A spectroscopic study of the membrane interaction of tuberoinfundibular peptide of 39 residues (TIP39)

The membrane interaction of tuberoinfundibular peptide of 39 residues (TIP39), which selectively activates the parathyroid hormone 2 (PTH2) receptor (PTH2-R), has been studied by fluorescence and NMR spectroscopic techniques. Membrane binding would be the first step of a potential membrane-bound activation pathway which has been discussed for a number of neuropeptides and G-protein coupled receptors (GPCRs). Here, the orientation of TIP39 on the surface of membrane mimicking dodecyl-phosphocholine (DPC) micelles was monitored by Photo-CIDNP (chemically-induced dynamic nuclear polarization) NMR which indicates that both Trp25 and Tyr29 face the membrane surface. However, the PTH2 receptor is located in the hypothalamus membrane, for which a more realistic model is required. Therefore, liposomes containing different mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS) and cholesterol were used for fluorescence and solid-state NMR spectroscopy. Fluorescence spectroscopy showed that a large proportion of TIP39 added to these liposomes binds to the membrane surface. Proton-decoupled ³¹P-MAS NMR is used to investigate the potential role of individual lipid headgroups in peptide binding. Significant line-broadening in POPC/cholesterol and POPC/POPS liposomes upon TIP39 association supports a surface binding model and indicates an interaction which is slightly mediated by the presence of POPS and cholesterol. Furthermore, smoothed order parameter profiles obtained from ²H powder spectra of liposomes containing POPC-d31 as bulk lipid in addition to POPS and cholesterol show that TIP39 does not penetrate beyond the headgroup region. Spectra of similar bilayers with POPS-d31 show a small increase in segmental chain order parameters which is interpreted as a small but specific interaction between the peptide and POPS. Our data demonstrate that TIP39 belongs to a class of signaling peptides that associate weakly with the membrane surface but do not proceed to insert into the membrane hydrophobic compartment.     

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.     

Solution structure and membrane interaction mode of an antimicrobial peptide gaegurin 4

We have applied NMR spectroscopy to determine the high-resolution structure of gaegurin 4, a 37-residue antimicrobial peptide from Rana rugosa, under varying hydrophobic conditions. Even in 100% H2O, gaegurin 4 contains a nascent turn near its C-terminal Rana box. Under a more hydrophobic condition it forms two amphipathic helices, one long encompassing residues 2-23 and the other consisting of residues 25-34, similar to what has been observed in cecropin A. Functional implication of the helix-breaking kink at Gly24 in gaegurin 4 was investigated by preparing several analogs. Based upon the current and previous results, we propose a novel seaanemone-like ion pore-forming model for gaegurin 4.     

Model membrane interaction and DNA-binding of antimicrobial peptide Lasioglossin II derived from bee venom

Lasioglossins, a new family of antimicrobial peptide, have been shown to have strong antimicrobial activity with low haemo-lytic and mast cell degranulation activity, and exhibit cytotoxic activity against various cancer cells in vitro. In order to understand the active conformation of these pentadecapeptides in membranes, we have studied the interaction of Lasioglossin II (LL-II), one of the members of Lasioglossins family with membrane mimetic micelle Dodecylphosphocholine (DPC) by fluorescence, Circular Dichroism (CD) and two dimensional (2D) ¹H NMR spectroscopy. Fluorescence experiments provide evidence of interaction of the N-terminal tryptophan residue of LL-II with the hydrophobic core of DPC micelle. CD results show an extended chain conformation of LL-II in water which is converted to a partial helical conformation in the presence of DPC micelle. Moreover we have determined the first three-dimensional NMR structure of LL-II bound to DPC micelle with rmsd of 0.36 Å. The solution structure of LL-II shows hydrophobic and hydrophilic core formation in peptide pointing towards different direction in the presence of DPC. This amphipathic structure may allow this peptide to penetrate deeply into the interfacial region of negatively charged membranes and leading to local membrane destabilization. Further we have elucidated the DNA binding ability of LL-II by agarose gel retardation and fluorescence quenching experiments.     

Simulation study of interaction mechanism between peptide and asymmetric membrane

Cell-penetrating peptides (CPPs) are widely used as drug carriers, owing to their superior ability to cross cell membrane both alone and with cargos, such as genes and other particles. Understanding the translocation mechanism of CPP is significant for many therapeutic purposes, including targeting drug and gene delivery. In this study, we performed a coarse-grained molecular dynamics simulation to investigate the interaction mechanism between polyarginine peptides and asymmetric membranes. Results showed that peptides can penetrate through the lipid bilayer by inducing a hydrophilic hole formation in the asymmetric membrane. Furthermore, the lengthy peptide chain length (R4–R16 peptides) and high membrane asymmetry positively affect peptide penetration. Our study provides insights into the molecular-level interactions between peptides and asymmetric membranes, as well as suggestions for targeted gene and drug delivery.     

A two-dimensional NMR study of the antimicrobial peptide magainin 2

Using two-dimensional NMR spectroscopy, a complete 1H resonance assignment has been obtained for the peptide magainin 2 recently isolated from Xenopus laevis. It is demonstrated that this peptide adopts an alpha-helical structure with amphiphilic character when dissolved in a mixture of trifluoroethanol (TFE) and H2O. The transition to the alpha-helical conformation occurs at very low concentrations of TFE.     

Ceruloplasmin-anion interaction. A circular dichroism spectroscopic study.

The effect of anion binding to ceruloplasmin has been studied using absorption and cirbular dichroism spectral data. At anion to ceruloplasmin molar ratios approaching infinite, OCN-, N3- and SCN- bind to ceruloplasmin giving rise to similar alterations in circular dichroism and absorption spectra. The positive bands at 610 and 520 nm in circular dichroism spectra disappear, a negative one apperars at 600 nm and the peak at 450 nm is only slightly modified. There is a new negative band at 410 nm well-defined in OCN- ceruloplasmin spectra. The decrease in absorption at 610 nm is ascribed to the disruption of one type I Cu-S(cysteine) bond owing presumably to the changes induced by anions in the protein secondary structure. The new band at 410 nm is assigned to a charge transfer transition from the ligand replacing cysteine at its binding site. Both absorption and circular dichroism spectra show isobestic points indicating that anion binding to the enzyme, disruption of one of the two type I Cu-S bonds and coordination of this Cu to another protein residue take place simultaneously.     

Optical spectroscopic study of the ADP-myosin interaction.

The binding of ADP to heavy meromyosin, and the separated subfragment 1 components S-1(A1) and S-1 (A2), has been observed by ultraviolet spectrophotometry. The results are compatible with the presence of spectroscopically equivalent and independent sites, one per head, at both 10 degrees C and 25 degrees C. We do not observe the heterogeneity of binding and of the spectroscopic response that has been reported. The binding has also been followed by other methods sensitive to the effect of ligand on the aromatic residues of the protein, viz. intrinsic fluorescence of heavy meromyosin and changes in the near-ultraviolet Cotton effects of myosin, and its active fragments. Within the limits of our experimental precision, the binding profiles, based on concentration of myosin heads, are the same for myosin as for subfragment 1. A perturbation in the circular dichroism is also generated by pyrophosphate, which competes with ADP. The spectra suggest that subsites for the purine ring and the diphosphate can be recognized. The sensitivity of binding profiles obtained by methods of the kind used here to cooperative or antagonistic interactions between the binding sites has been analysed. It is clear that sizeable effects of this nature could be concealed by the binding curves, even for high experimental precision.     

Investigations into the membrane activity of arenicin antimicrobial peptide AA139

Arenicin-3 is an amphipathic β-hairpin antimicrobial peptide that is produced by the lugworm Arenicola marina. In this study, we have investigated the mechanism of action of arenicin-3 and an optimized synthetic analogue, AA139, by studying their effects on lipid bilayer model membranes and Escherichia coli bacterial cells. The results show that simple amino acid changes can lead to subtle variations in their interaction with membranes and therefore alter their pre-clinical potency, selectivity and toxicity. While the mechanism of action of arenicin-3 is primarily dependent on universal membrane permeabilization, our data suggest that the analogue AA139 relies on more specific binding and insertion properties to elicit its improved antibacterial activity and lower toxicity, as exemplified by greater selectivity between lipid composition when inserting into model membranes i.e. the N-terminus of AA139 seems to insert deeper into lipid bilayers than arenicin-3 does, with a clear distinction between zwitterionic and negatively charged lipid bilayer vesicles, and AA139 demonstrates a cytoplasmic permeabilization dose response profile that is consistent with its greater antibacterial potency against E. coli cells compared to arenicin-3.     

Dermaseptin 01 as antimicrobial peptide with rich biotechnological potential: study of peptide interaction with membranes containing Leishmania amazonensis lipid‐rich extract and membrane models

This article addresses the interactions of the synthetic antimicrobial peptide dermaseptin 01 (GLWSTIKQKGKEAAIAAA‐ KAAGQAALGAL‐NH₂, DS 01) with phospholipid (PL) monolayers comprising (i) a lipid‐rich extract of Leishmania amazonensis (LRE‐La), (ii) zwitterionic PL (dipalmitoylphosphatidylcholine, DPPC), and (iii) negatively charged PL (dipalmitoylphosphatidylglycerol, DPPG). The degree of interaction of DS 01 with the different biomembrane models was quantified from equilibrium and dynamic liquid‐air interface parameters. At low peptide concentrations, interactions between DS 01 and zwitterionic PL, as well as with the LRE‐La monolayers were very weak, whereas with negatively charged PLs the interactions were stronger. For peptide concentrations above 1 µg/ml, a considerable expansion of negatively charged monolayers occurred. In the case of DPPC, it was possible to return to the original lipid area in the condensed phase, suggesting that the peptide was expelled from the monolayer. However, in the case of DPPG, the average area per lipid molecule in the presence of DS 01 was higher than pure PLs even at high surface pressures, suggesting that at least part of DS 01 remained incorporated in the monolayer. For the LRE‐La monolayers, DS 01 also remained in the monolayer. This is the first report on the antiparasitic activity of AMPs using Langmuir monolayers of a natural lipid extract from L. amazonensis. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

A spectroscopic study of the interaction of isoflavones with human serum albumin

Genistein and daidzein, the major isoflavones present in soybeans, possess a wide spectrum of physiological and pharmacological functions. The binding of genistein to human serum albumin (HSA) has been investigated by equilibrium dialysis, fluorescence measurements, CD and molecular visualization. One mole of genistein is bound per mole of HSA with a binding constant of 1.5 +/- 0.2 x 10(5) m(-1). Binding of genistein to HSA precludes the attachment of daidzein. The ability of HSA to bind genistein is found to be lost when the tryptophan residue of albumin is modified with N-bromosuccinimide. At 27 degrees C (pH 7.4), van't Hoff's enthalpy, entropy and free energy changes that accompany the binding are found to be -13.16 kcal x mol(-1), -21 cal x mol(-1) K(-1) and -6.86 kcal x mol(-1), respectively. Temperature and ionic strength dependence and competitive binding measurements of genistein with HSA in the presence of fatty acids and 8-anilino-1-naphthalene sulfonic acid have suggested the involvement of both hydrophobic and ionic interactions in the genistein-HSA binding. Binding measurements of genistein with BSA and HSA, and those in the presence of warfarin and 2,3,5-tri-iodobenzoic acid and F?rster energy transfer measurements have been used for deducing the binding pocket on HSA. Fluorescence anisotropy measurements of daidzein bound and then displaced with warfarin, 2,3,5-tri-iodobenzoic acid or diazepam confirm the binding of daidzein and genistein to subdomain IIA of HSA. The ability of HSA to form ternery complexes with other neutral molecules such as warfarin, which also binds within the subdomain IIA pocket, increases our understanding of the binding dynamics of exogenous drugs to HSA.     

Selective acylation enhances membrane charge sensitivity of the antimicrobial peptide mastoparan-x

The partitioning of the wasp venom peptide mastoparan-X (MPX) into neutral and negatively charged lipid membranes has been compared with two new synthetic analogs of MPX where the N^α-terminal of MPX was acylated with propanoic acid (PA) and octanoic acid (OA). The acylation caused a considerable change in the membrane partitioning properties of MPX and it was found that the shorter acylation with PA gave improved affinity and selectivity toward negatively charged membranes, whereas OA decreased the selectivity. Based on these findings, we hypothesize that minor differences in the embedding and positioning of the peptide in the membrane caused by either PA or OA acylation play a critical role in the fine-tuning of the effective charge of the peptide and thereby the fine-tuning of the peptide's selectivity between neutral and negatively charged lipid membranes. This finding is unique compared to previous reports where peptide acylation enhanced membrane affinity but also resulted in impaired selectivity. Our result may provide a method of enhancing selectivity of antimicrobial peptides toward bacterial membranes due to their high negative charge—a finding that should be investigated for other, more potent antimicrobial peptides in future studies.     

Analogs of the antimicrobial peptide trichogin having opposite membrane properties.

Four analogs of the antimicrobial peptide trichogin GA IV were studied. Their sequences are as follows: GT, n-octanoyl-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-OMe; ST, n-octanoyl-Aib-Ser-Leu-Aib-Ser-Ser-Leu-Aib-Ser-Ile-Leu-OMe; BT, n-octanoyl-Aib-Ser(tBu)-Leu-Aib-Ser(tBu)-Ser(tBu)-Leu-Aib-Ser(tBu)-Ile-Leu-OMe; and DT, n-octanoyl-Aib-Ser(tBu)-Leu-Aib-Ser(tBu)-Ser(tBu)-Leu-Aib-Ser(tBu)-Ile-Leu-Aib-Ser(tBu)-Leu-Aib-Ser(tBu)-Ser(tBu)-Leu-Aib-Ser(tBu)-Ile-Leu-OMe. The trichogin GA IV differs from GT only in the nature of the C-terminal residue, being a 1,2 aminoalcohol (leucinol) in the case of the parent peptide. Compared with GT, ST has an increased amphiphilicity. In contrast, BT has little amphiphilicity being composed only of hydrophobic amino acids. DT is an octanoylated head-to-tail dimer of BT. We show that BT and DT lower the bilayer-to-hexagonal phase transition temperature (T(H)) of dipalmitoleoylphosphatidylethanolamine, indicating that the peptides promote negative curvature. These two peptides, composed of only hydrophobic amino acids, have their bulkier groups on one face of the helix, suggesting that they may penetrate membranes at an oblique angle. In contrast, GT and ST, like trichogin itself, increase TH, promoting positive curvature. These peptides have contrasting membrane lytic activities. Whereas DT and BT did not produce leakage of aqueous contents, GT and ST, like trichogin, did cause rapid leakage. The leakage activity with liposomes also correlates with the greater potency of GT and ST, compared with the hydrophobic analogs, in their hemolytic and bacteriostatic action. ST has greater lytic ability than GT in liposomal leakage as well as hemolysis. We also measured the rate of peptide-promoted lipid mixing as an indication of membrane fusion. BT produced lipid mixing only with large unilamellar vesicles enriched with dioleoylphosphatidylethanolamine; ST did not produce lipid mixing, as its apparent reduction of energy transfer proved to be artifactual. Quasi-elastic light scattering of large unilamellar vesicles was also carried out after adding ST and BT. Peptide BT, but not ST, was able to aggregate large unilamellar vesicles. Thus, one of the properties of BT that leads to the induction of lipid mixing is that it is able to aggregate vesicles, placing the bilayers in juxtaposition. Thus, the two pairs of peptides, BT and DT vs GT and ST, exhibit contrasting behaviour with respect to a number of membrane biophysical properties. This occurs despite the fact that the chemical structures of the peptides are rather similar. Such distinct behavior is also reflected in their hemolytic and bacteriostatic actions.     

Protein-flavonol interaction: fluorescence spectroscopic study

Recent studies have shown that various synthetic as well as therapeutically active naturally occurring flavonols possess novel luminescence properties that can potentially serve as highly sensitive monitors of their microenvironments in biologically relevant systems. We report a study on the interactions of bovine serum albumin (BSA) with the model flavonol 3-hydroxyflavone (3HF), using the excited-state proton-transfer (ESPT) luminescence of 3HF as a probe. Upon addition of BSA to the flavonoid solutions, we observe remarkable changes in the absorption, ESPT fluorescence emission and excitation profiles as well as anisotropy (r) values. Complexation of 3HF with protein results in a pronounced shift (20 nm) of the ESPT emission maximum of the probe (from lambda(max)(em) = 513 nm to lambda(max)(em) = 533 nm) accompanied by a significant increase in fluorescence intensity. The spectral data also suggest that, in addition to ESPT, the protein environment induces proton abstraction from 3HF leading to formation of anionic species in the ground state. Fairly high values of anisotropy are observed in the presence of BSA for the tautomer (r = 0.25) as well as anion (r = 0.35) species of 3HF, implying that both the species are located in motion-restricted environments of BSA molecules. Analysis of relevant spectroscopic data leads to the conclusions that two binding sites are involved in BSA-3HF interaction, and the interaction is slightly positively cooperative in nature with a similar binding constant of 1.1 - 1.3 x 10(5) M(-1) for both these sites. Proteins 2001;43:75-81.     

Molecular dynamics study of the membrane interaction of a membranotropic dengue virus C protein-derived peptide

Dengue virus C protein, essential in the dengue virus life cycle, possesses a segment, peptide PepC, known to bind membranes composed of negatively charged phospholipids. To characterize its interaction with the membrane, we have used the molecular dynamics HMMM membrane model system. This approach is capable of achieving a stable system and sampling the peptide/lipid interactions which determine the orientation and insertion of the peptide upon membrane binding. We have been able to demonstrate spontaneous binding of PepC to the 1,2-divaleryl-sn-glycero-3-phosphate/1,2-divaleryl-sn-glycero-3-phosphocholine membrane model system, whereas no binding was observed at all for the 1,2-divaleryl-sn-glycero-3-phosphocholine one. PepC, adopting an α-helix profile, did not insert into the membrane but did bind to its surface through a charge anchor formed by its three positively charged residues. PepC, maintaining its three-dimensional structure along the whole simulation, presented a nearly parallel orientation with respect to the membrane when bound to it. The positively charged amino acid residues Arg-2, Lys-6, and Arg-16 are mainly responsible for the peptide binding to the membrane stabilizing the structure of the bound peptide. The segment of dengue virus C protein where PepC resides is a fundamental protein–membrane interface which might control protein/membrane interaction, and its positive amino acids are responsible for membrane binding defining its specific location in the bound state. These data should help in our understanding of the molecular mechanism of DENV life cycle as well as making possible the future development of potent inhibitor molecules, which target dengue virus C protein structures involved in membrane binding.     

Immunogobulin-lipid interaction: A model membrane study

We recently presented evidence (Vandenbranden, M., De Coen, J.L., Jeener, R., Kanarek, L. and Ruysschaert, J.M. (1981) Mol. Immunol. 8, 621–631) for the existence of two conformational rabbit serum IgG immunoglobulin isomers. In the present report, their capacity to interact with lipid is investigated in model membranes. (1) One isomer, IgG(H), behaves like several intrinsic membrane proteins: it induces a large surface pressure increase when injected under a lipid monolayer in the close packed state and increases by 20-fold the conductance of a planar bilayer. The other isomer, IgG(S) doesn't interact significantly with the lipids. (2) IgG(H) marked a preference for monolayers made of lipids with a negatively charged polar headgroup and bearing unsaturations in their acyl chains. Penetration is stronger with lipid monolayer in the fluid state than in the rigid state. (3) As previously shown (Vandenbranden, M., De Coen, J.L., Jeener, R., Kanarek, L. and Ruysschaert, J.M. (1981) Mol. Immunol. 18, 621–631), circular dichroïsm spectra and antigen precipitation tests don't allow to detect any difference in the overall secondary conformation and antigen recognition properties of the two isomers. (4) Papaïn cleavage of the hinge region suppresses the hydrophobic properties of IgG towards lipid monolayers. (5) The hypothesis of a binding of the hinge region with the lipid bilayer is discussed.     

Structure of the antimicrobial peptide tachystatin A

The solution structure of antimicrobial peptide tachystatin A from the Japanese horseshoe crab (Tachypleus tridentatus) was determined by two-dimensional nuclear magnetic resonance measurements and distance-restrained simulated annealing calculations. The correct pairs of disulfide bonds were also confirmed in this study. The obtained structure has a cysteine-stabilized triple-stranded beta-sheet as a dominant secondary structure and shows an amphiphilic folding observed in many membrane-interactive peptides. Interestingly, tachystatin A shares structural similarities with the calcium channel antagonist omega-agatoxin IVA isolated from spider toxin and mammalian defensins, and we predicted that omega-agatoxin IVA also have the antifungal activity. These structural comparisons and functional correspondences suggest that tachystatin A and omega-agatoxin IVA may exert the antimicrobial activity in a manner similar to defensins, and we have confirmed such activity using fungal culture assays. Furthermore, tachystatin A is a chitin-binding peptide, and omega-agatoxin IVA also showed chitin-binding activities in this study. Tachystatin A and omega-agatoxin IVA showed no structural homology with well known chitin-binding motifs, suggesting that their structures belong to a novel family of chitin-binding peptides. Comparison of their structures with those of cellulose-binding proteins indicated that Phe(9) of tachystatin A might be an essential residue for binding to chitin.     

Spectroscopic and thermodynamic evidence for antimicrobial peptide membrane selectivity

In our laboratory we developed a series of antimicrobial peptides that exhibit selectivity and potency for prokaryotic over eukaryotic cells (Hicks et al., 2007). Circular dichroism (CD), isothermal calorimetry (ITC) and calcein leakage assays were conducted to determine the mechanism of lipid binding of a representative peptide 1 (Ac-GF-Tic-Oic-GK-Tic-Oic-GF-Tic-Oic-GK-Tic-KKKK-CONH₂) to model membranes. POPC liposomes were used as a simple model for eukaryotic membranes and 4:1 POPC:POPG liposomes were used as a simple model for prokaryotic membranes. CD, ITC and calcein leakage data clearly indicate that compound 1 interacts via very different mechanisms with the two different liposome membranes. Compound 1 exhibits weaker binding and induces less calcein leakage in POPC liposomes than POPC:POPG (4:1 mole ratio) liposomes. The predominant binding mechanism to POPC appears to be limited to surface interactions while the mechanism of binding to 4:1 POPC:POPG most likely involves some type of pore formation.