helix and β-sheets components with a predominance of the helix. The shapes, intensities and positions of the amide I and II bands suggest for the helix an orientation perpendicular to the interface after a period of molecular reorganisation.     

Penetration of mercury-adsorbed phospholipid monolayers by polynuclear aromatic hydrocarbons

The penetration of phospholipid monolayers (dioleoyl lecithin) adsorbed on mercury by polynuclear aromatic hydrocarbons (PAH) is described. The PAH studied were anthracene, phenanthrene, pyrene, benzo[a]anthracene, fluoranthene and perylene. The penetration is monitored by measuring the differential capacitance of the monolayer; the uptake of PAH causes a potential shift (up to ?0.25 V) in the cathodic capacitance peaks. This occurs without displacement of the lipid from the mercury. The differential capacitance is measured by out-of-phase (90°) a.c. voltammetry and rapid cyclic voltammetry. The PAH permeate the mercury-adsorbed lipid layers from dilute aqueous solution; the order of affinity is benzo[a]anthracene > fluoranthrene = pyrene > anthracene = phenanthrene. The rates of penetration vary for the different compounds and depend on their water solubility.     

Study of the diclofenac/phospholipid interactions with liposomes and monolayers

The interaction of diclofenac sodium (SD) with soya phosphatidylcholine (SPC) has been studied with floating Langmuir monolayers and liposomes. SD was either introduced into the subphase of SPC monolayers or co-spread with SPC on an aqueous subphase. In both cases, SD caused the surface pressure isotherm to become more expanded, thus demonstrating the affinity between SD and SPC. The incorporation of SD caused SPC liposomes to have a decreased diameter according to light scattering experiments. When SPC liposomes were injected into an aqueous subphase, their destruction yielding surface-active monomers could be monitored by changes in surface pressure. SD-loaded liposomes displayed a much faster kinetics when the surface density of surface-active monomers was plotted against time, with rate constants increasing significantly with the SD concentration. The kinetic profile can be quantitatively analyzed by plotting ln[1 - (gamma/gamma infinity)] versus t1/2.     

Sequence diversity in the lasso peptide framework: discovery of functional microcin J25 variants with multiple amino acid substitutions

Microcin J25 (MccJ25) is a ribosomally synthesized antimicrobial peptide that has an unusual threaded lasso structure in which the C-terminal "tail" of the peptide is fed through a macrocyclic "ring" formed by the N-terminal residues. Production of MccJ25 in Escherichia coli is dependent upon a four-gene cluster encoding the structural gene mcjA, two maturation enzymes mcjB and mcjC, and an immunity factor, mcjD, in the form of an MccJ25 export pump. Here we have developed a system for orthogonal control of the expression of mcjA and mcjD, thus permitting independent control of MccJ25 production and export/immunity in E. coli. We used this system to screen saturation mutagenesis libraries targeted to either the ring or tail portions of MccJ25 and discovered nearly 100 new MccJ25 variants that retain antimicrobial function. While multiple amino acid substitutions in the tail portion of the peptide are well-tolerated, mutagenesis of the ring portion of the peptide is detrimental to the antimicrobial function of MccJ25. We demonstrated that the decreased function of the ring variants is due to the inability of these variants to be transported to the cytoplasm of susceptible strains. Additionally, we found several MccJ25 variants from the tail library with improved efficacy toward the MccJ25-sensitive strains E. coli and Salmonella enterica serovar Newport with the best variants exhibiting a nearly 5-fold increase in potency. The results described here provide further evidence that diverse amino acid sequences can be tolerated by the rigid lasso peptide fold.     

Penetration behaviour of alkylbetainate chlorides into lipid monolayers

In this paper, the penetration behaviour of the alkylbetainate chloride surfactants (C(n)BC, n=10-16) into lipid monolayers of dipalmitoylphosphatidylserine (DPPS), dipalmitoylphosphatidic acid (DPPA), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyoleoylphosphatidylcholine (POPC) and cholesterol (CHOL) is investigated using the Langmuir trough technique. The penetration of C(n)BC is followed by measurement of the surface pressure increase (Δπ) at a constant surface area after the injection of C(n)BC into the aqueous phase, underneath the lipid monolayer previously spread at the air-water interface at 25°C and at different initial surface pressures (π(i)). The influence of both the lipid head group and the surfactant hydrocarbon chain length on the effectiveness of C(n)BC penetration into these monolayers is discussed. The results have shown that C(n)BC adsorb at the air-water interface giving evidence of their surface-active properties. The adsorption kinetics of C16BC into different lipid monolayers are lipid head charge and lipid head volume-dependent. The magnitude of the surface pressure increase (Δπ) arises in the following order: DPPA>DPPS?CHOL≈DPPE>POPC. C(n)BC penetration into negatively-charged (DPPS and DPPA) monolayers does not seem to depend on surfactant alkyl-chain length compared to uncharged (CHOL) and zwitterionic (DPPE and POPC) monolayers for which Δπ increases with a larger alkyl-chain length. Electrostatic interactions are mainly involved in the affinity of C(n)BC with monolayers but the hydrophobic effect plays also a role.     

Penetration of dissolved amphiphiles into two-dimensional aggregating lipid monolayers

The review demonstrates the recent theoretical and experimental progress in the understanding of penetration systems at the air-water interface in which a dissolved amphiphile (surfactant, protein) penetrates into a Langmuir monolayer. The critical review of the existing theoretical models which describe the thermodynamics of the penetration are critically reviewed. Although a rigorous thermodynamic analysis of penetration systems is unavailable due to their complexity, some model assumptions, e.g. the invariability of the activity coefficient of the insoluble component of the monolayer during the penetration of the soluble component results in reasonable solutions. New theoretical models describing the equilibrium behaviour of the insoluble monolayers which undergo the 2D aggregation in the monolayer, and the equations of state and adsorption isotherms which assume the existence of multiple states (conformations) of a protein molecule within the monolayer and the non-ideality of the adsorbed monolayers are now available. The theories which describe the penetration of a soluble surfactant into the main phases of Langmuir monolayers were presented first for the case of the mixture of the molecules possessing equal partial molar surfaces (the mixture of homologues), with further extension of the models to include the interesting process of the protein penetration into the monolayer of 2D aggregating phospholipid. This extension was based on a concept which subdivides the protein molecules into independent fragments with areas equal to those of the phospholipid molecule. Various mechanisms for the effect of the soluble surfactant on the aggregation of the insoluble component were considered in the theoretical models: (i) no effect on the aggregate formation process; (ii) formation of mixed aggregates; and (iii) the influence on the aggregating process via the change of aggregation constant, but without any formation of mixed aggregates. Accordingly depending on the mechanism, different forms of the equations of state of the monolayer and of the adsorption isotherms of soluble surfactant are predicted. Based on the shape of the experimental pi-A isotherms, interesting conclusions can be drawn on the real mechanism. First experimental evidence has been provided that the penetration of different proteins and surfactants into a DPPC monolayer in a fluid-like state induces a first order main phase transition of pure DPPC. The phase transition is indicated by a break point in the pi(t) penetration kinetics curves and the domain formation by BAM. Mixed aggregates of protein with phospholipid are not formed. These results agree satisfactorily with the predictions of the theoretical models. New information on phase transition and phase properties of Langmuir monolayers penetrated by soluble amphiphiles are obtained by coupling of the pi(t) penetration kinetics curves with BAM and GIXD measurements. The GIXD results on the penetration of beta-lactoglobulin into DPPC monolayers have shown that protein penetration occurs without any specific interactions with the DPPC molecules and the condensed phase consists only of DPPC.     

Study of adsorption and penetration of E2(279-298) peptide into Langmuir phospholipid monolayers

The peptide corresponding to the sequence (279-298) of the Hepatitis G virus (HGV/GBV-C) E2 protein was synthesized, and surface activity measurements, pi-A compression isotherms, and penetration of E2(279-298) into phospholipid monolayers spread at the air-water interface were carried out on water and phosphate buffer subphases. The results obtained indicated that the pure E2(279-298) Langmuir monolayer exhibited a looser packing on saline-buffered than on pure water subphase and suggest that the increase in subphase ionic strength stabilizes the peptide monolayer. To better understand the topography of the monolayer, Brewster angle microscopy (BAM) images of pure peptide monolayers were obtained. Penetration of the peptide into the pure lipid monolayers of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) and into mixtures of dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) at various initial surface pressures was investigated to determine the ability of these lipid monolayers to host the peptide. The higher penetration of peptide into phospholipids is attained when the monolayers are in the liquid expanded state, and the greater interaction is observed with DMPC. Furthermore, the penetration of the peptide dissolved in the subphase into these various lipid monolayers was investigated to understand the interactions between the peptide and the lipid at the air-water interface. The results obtained showed that the lipid acyl chain length is an important parameter to be taken into consideration in the study of peptide-lipid interactions.     

Interaction of the cationic peptide bactenecin with mixed phospholipid monolayers at the air-water interface

The initial mechanism by which antimicrobial peptides target microbes occurs via electrostatic interactions; however, the mechanism is not well understood. We investigate the interaction of the antimicrobial peptide bactenecin with a 50:50 w:w% 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG) phospholipid mixture at the air-water interface with different NaCl concentrations (0.01, 0.05, 0.1, 0.5 M) in the subphase. A larger shift of DPPC:DMPG isotherms was obtained for 0.1 M salt concentration at lower and higher pressures, demonstrating the influence of the negative charge of DMPG molecules and the screening of the electrostatic interaction by the salt concentration. Raman spectroscopy of monolayers demonstrated the presence of cysteine-cysteine bridges in bactenecin loops. The peptide adsorption in DPPC:DMPG monolayers observed by AFM images suggests a self-assembled aggregation process, starting with filament-like networks. Domains similar to carpets were formed and pore structures were obtained after a critical peptide concentration, according to the carpet model.     

Penetration of sodium cetylsulfate into monolayers of 1,2-dipalmitoyl- and 1,2-dimyristoyl-phosphatidylethanolamine

The penetration of sodium cetylsulfate into monolayers of dipalmitoyl- and dimyristoyl-phosphatidylethanolamine was studied by the measurement of surface and penetration pressures using the vertical plate method of Wilhelmy. The penetration isotherms in two systems were investigated at different initial molecular areasA _M :System I: Sodium cetylsulfate/1,2-dipalmitoyl-phosphatidylethanolamine atA _M = 0.85; 0.75; 0.65; 0.55; 0.50; 0.46 and 0.44 nm² · molecule^–1.System II: Sodium cetylsulfate/1,2-dimyristoyl-phosphatidylethanolamine atA _M = 0.85; 0.75; 0.60 and 0.55 nm² · molecule^–1.(T=295 K; substrate 0.1 M NaCl)The penetration isotherms (F _t vs. logc _s ) increase linearly atF _t > 10 mN · m^–1 in system I and atF _t >25 mN · m^–1 in system II. The isotherms of both systems are shifted to lower surfactant concentrations with decreasing molecular area of spread monolayer. A maximum of the slopes (dF_t/d logc _s )occurs at A_M=0.50 nm² · molecule^–1. This behavior is also reflected in the dependenceG _p ⁰ (free standard penetration enthalpy) and _s (relative surface excess concentration of surfactant) onA _M . These changes are related to a different packing of the compounds in the binary penetrated monolayers.In the high pressure region both system are nearly identical. Differences in the low pressure region arise from the penetration into different monolayer states.Nomenclature _M effective cross sectional area of monolayer molecule - a _M partial molecular area of monolayer molecule - a _s partial molecular area of surfactant molecule in the penetrated film - a _s ⁰ molecular area of surfactant molecule at definite film pressure (eq. (3)) - A _M molecular area of theF/A-isotherm - A _N constant in equation (2) - A _K collapse area - b penetration coefficient in equations (2); (2 a) - c _s bulk concentration of surfactant - logc _s relative shift of penetration isotherm with regard to the adsorption isotherm at constantF _t - F film pressure of monolayer component in absence of surfactant - F _t total film pressure - F _p film pressure change due to penetration - F _p,max constant in equation (1) - G _p ⁰ free standard penetration energy - k Boltzmann constant - K constant in equation (1) - R gas constant - T temperature - x _s ⁰ mole fraction of surfactant in the penetrated film - _M surface concentration of monolayer molecules - _s relative adsorption of surfactant - _w ⁰ surface concentration of surfactant in monolayer-free surface - factor in equation (6 a) - surface tension     

Chain dependence in phospholipid interactions: A thermodynamic study of mixed monolayers

 The interactions between three of the major phospholipids in biomembranes, distearoylphos-phatidylethanolamine, distearoylphosphatidylcholine and sphyngomyelin, forming monolayers at the air–water interface, are studied. Following the Goodrich [1] thermo-dynamic formulation, a quantitative analysis on these interactions is carried out. The general conclusion reached is that, depending on the molecular structure of the lipid and the experimental temperature, significant interactions occur between lipid molecules. One hypothesis is the possibility that, the usual miscibility analysis, in mixed monolayers, could be inadequate to provide information on the interactions in the systems which the hydration forces are significant, is advanced. Received: 30 May 1997 Accepted: 5 October 1997     

Adsorption of a phospholipid-hydroperoxide glutathione peroxidase into phospholipid monolayers at the air-water interface

The interfacial behavior differences of two glutathione peroxidase isoforms have been investigated. The first isoform is the phospholipid-hydroperoxide glutathione peroxidase (EC 1.11.1.12) (GPx-4) isolated from rat testes and the second one is the cytosolic glutathione peroxidase (EC 1.11.1.9) (GPx-1) from bovine erythrocytes. Injected in the subphase buffer of a Langmuir trough, GPx-4 was able to adsorb quickly at the air-water interface whereas the GPx-1 was not. Then, the protein interaction with phospholipid monolayers was explored. Indeed, a monolayer of phospholipids containing a different number of polyunsaturated fatty acyl chains was prepared at the air-water interface. Under each kind of monolayer, the protein solution was injected and its adsorption was visualized by the measurement of successive pressure-area isotherms. We have, then, determined the molecular area increase due to the protein adsorption. It was found that the GPx-4 is adsorbed in each kind of monolayer tested whereas no molecular area increase was detected with the GPx-1. This indicates that the GPx-4 has a higher affinity for the interface, recovered or not by lipids, than the GPx-1. Moreover, the GPx-4 presents a different affinity for the phospholipid monolayers depending on the number of polyunsaturated fatty acyl chains.     

Phase behavior of mixed Langmuir monolayers from amphiphilic block copolymers and an antimicrobial peptide

The behavior of binary monolayers from PMOXA-PDMS-PMOXA triblock copolymers and alamethicin, an antimicrobial peptide, was investigated in the context of formation of novel biocomposite nanostructured materials. The properties of mixed monolayers were studied by surface pressure-area isotherms and Brewster angle imaging. As reported previously, functionality of alamethicin relies on its aggregation properties in lipid mono- and bilayers. This is also the case in polymer matrixes, however, here the mixing properties differ from lipid-peptide systems due to the polymers' structural specificity. The peptide influence on the polymer films is provided in detail for the first time, and supported by the compressibility data to asses the elastic properties of such composite membranes.     

Aggregation of a peptide antibiotic alamethicin at the air-water interface and its influence on the viscoelasticity of phospholipid monolayers

The aggregation properties of an antibiotic membrane-active peptide alamethicin at the air-water interface have been studied using interfacial rheology and fluorescence microscopy techniques. Fluorescence microscopy of alamethicin monolayers revealed a coexistence of liquid expanded (LE) and solid phases at the surface concentrations studied. Interfacial oscillatory shear measurements on alamethicin monolayers indicate that its viscoelastic properties are determined by the area fraction of the solid domains. The role of zwitterionic phospholipids dioleoylphosphatidyl choline (DOPC) and dioleoylphosphatidyl ethanolamine (DOPE) on the peptide aggregation behavior was also investigated. Fluorescence microscopy of alamethicin/phospholipid monolayers revealed an intermediate phase (I) in addition to the solid and LE phase. In mixed monolayers of phospholipid (L)/alamethicin (P), with increase in L/P, the monolayer transforms from a viscoelastic to a viscous fluid with the increase in area fraction of the intermediate phase. Further, a homogeneous mixing of alamethicin/lipid molecules is observed at L/P > 4. Our studies also confirm that the viscoelasticity of alamethicin/phospholipid monolayers is closely related to the alamethicin/phospholipid interactions at the air-water interface.     

Sequence, structure, and function of peptide self-assembled monolayers

Cysteine is commonly used to attach peptides onto gold surfaces. Here we show that the inclusion of an additional linker with a length of four residues (-PPPPC) and a rigid, hydrophobic nature is a better choice for forming peptide self-assembled monolayers (SAMs) with a well-ordered structure and high surface density. We compared the structure and function of the nonfouling peptide EKEKEKE-PPPPC-Am with EKEKEKE-C-Am. Circular dichroism, attenuated total internal reflection Fourier transform IR spectroscopy, and molecular dynamics results showed that EKEKEKE-PPPPC-Am forms a secondary structure while EKEKEKE-C-Am has a random structure. Surface plasmon resonance sensor results showed that protein adsorption on EKEKEKE-PPPPC-Am/gold is very low with small variation while protein adsorption on EKEKEKE-C-Am/gold is high with large variation. X-ray photoelectron spectroscopy results showed that both peptides have strong gold-thiol binding with the gold surface, indicating that their difference in protein adsorption is due to their assembled structures. Further experimental and simulation studies were performed to show that -PPPPC is a better linker than -PC, -PPC, and -PPPC. Finally, we extended EKEKEKE-PPPPC-Am with the cell-binding sequence RGD and demonstrated control over specific versus nonspecific cell adhesion without using poly(ethylene glycol). Adding a functional peptide to the nonfouling EK sequence avoids complex chemistries that are used for its connection to synthetic materials.