Expression, purification and structural studies of a short antimicrobial peptide

We have produced a small antimicrobial peptide PFWRIRIRR in bacteria utilizing production in the form of insoluble fusion protein with ketosteroid isomerase. The recombinant peptide was rapidly and efficiently isolated by acidic cleavage of the fusion protein based on the acid labile Asp-Pro bond at the N-terminus of the peptide. The peptide has antibacterial activity and neutralizes macrophage activation by LPS. The selectivity of the peptide against bacteria correlates with preferential binding to acidic phospholipid vesicles. Solution structure of the peptide in SDS and DPC micelles was determined by NMR. The peptide adopts a well-defined structure, comprising a short helical segment. Cationic and hydrophobic clusters are segregated along the molecular axis of the short helix, which is positioned perpendicular to the membrane plane. The position of the helix is shifted in two micellar types and more nonpolar surface is exposed in anionic micelles. Overall structure explains the advantageous role of the N-terminal proline residue, which forms an integral part of the hydrophobic cluster.     

Design and mechanism of action of a novel bacteria-selective antimicrobial peptide from the cell-penetrating peptide Pep-1

Here, we report the successful design of a novel bacteria-selective antimicrobial peptide, Pep-1-K (KKTWWKTWWTKWSQPKKKRKV). Pep-1-K was designed by replacing Glu-2, Glu-6, and Glu-11 in the cell-penetrating peptide Pep-1 with Lys. Pep-1-K showed strong antibacterial activity against reference strains (MIC = 1-2 microM) of Gram-positive and Gram-negative bacteria as well as against clinical isolates (MIC = 1-8 microM) of methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa. In contrast, Pep-1-K did not cause hemolysis of human erythrocytes even at 200 microM. These results indicate that Pep-1-K may be a good candidate for antimicrobial drug development, especially as a topical agent against antibiotic-resistant microorganisms. Tryptophan fluorescence studies indicated that the lack of hemolytic activity of Pep-1-K correlated with its weak ability to penetrate zwitterionic phosphatidylcholine/cholesterol (10:1, w/w) vesicles, which mimic eukaryotic membranes. Furthermore, Pep-1-K caused little or no dye leakage from negatively charged phosphatidylethanolamine/phosphatidylglycerol (7:3, w/w) vesicles, which mimic bacterial membranes but had a potent ability to cause depolarization of the cytoplasmic membrane potential of intact S. aureus cells. These results suggested that Pep-1-K kills microorganisms by not the membrane-disrupting mode but the formation of small channels that permit transit of ions or protons but not molecules as large as calcein.     

The interaction of the antimicrobial peptide cLEAP-2 and the bacterial membrane

Chicken Liver Expressed Antimicrobial Peptide-2 (cLEAP-2) is known to have killing activities against Salmonella spp., but the mechanism by which killing occurs remains to be elucidated. The ability of cLEAP-2 to disrupt the outer membrane of several Salmonella spp. was assessed using the fluorescent probe N-phenyl-1-naphthylamine (NPN). A rapid dose-dependent permeabilization of the outer membranes of Salmonella enterica serovar Typhimurium phoP, and S. enterica serovar Typhimurium SL1344 was observed although no significant permeabilisation of the S. enteriditis membrane was detected. These data suggested that the ability of the mature cLEAP-2 peptide to permeabilise the Salmonella outer membrane is important in mediating its killing activities. The ability of the peptide to kill Gram-positive bacteria, specifically Streptococcus spp. and Staphylococcus spp. was also investigated using recombinant peptide and a time-kill assay. Of the strains analysed the Streptococcus pyogenes M1 strain appeared the most resistant to LEAP-2 killing although S. pyogenes mutants deficient in Sortase and M1 activities showed increased sensitivity to the mature peptide. This suggested the involvement of specific Streptococcus cell wall proteins including M1 in the resistance of the bacteria to cLEAP-2 killing. cLEAP-2 showed no significant toxicity towards mammalian erythrocytes indicating selectivity for bacterial over eukaryote cell membranes. These data provide further support for mature cLEAP-2 functioning in protecting the chicken against microbial attack.     

Structure, membrane orientation, mechanism, and function of pexiganan — A highly potent antimicrobial peptide designed from magainin

The growing problem of bacterial resistance to conventional antibiotic compounds and the need for new antibiotics have stimulated interest in the development of antimicrobial peptides (AMPs) as human therapeutics. Development of topically applied agents, such as pexiganan (also known as MSI-78, an analog of the naturally occurring magainin2, extracted from the skin of the African frog Xenopus laevis) has been the focus of pharmaceutical development largely because of the relative safety of topical therapy and the uncertainty surrounding the long-term toxicology of any new class of drug administered systemically. The main hurdle that has hindered the development of antimicrobial peptides is that many of the naturally occurring peptides (such as magainin), although active in vitro, are effective in animal models of infection only at very high doses, often close to the toxic doses of the peptide, reflecting an unacceptable margin of safety. Though MSI-78 did not pass the FDA approval, it is still the best-studied AMP to date for therapeutic purposes. Biophysical studies have shown that this peptide is unstructured in solution, forms an antiparallel dimer of amphipathic helices upon binding to the membrane, and disrupts membrane via toroidal-type pore formation. This article covers functional, biophysical, biochemical and structural studies on pexiganan.     

Fluorescence anisotropy analysis of the mechanism of action of mesenterocin 52A: speculations on antimicrobial mechanism

Mesenterocin 52A (Mes 52A) is a class IIa bacteriocin produced by Leuconostoc mesenteroides subsp. mesenteroides FR52, active against Listeria sp. The interaction of Mes 52A with bacterial membranes of two sensitive Listeria strains has been investigated. The Microbial Adhesion to Solvents test used to study the physico-chemical properties of the surface of the two strains indicated that both surfaces were rather hydrophilic and bipolar. The degree of insertion of Mes 52A in phospholipid bilayer was studied by fluorescence anisotropy measurements using two probes, 1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and DPH, located at different positions in the membrane. TMA-DPH reflects the fluidity at the membrane surface and DPH of the heart. With Listeria ivanovii CIP 12510, Mes 52A induced an increase only in the TMA-DPH fluorescence anisotropy, indicating that this bacteriocin affects the membrane surface without penetration into the hydrophobic core of the membrane. No significant K⁺ efflux was measured, whereas the ΔΨ component of the membrane potential was greatly affected. With Listeria innocua CIP 12511, Mes 52A caused an increase in the fluorescence of TMA-DPH and DPH, indicating that this peptide inserts deeply in the cytoplasmic membrane of this sensitive strain. This insertion led to K⁺ efflux, without perturbation of ΔpH and a weak modification of ΔΨ, and is consistent with pore formation. These data indicate that Mes 52A interacts at different positions of the membrane, with or without pore formation, suggesting two different mechanisms of action for Mes 52A depending on the target strain.     

Role of non-electrostatic forces in antimicrobial potency of a dengue-virus derived fusion peptide VG16KRKP: Mechanistic insight into the interfacial peptide-lipid interactions

Cationic antimicrobial peptides (AMPs) are emerging as effective alternatives to conventional therapeutics that are used against the ever-rising number of multidrug-resistant microbial strains. Most studies established the peptide's amphipathicity and electrostatic interaction with the membrane as the basis for their antimicrobial effect. However, the interplay between the stoichiometric ratio of lipids, local geometry, diverse physicochemical properties of the host membranes and antimicrobial peptide efficacy is still poorly understood. In the present study, we investigate the mechanism of interaction of VG16KRKP (VARGWKRKCPLFGKGG), a novel AMP designed from the dengue-virus fusion peptide, with bacterial/fungal membrane mimics. Fluorescence based dye leakage assays show that membrane disruption is not solely induced by electrostatic interaction but also driven by stoichiometric ratio of the lipids that dictates the net surface charge, amount of lipid defects and local geometry of the membrane. Solid-state ¹⁴N and ³¹P NMR experiments show that peptide interaction results in lowering of lipid order around both the headgroups and acyl chains, suggesting deep peptide insertion. Further, an increase or decrease in membrane stability of the host membrane was observed in differential scanning calorimetry (DSC) thermograms, dictated by the overall stoichiometric ratio of the lipids and the sterol present. In general, our results help understand the diverse fates of host membranes against an antimicrobial peptide.     

Antibiotic activity and structural analysis of the scorpion-derived antimicrobial peptide IsCT and its analogs

IsCT is a non-cell-selective antimicrobial peptide isolated from the scorpion Opisthacanthus madagascariensis that has potent cytolytic activity against both mammalian and bacterial cells. To investigate the structure-activity relationships of IsCT and to design novel peptide antibiotics with bacterial cell selectivity, we synthesized several analogs of IsCT and determined their three-dimensional structures in solution by 2D-NMR spectroscopy. IsCT has a linear alpha-helical structure from Gly3 to Phe13, and [K7]-IsCT has a linear alpha-helical structure from Leu2 to Phe13. [K7, P8, K11]-IsCT, which has a bend in its middle region, exhibited the highest antibacterial activity without hemolytic activity, suggesting that its proline-induced bend is an important determinant of this selectivity. Tryptophan fluorescence showed that the high selectivity of [K7, P8, K11]-IsCT toward bacterial cells is closely correlated with its highly selective interaction with negatively charged phospholipids. Its potent activity against antibiotic-resistant bacteria suggests that [K7, P8, K11]-IsCT may serve as a promising lead candidate in the development of new peptide antibiotics.     

Conformational studies of alanine-rich peptide using CD and FTIR spectroscopy.

The circular dichroism (CD) and Fourier transform infrared (FTIR) methods were applied to the conformational studies of alanine-rich peptide Ac-K-[A]11-KGGY-NH2 (where K is lysine, A is alanine, G is glycine and Y is tyrozyne) in water, methanol (MeOH) and trifluoroethanol (TFE). The analysis of CD-spectra of the peptide in water at different concentrations revealed that the secondary structure content depends on the peptide concentration and pH of the solution. The increase of the peptide concentration causes a decrease of alpha-helix content and, simultaneously, an increase of beta-sheet structure, while the unordered structure is the predominant one. Additional elements are discovered in MeOH and TFE but alpha-helix and beta-turns predominate. Moreover, in these solutions the percentage content of the secondary structure does not depend on the temperature. FTIR measurements, carried out at higher peptide concentration (about one order of magnitude) than these CD measurements mentioned above, revealed that in water solution the solid state beta-sheet, and aggregated structures, dominate. However, in TFE the most abundant are alpha-helix and beta-turns structures. The thioflavine T assay showed the tendency of the studied peptide for aggregate.     

Biophysical studies of the interactions between 14-mer and 21-mer model amphipathic peptides and membranes: Insights on their modes of action

We have investigated the interactions between synthetic amphipathic peptides and zwitterionic model membranes. Peptides with 14 and 21 amino acids composed of leucines and phenylalanines modified by the addition of crown ethers have been synthesized. The 14-mer and 21-mer peptides both possess a helical amphipathic structure as revealed by circular dichroism. To shed light on their mechanism of membrane interaction, different complementary biophysical techniques have been used such as circular dichroism, fluorescence, membrane conductivity measurement and NMR spectroscopy. Results obtained by these different techniques show that the 14-mer peptide is a membrane perturbator that facilitate the leakage of species such as calcein and Na ions, while the 21-mer peptide acts as an ion channel. ³¹P solid-state NMR experiments on multilamellar vesicles reveal that the dynamics and/or orientation of the polar headgroups are greatly affected by the presence of the peptides. Similar results have also been obtained in mechanically oriented DLPC and DMPC bilayers where different acyl chain lengths seem to play a role in the interaction. On the other hand, ²H NMR experiments on multilamellar vesicles demonstrate that the acyl chain order is affected differently by the two peptides. Based on these studies, mechanisms of action are proposed for the 14-mer and 21-mer peptides with zwitterionic membranes.     

Single-molecule investigation of the interactions between reconstituted planar lipid membranes and an analogue of the HP(2-20) antimicrobial peptide

In this study, we employed electrophysiology experiments carried out at the single-molecule level to study the mechanism of action of the HPA3 peptide, an analogue of the linear antimicrobial peptide, HP(2–20), isolated from the N-terminal region of the Helicobacter pylori ribosomal protein. Amplitude analysis of currents fluctuations induced by HPA3 peptide at various potentials in zwitterionic lipid membranes reveal the existence of reproducible conductive states in the stochastic behavior of such events, which directly supports the existence of transmembrane pores induced the peptide. From our data recorded both at the single-pore and macroscopic levels, we propose that the HPA3 pore formation is electrophoretically facilitated by trans-negative transmembrane potentials, and HPA3 peptides translocate into the trans monolayers after forming the pores. We present evidence according to which the decrease in the membrane dipole potential of a reconstituted lipid membranes leads to an augmentation of the membrane activity of HPA3 peptides, and propose that a lower electric dipole field of the interfacial region of the membrane caused by phloretin facilitates the surface-bound HPA3 peptides to break free from one leaflet of the membrane, insert into the membrane and contribute to pore formation spanning the entire thickness of the membrane.     

Kissper, a kiwi fruit peptide with channel-like activity: structural and functional features.

Kissper is a 39-residue peptide isolated from kiwi fruit (Actinidia deliciosa). Its primary structure, elucidated by direct protein sequencing, is identical to the N-terminal region of kiwellin, a recently reported kiwi fruit allergenic protein, suggesting that kissper derives from the in vivo processing of kiwellin. The peptide does not show high sequence identity with any other polypeptide of known function. However, it displays a pattern of cysteines similar, but not identical, to those observed in some plant and animal proteins, including toxins involved in defence mechanisms. A number of these proteins are also active on mammalian cells. Functional characterization of kissper showed pH-dependent and voltage-gated pore-forming activity, together with anion selectivity and channeling in model synthetic PLMs, made up of POPC and of DOPS:DOPE:POPC. A 2DNMR analysis indicates that in aqueous solution kissper has only short regions of regular secondary structure, without any evident similarity with other bioactive peptides. Comparative analysis of the structural and functional features suggests that kissper is a member of a new class of pore-forming peptides with potential effects on human health.     

Conformational analysis of peptide analogues of silkmoth chorion protein segments using CD, NMR and molecular modelling.

Silkmoth proteins secreted from the follicular cells that surround the oocyte form a large extracellular assembly which is important for protecting and sustaining the structure of the oocyte and the developing embryo. These proteins have been classified into two major families (A and B). Sequence analysis showed conservation of a central domain containing long stretches of six amino acid residue repeats in both families, which have been suggested to be organized in beta-sheet structures. In this work NMR and CD spectra, as well as molecular calculations, have been used to investigate the conformational properties of two synthetic peptides (A and B), analogues of parts of the central domain of silkmoth chorion proteins of the A and B families, respectively. These peptides consist of three tandem repeats of the six-residue basic motif. Analysis of CD spectra of the two peptides in aqueous solutions and mixtures with organic solvents revealed beta-sheet and turn structural elements with a percentage higher than 40%. NOESY spectra at low temperatures (263-273 K) show sequential nOe connectivities (i, i + 1), indicative of a relative flexibility. The presence of HNi-HNi+1 cross-peaks and medium Halphai-HNi+1 connectivities, chemical shift deviations and temperature coefficient data provide, for the first time, experimental evidence that local folded structures around Gly residues occur in peptide segments of chorion proteins in solution. Simulated annealing calculations were used to examine the conformational space of the peptides and to probe the initial steps of amyloid fibril formation in the case of chorion proteins.     

Insertion selectivity of antimicrobial peptide protegrin-1 into lipid monolayers: Effect of head group electrostatics and tail group packing

The ability to selectively target the harmful microbial membrane over that of the host cell is one of the most important characteristics of the antimicrobial peptides (AMPs). This selectivity strongly depends on the chemical and structural properties of the lipids that make up the cell membrane. A systematic study of the initial membrane selectivity of protegrin-1 (PG-1), a β-sheet AMP, was performed using Langmuir monolayers. Constant pressure insertion assay was used to quantify the amount of PG-1 insertion and fluorescence microscopy was employed to observe the effect of PG-1 on lipid ordering. Charge and packing properties of the monolayer were altered by using lipids with different head groups, substituting saturated with unsaturated lipid tail group(s) and incorporating spacer molecules. PG-1 inserted most readily into anionic films composed of phosphatidylglycerol (PG) and lipid A, consistent with its high selectivity for microbial membranes. It also discriminated between zwitteranionic phospholipids, inserting more readily into phosphatidylcholine (PC) monolayers than those composed of phosphatidylethanolamine, potentially explaining why PG-1 is hemolytic for PC-rich human erythrocytes and not for the PE-rich erythrocytes of ruminants. Increased packing density of the monolayer by increased surface pressure, increased tail group saturation or incorporation of dihydrocholesterol diminishes the insertion of PG-1. Fluorescence microscopy shows that lipid packing is disordered upon PG-1 insertion. However, the presence of PG-1 can still affect lipid morphology even with no observed PG-1 insertion. These results show the important role that lipid composition of the cell membrane plays in the activity of AMPs.     

Investigating the effects of positive charge and hydrophobicity on the cell selectivity, mechanism of action and anti-inflammatory activity of a Trp-rich antimicrobial peptide indolicidin

To investigate the effects of positive charge and hydrophobicity on the cell selectivity, mechanism of action and anti-inflammatory activity of a Trp-rich antimicrobial peptide indolicidin (IN), a series of IN analogs with Trp→Lys substitution were synthesized. All IN analogs displayed an approximately 7- to 18-fold higher cell selectivity, compared with IN. IN, IN-1 and IN-2 depolarized (50−90%) the cytoplasmic membrane potential of Staphylococcus aureus close to minimal inhibitory concentration (5–10 μg mL⁻¹). However, other IN analogs (IN-3 and IN-4) displayed very low ability in membrane depolarization even at 40 μg mL⁻¹. Confocal laser-scanning microscopy revealed that IN-3 and IN-4 penetrated the Escherichia coli cell membrane, whereas IN, IN-1 and IN-2 did not enter the cell membrane. In the gel retardation assay, IN-3 and IN-4 bound more strongly to DNA compared with IN, IN-1 and IN-2. These findings suggest that the mechanism of antimicrobial action of IN-3 and IN-4 may be involved in the inhibition of intracellular functions via interference with DNA/RNA synthesis. Unlike IN, all IN analogs did not inhibit nitric oxide production or inducible nitric oxide synthase mRNA expression in lipopolysaccharide-stimulated mouse macrophage RAW264.7 cells, indicating that the hydrophobicity of IN is more important for anti-inflammatory activity in lipopolysaccharide-treated macrophage cells than the positive charge.     

CD and NMR conformational studies of a peptide encompassing the Mid Loop interface of Ship2–Sam

The lipid phosphatase Ship2 is a protein that intervenes in several diseases such as diabetes, cancer, neurodegeneration, and atherosclerosis. It is made up of a catalytic domain and several protein docking modules such as a C‐terminal Sam (Sterile alpha motif) domain. The Sam domain of Ship2 (Ship2–Sam) binds to the Sam domains of the EphA2 receptor (EphA2–Sam) and the PI3K effector protein Arap3 (Arap3–Sam). These heterotypic Sam–Sam interactions occur through formation of dimers presenting the canonical “Mid Loop/End Helix” binding mode. The central region of Ship2–Sam, spanning the C‐terminal end of α2, the α3 and α4 helices together with the α2α3 and α3α4 interhelical loops, forms the Mid Loop surface that is needed to bind partners Sam domains. A peptide encompassing most of the Ship2–Sam Mid Loop interface (Shiptide) capable of binding to both EphA2–Sam and Arap3–Sam, was previously identified. Here we investigated the conformational features of this peptide, through solution CD and NMR studies in different conditions. These studies reveal that the peptide is highly flexible in aqueous buffer, while it adopts a helical conformation in presence of 2,2,2‐trifluoroethanol. The discovered structural insights and in particular the identification of a helical motif, may lead to the design of more constrained and possibly cell permeable Shiptide analogs that could work as efficient antagonists of Ship2–Sam heterotypic interactions and embrace therapeutic applications. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1088–1098, 2014.     

Deuterated detergents for structural and functional studies of membrane proteins: Properties,chemical synthesis and applications

Abstract

Detergents are amphiphilic compounds that have crucial roles in the extraction, purification and stabilization of integral membrane proteins and in experimental studies of their structure and function. One technique that is highly dependent on detergents for solubilization of membrane proteins is solution-state NMR spectroscopy, where detergent micelles often serve as the best membrane mimetic for achieving particle sizes that tumble fast enough to produce high-resolution and high-sensitivity spectra, although not necessarily the best mimetic for a biomembrane. For achieving the best quality NMR spectra, detergents with partial or complete deuteration can be used, which eliminate interfering proton signals coming from the detergent itself and also eliminate potential proton relaxation pathways and strong dipole-dipole interactions that contribute line broadening effects. Deuterated detergents have also been used to solubilize membrane proteins for other experimental techniques including small angle neutron scattering and single-crystal neutron diffraction and for studying membrane proteins immobilized on gold electrodes. This is a review of the properties, chemical synthesis and applications of detergents that are currently commercially available and/or that have been synthesized with partial or complete deuteration. Specifically, the detergents are sodium dodecyl sulphate (SDS), lauryldimethylamine-oxide (LDAO), n-octyl-β-D-glucoside (β-OG), n-dodecyl-β-D-maltoside (DDM) and fos-cholines including dodecylphosphocholine (DPC). The review also considers effects of deuteration, detergent screening and guidelines for detergent selection. Although deuterated detergents are relatively expensive and not always commercially available due to challenges associated with their chemical synthesis, they will continue to play important roles in structural and functional studies of membrane proteins, especially using solution-state NMR.     

Structure–activity study of macropin,a novel antimicrobial peptide from the venom of solitary bee Macropis fulvipes (Hymenoptera: Melittidae)

A novel antimicrobial peptide, designated macropin (MAC‐1) with sequence Gly‐Phe‐Gly‐Met‐Ala‐Leu‐Lys‐Leu‐Leu‐Lys‐Lys‐Val‐Leu‐NH₂, was isolated from the venom of the solitary bee Macropis fulvipes. MAC‐1 exhibited antimicrobial activity against both Gram‐positive and Gram‐negative bacteria, antifungal activity, and moderate hemolytic activity against human red blood cells. A series of macropin analogs were prepared to further evaluate the effect of structural alterations on antimicrobial and hemolytic activities and stability in human serum. The antimicrobial activities of several analogs against pathogenic Pseudomonas aeruginosa were significantly increased while their toxicity against human red blood cells was decreased. The activity enhancement is related to the introduction of either l ‐ or d ‐lysine in selected positions. Furthermore, all‐d analog and analogs with d ‐amino acid residues introduced at the N‐terminal part of the peptide chain exhibited better serum stability than did natural macropin. Data obtained by CD spectroscopy suggest a propensity of the peptide to adopt an amphipathic α‐helical secondary structure in the presence of trifluoroethanol or membrane‐mimicking sodium dodecyl sulfate. In addition, the study elucidates the structure–activity relationship for the effect of d ‐amino acid substitutions in MAC‐1 using NMR spectroscopy. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Investigation of the mechanism of action of novel amphipathic peptides: Insights from solid-state NMR studies of oriented lipid bilayers

We have investigated in the present study the effect of both non-selective and selective cationic 14-mer peptides on the lipid orientation of DMPC bilayers by ³¹P solid-state nuclear magnetic resonance (NMR) spectroscopy. Depending on the position of substitution, these peptides adopt mainly either an α-helical structure able to permeabilize DMPC and DMPG vesicles (non-selective peptides) or an intermolecular β-sheet structure only able to permeabilize DMPG vesicles (selective peptides). Several systems have been investigated, namely bilayers mechanically oriented between glass plates as well as bicelles oriented with their normal perpendicular or parallel to the external magnetic field. The results have been compared with spectral simulations with the goal of elucidating the difference in the interaction of these two types of peptides with zwitterionic lipid bilayers. The results indicate that the perturbation induced by selective peptides is much greater than that induced by non-selective peptides in all the lipid systems investigated, and this perturbation has been associated to the aggregation of the selective β-sheet peptides in these systems. On the other hand, the oriented lipid spectra obtained in the presence of non-selective peptides suggest the presence of toroidal pores. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.