Amphipathic Alpha-helical Structure Research Articles

Teleost Piscidins-In Silico Perspective of Natural Peptide Antibiotics from Marine Sources.

Fish, like all other animals, are exposed to constant contact with microbes, both on their skin and on the surfaces of their respiratory and digestive systems. Fish have a system of non-specific immune responses that provides them with initial protection against infection and allows them to survive under normal conditions despite the presence of these potential invaders. However, fish are less protected against invading diseases than other marine vertebrates because their epidermal surface, composed primarily of living cells, lacks the keratinized skin that serves as an efficient natural barrier in other marine vertebrates. Antimicrobial peptides (AMPs) are one type of innate immune protection present in all life forms. AMPs have been shown to have a broader range of biological effects than conventional antibiotics, including antibacterial, antiviral, antiprotozoal, and antifungal effects. Although other AMPs, such as defensins and hepcidins, are found in all vertebrates and are relatively well conserved, piscidins are found exclusively in Teleost fish and are not found in any other animal. Therefore, there is less information on the expression and bioactivity of piscidins than on other AMPs. Piscidins are highly effective against Gram-positive and Gram-negative bacteria that cause disease in fish and humans and have the potential to be used as pharmacological anti-infectives in biomedicine and aquaculture. To better understand the potential benefits and limitations of using these peptides as therapeutic agents, we are conducting a comprehensive study of the Teleost piscidins included in the "reviewed" category of the UniProt database using bioinformatics tools. They all have amphipathic alpha-helical structures. The amphipathic architecture of piscidin peptides and positively charged residues influence their antibacterial activity. These alpha-helices are intriguing antimicrobial drugs due to their stability in high-salt and metal environments. New treatments for multidrug-resistant bacteria, cancer, and inflammation may be inspired by piscidin peptides.

A histone H2A amino terminal derived peptide Etroplusin from Pearl spot, Etroplus suratensis (Bloch, 1790): Phylogenetic and functional characterization

Antimicrobial peptides (AMPs) derived from histone precursors form a significant class of therapeutic molecules. The study explores the comprehensive molecular characterization of core histone H2A-derived antimicrobial peptide, Etroplusin from Pearl spot, Etroplus suratensis. Etroplusin exhibited characteristic features of AMP such as net +11 charge and amphipathic alpha-helical structure. Multiple sequence alignment showed the homologous nature of etroplusin with previously reported H2A peptides. Phylogenetic analysis revealed that etroplusin is similar to Carassius auratus H2A-derived peptide and comes under the fish H2A peptide clade. Structure modelling with bioinformatics tools predicted alpha-helical structure and random coils with a proline hinge. The functional characterization of etroplusin revealed its antibacterial, antifungal, and anticancer activities. Significant upregulation of etroplusin was observed in the systemic tissues of E. suratensis on a challenge with the bacterial fish pathogen, Streptococcus agalactiae. The present study will shed light on fish innate immune responses and the importance of etroplusin, the H2A-derived peptide as a defense molecule.

Effects of charge on the interaction of the huntingtin N-terminal sequence with model cell membranes

Huntington’s Disease (HD) is a genetic neurodegenerative disorder caused by an expansion in the polyglutamine (poly-Q) region near the N-terminus of the huntingtin (htt) protein. When the poly-Q expansion surpasses a threshold of 35-40 glutamine repeats, the protein misfolds into a beta sheet rich structure that forms insoluble neuronal aggregates. The poly-Q region of htt is flanked by a 17 amino acid sequence (Nt17) that facilitates insertion of htt into lipid membranes via an amphipathic alpha helical structure, a function that is proposed to anchor the protein and may affect the aggregation process.

Membrane binding properties of the C-terminal segment of retinol dehydrogenase 8

Light absorption by rhodopsin leads to the release of all-trans retinal (ATRal) in the lipid phase of photoreceptor disc membranes. Retinol dehydrogenase 8 (RDH8) then reduces ATRal into all-trans retinol, which is the first step of the visual cycle. The membrane binding of RDH8 has been postulated to be mediated by one or more palmitoylated cysteines located in its C-terminus. Different peptide variants of the C-terminus of RDH8 were thus used to obtain information on the mechanism of membrane binding of this enzyme. Steady-state and time-resolved fluorescence measurements were performed using short and long C-terminal segments of bovine RDH8, comprising one or two tryptophan residues. The data demonstrate that the amphipathic alpha helical structure of the first portion of the C-terminus of RDH8 strongly contributes to its membrane binding, which is also favored by palmitoylation of at least one of the cysteines located in the last portion of the C-terminus.

Influence of adding terminal tags on the structural and antimicrobial properties of the peptide caerin 1.1

Aquaculture is threatened by severe bacterial and viral outbreaks which can cause massive mortality and limit productivity. Antimicrobial peptides are a promising tool for the control of infectious diseases; however, their use in the aquaculture sector has been poorly explored. Recombinant production of peptides is an interesting alternative to their chemical synthesis, which has still important challenges to address. Although extra amino acid tags are usually added to the ends of recombinant proteins to enhance their stability and facilitate their purification or their translational processing, the effect of these tags on the function and the stability of antimicrobial peptides is completely unknown. This is particularly important in the case of the short cationic antimicrobial peptide caerin, originally isolated from frog skin secretions, and other peptides which bioactivity has been directly related with its ability to adopt an amphipathic alpha-helical structure upon interaction with the microbial membranes. Caerin has demonstrated bactericidal activity against several important human pathogens, however its antimicrobial activity over usual aquaculture pathogens remains practically unexplored. In the present work, we have studied the effect of adding the terminal tag polyhistidine (6xHis) and the 2A peptide from the foot and mouth disease virus (FMDV-2A) to the N- or the C- terminus of caerin on its activity against a collection of bacterial and viral pathogens common to aquaculture species. In addition, the antimicrobial activity of a peptide composed of two tandem consecutive caerin sequences has been investigated. We have concluded that the addition of terminal tags to caerin causes a drastic reduction of its antiviral activity against the studied viruses and that the effect of the added tags on the bactericidal activity of the peptide depends of the target bacterial species. However, caerin with a 6xHis extension at its N terminus maintains or increases its antimicrobial activity against most of the fish pathogenic bacteria tested. According to our data, there is only a moderate agreement between the theoretical predictions obtained from in silico models and the experimental observations. This information is essential for the rational design of strategies to aim the recombinant production of caerin and other antimicrobial peptides, which can be used as anti-infective agents in aquaculture.

Abstract 2624: NRP2b promotes migration, drug resistance and AKT activation in lung cancers by recruiting GSK3â to phosphorylate and destabilize PTEN

Abstract Background: Neuropilins (NRPs) are cell surface co-receptors known to enhance signaling from multiple ligands, including growth factors and their cognate receptors, such as HGF/MET. We have previously shown that the novel “b” isoform of NRP2 (NRP2b) is specifically associated with aggressive NSCLC and is required for TGFβ-induced EMT. We also found that NRP2b promotes AKT activation, drug resistance and metastatic spread, while the canonical NRP2a isoform inhibits these processes. However, the mechanisms supporting these differential functions remain obscure. Results: Live cell imaging revealed that NRP2b expression led to slow internalization/rapid surface recycling of HGF-bound MET compared to NRP2a, which promoted rapid internalization and little/no recycling. Co-immunoprecipitation experiments showed that GSK3β interacted robustly with NRP2b, but only weakly with NRP2a. Our previous work demonstrated analogous differential recruitment of PTEN, but with reversed preferences; i.e., robust binding to NRP2a, but weak binding to NRP2b. Importantly, GSK3β inhibition blocked HGF-dependent migration of lung cancer cells expressing NRP2b, but not in cells expressing NRP2a. Given that PTEN is an established target of GSK3β, and that modification by GSK3β leads to its proteasome-mediated degradation, PTEN levels were examined in cells expressing individual NRP2 isoforms. PTEN was significantly reduced by expression of NRP2b, but not by NRP2a. The reduction of PTEN in the presence of NRP2b was blocked by inhibition of GSK3β, and by proteasome inhibition with MG132. Despite lower PTEN levels, phosphorylation of the GSK3β target site on Thr366 was specifically increased by NRP2b. Co-immunoprecipitation of GSK3β with NRP2b deletion mutants identified a C-terminal 15 amino acid peptide as the site of interaction. Mutants without this sequence failed to recruit GSK3β and failed to reduce PTEN levels. In silico modeling of this peptide suggested an amphipathic alpha-helical structure with a high affinity docking site (ΔG = -45 kcal) on the atomic structure of GSK3β. Conclusion: Our results suggest that NRP2b promotes receptor signaling to AKT by differential endosomal trafficking and by recruitment of GSK3β. This kinase will phosphorylate and destabilize active PTEN recruited into the complex by NRP2a, which heterodimerizes with NRP2b. Increased AKT signaling following PTEN loss is likely responsible for the pro-tumorigenic properties of NRP2b, including resistance to targeted agents and metastatic spread. This novel mechanism for NRP2b function suggests that targeting its interaction with GSK3β might be a fruitful therapeutic strategy to reduce drug resistance and/or metastatic spread in lung cancer. Citation Format: Anastasios Dimou, Cecile Nasarre, Monika Gooz, Rose Pagano, Yuri Peterson, Kent Armeson, Harry A. Drabkin, Patrick Nasarre, Chadrick E. Denlinger, Robert M. Gemmill. NRP2b promotes migration, drug resistance and AKT activation in lung cancers by recruiting GSK3â to phosphorylate and destabilize PTEN [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2624.

An inhibitory mechanism of action of coiled-coil peptides against type three secretion system from enteropathogenic Escherichia coli.

Human pathogenic gram-negative bacteria, such as enteropathogenic Escherichia coli (EPEC), rely on type III secretion systems (T3SS) to translocate virulence factors directly into host cells. The coiled-coil domains present in the structural proteins of T3SS are conformed by amphipathic alpha-helical structures that play an important role in the protein-protein interaction and are essential for the assembly of the translocation complex. To investigate the inhibitory capacity of these domains on the T3SS of EPEC, we synthesized peptides between 7 and 34 amino acids based on the coiled-coil domains of proteins that make up this secretion system. This analysis was performed through in vitro hemolysis assays by assessing the reduction of T3SS-dependent red blood cell lysis in the presence of the synthesized peptides. After confirming its inhibitory capacity, we performed molecular modeling assays using combined techniques, docking-molecular dynamic simulations, and quantum-mechanic calculations of the various peptide-protein complexes, to improve the affinity of the peptides to the target proteins selected from T3SS. These techniques allowed us to demonstrate that the peptides with greater inhibitory activity, directed against the coiled-coil domain of the C-terminal region of EspA, present favorable hydrophobic and hydrogen bond molecular interactions. Particularly, the hydrogen bond component is responsible for the stabilization of the peptide-protein complex. This study demonstrates that compounds targeting T3SS from pathogenic bacteria can indeed inhibit bacterial infection by presenting a higher specificity than broad-spectrum antibiotics. In turn, these peptides could be taken as initial structures to design and synthesize new compounds that mimic their inhibitory pharmacophoric pattern.

The activity and action mechanism of novel short selective LL-37-derived anticancer peptides against clinical isolates of Escherichia coli.

Human cathelicidin LL-37 has recently attracted interest as a potential therapeutic agent, mostly because of its ability to kill a wide variety of pathogens and cancer cells. In this study, we aimed to investigate the antibacterial activity and cytotoxicity of previously designed LL-37 anticancer derivatives (i.e., P7, P22, and P38). Calcein release assay and field emission-scanning electron microscopy (FE-SEM) were performed to elucidate the possible mechanism of action of P38, the peptide with the highest bactericidal activity. In silico analysis demonstrated the amphipathic alpha-helical structure for three peptides. Antibacterial activity of P38 against multidrug-resistant (MDR) clinical isolates of Escherichia coli was higher than that of P7 and P22. P38 caused no hemolysis or cytotoxicity. Treating calcein-loaded E. coli with 4× MIC of P38 resulted in more than 96% leakage of calcein. Noticeably, FE-SEM revealed that P38 killed E. coli by disrupting the bacterial membrane. Molecular docking studies showed that P38 had a much higher affinity for the outer membrane of Gram-negative bacteria compared with both P22 and P7. Owing to the bactericidal activity of P38 against MDR E. coli isolates and its negligible cytotoxicity, P38 has the potential for further studies in a mouse model of infectious disease.

Alpha‐synuclein p.H50Q, a novel pathogenic mutation for Parkinson's disease

Alpha-synuclein plays a central role in the pathophysiology of Parkinson's disease. Three missense mutations in SNCA, the gene encoding alpha-synuclein, as well as genomic multiplications have been identified as causes for autosomal-dominantly inherited Parkinsonism. Here, we describe a novel missense mutation in exon 4 of SNCA encoding a H50Q substitution in a patient with dopa-responsive Parkinson's disease with a family history of parkinsonism and dementia. The variant was not observed in public databases or identified in unrelated subjects. The substitution's evolutionary conservation and protein modeling provide additional support for pathogenicity as the amino acid perturbs the same amphipathic alpha helical structure as the previously described pathogenic mutations.

Analysis of the solution structure of the human antibiotic peptide dermcidin and its interaction with phospholipid vesicles

Dermcidin is a human antibiotic peptide that is secreted by the sweat glands and has no homology to other known antimicrobial peptides. As an initial step toward understanding dermcidin's mode of action at bacterial membranes, we used homonuclear and heteronuclear NMR to determine the conformation of the peptide in 50% trifluoroethanol solution. We found that dermcidin adopts a flexible amphipathic alpha-helical structure with a helix-hinge-helix motif, which is a common molecular fold among antimicrobial peptides. Spin-down assays of dermcidin and several related peptides revealed that the affinity with which dermcidin binds to bacterial-mimetic membranes is primarily dependent on its amphipathic alpha-helical structure and its length (>30 residues); its negative net charge and acidic pI have little effect on binding. These findings suggest that the mode of action of dermcidin is similar to that of other membrane-targeting antimicrobial peptides, though the details of its antimicrobial action remain to be determined.

Novel antimicrobial peptides from the venom of the eusocial bee Halictus sexcinctus (Hymenoptera: Halictidae) and their analogs

Two novel antimicrobial peptides, named halictines, were isolated from the venom of the eusocial bee Halictus sexcinctus. Their primary sequences were established by ESI-QTOF mass spectrometry, Edman degradation and enzymatic digestion as Gly-Met-Trp-Ser-Lys-Ile-Leu-Gly-His-Leu-Ile-Arg-NH2 (HAL-1), and Gly-Lys-Trp-Met-Ser-Leu-Leu-Lys-His-Ile-Leu-Lys-NH2 (HAL-2). Both peptides exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria but also noticeable hemolytic activity. The CD spectra of HAL-1 and HAL-2 measured in the presence of trifluoroethanol or SDS showed ability to form an amphipathic alpha-helical secondary structure in an anisotropic environment such as bacterial cell membrane. NMR spectra of HAL-1 and HAL-2 measured in trifluoroethanol/water confirmed formation of helical conformation in both peptides with a slightly higher helical propensity in HAL-1. Altogether, we prepared 51 of HAL-1 and HAL-2 analogs to study the effect of such structural parameters as cationicity, hydrophobicity, alpha-helicity, amphipathicity, and truncation on antimicrobial and hemolytic activities. The potentially most promising analogs in both series are those with increased net positive charge, in which the suitable amino acid residues were replaced by Lys. This improvement basically relates to the increase of antimicrobial activity against pathogenic Pseudomonas aeruginosa and to the mitigation of hemolytic activity.

Geometric Curvature Sensing of Alpha-Helical Proteins

Understanding how specific proteins are recruited to membranes is crucial to understanding key biological functions. Membrane curvature sensing is increasingly recognized as a powerful means for guiding protein localization, coupling protein binding to the local membrane geometry. The mechanisms underlying curvature sensing remain poorly quantified, however. We focus on the two curvature-associated proteins that share an amphipathic alpha-helical structure. The first, SpoVM, is a small (26 residue) protein from Bacillus subtillus that has recently been shown to preferentially bind to convex surfaces [K.S. Ramamurthy et al., Science 323: 1354 (2009)]. The second is the 23 amino acid N-terminal helical domain of Sar1, which initiates the assembly of COPII coated vesicles at the endoplasmic reticulum and which recent work [Parthasarthy Lab, unpublished] has shown to dramatically alter membrane rigidity. Using both microfabricated surfaces that present controlled curvatures to the proteins and optical-trap based assays involving dynamic membrane deformation, we quantify the curvature dependence of the membrane binding affinity of these structurally similar yet functionally disparate proteins.

Apoprotein A-I mimetic peptides and their potential anti-atherogenic mechanisms of action

Peptides that resemble in physicochemical properties the helices of apoprotein A-I, the major protein of atheroprotective HDL, show promise for the treatment of atherosclerosis-related vascular disease. The properties and promise of these so-called mimetic peptides will be explored in this review. Both HDL and mimetic peptides are able to scavenge and sequester oxidized lipids and hence protect endothelial cells and arteries from the pro-inflammatory action of oxidized LDL. Active mimetic peptides have an amphipathic alpha-helical secondary structure, whose hydrophobic face is particularly important for its bioactivity. The most frequently employed peptide is 4F. The comparative bioactivity of variants of 4F, particularly tandem helical peptides, has been explored. The recent observation of the very high affinity of bioactive peptides for oxidized fatty acids and phospholipids provides a likely mechanism for the action of these peptides in inhibiting early atherosclerosis formation. It is not clear that these peptides alone are effective in reversing established atherosclerosis, although they may achieve this outcome in synergy with statin therapy. Recent observations of mimetic peptides have pointed to promising therapies for patients with cardiovascular disease. The peptides appear to be well tolerated and effective in promoting the anti-inflammatory properties of HDL.

Structural characterization of two peptides corresponding to Helix 4 of Apolipoprotein E

Apolipoprotein E (ApoE) is a ligand for the low density lipoprotein (LDL) receptor. The amino-terminal domain of ApoE is a 4-helix bundle and helix 4 is involved in LDL receptor binding. This region associates with lipids and undergoes structural changes which allow a key arginine residue, Arg172 to participate in receptor binding. To characterize this region and to study lipid-induced conformational changes we developed 2 synthetic peptides: one corresponding to residues 128-164 (ApoE-164) and the other corresponding to residues 128-183 (ApoE-183). Secondary structure prediction indicates amphipathic alpha-helical structure for both peptides. Circular dichroism spectroscopy (CD) studies show that ApoE-164 exhibits concentration-dependent self association whereas ApoE-183 does not. To study the effect of lipids on the two peptides we used three detergents: 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), dodecyl phosphocholine(DPC), and octyl-beta-d-glucopyranoside(BetaOG). CD shows that both peptides exhibit an increase in alpha-helix content at the critical micelle concentration (CMC) for all three detergents. The alpha-helix content for both peptides in the absence of detergent is about 30%. Upon the addition of DHPC, DPC or BetaOG there is a 30-40% increase in alpha-helicity at the CMC. This approach is directed towards understanding (i) how lipids induce a conformational change in the 165-183 region, and (ii) how the relative orientation of Arg172 is changed such that it contributes to LDL receptor binding.

Effect of Leucine to Phenylalanine Substitution on the Nonpolar Face of a Class A Amphipathic Helical Peptide on Its Interaction with Lipid: HIGH RESOLUTION SOLUTION NMR STUDIES OF 4F-DIMYRISTOYLPHOSPHATIDYLCHOLINE DISCOIDAL COMPLEX

Model class A amphipathic helical peptides mimic several properties of apolipoprotein A-I (apoA-I), the major protein component of high density lipoproteins. Previously, we reported the NMR structures of Ac-18A-NH(2) (renamed as 2F because of two phenylalanines), the base-line model class A amphipathic helical peptide in the presence of lipid ( Mishra, V. K., Anantharamaiah, G. M., Segrest, J. P., Palgunachari, M. N., Chaddha, M., Simon Sham, S. W., and Krishna, N. R. (2006) J Biol. Chem. 281, 6511-6519 ). Substitution of two Leu residues on the nonpolar face (Leu(3) and Leu(14)) with Phe residues produced the peptide 4F (so named because of four phenylalanines), which has been extensively studied for its anti-inflammatory and antiatherogenic properties. Like 2F, 4F also forms discoidal nascent high density lipoprotein-like particles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Since subtle structural changes in the peptide-lipid complexes have been shown to be responsible for their antiatherogenic properties, we undertook high resolution NMR studies to deduce detailed structure of 4F in 4F.DMPC discs. Like 2F, 4F adopts a well defined amphipathic alpha-helical structure in association with the lipid at a 1:1 peptide/lipid weight ratio. Nuclear Overhauser effect (NOE) spectroscopy revealed a number of intermolecular close contacts between the aromatic residues in the hydrophobic face of the helix and the lipid acyl chain protons. Similar to 2F, the pattern of observed peptide-lipid NOEs is consistent with a parallel orientation of the amphipathic alpha helix, with respect to the plane of the lipid bilayer, on the edge of the disc (the belt model). However, in contrast to 2F in 2F.DMPC, 4F in the 4F.DMPC complex is located closer to the lipid headgroup as evidenced by a number of NOEs between 4F and DMPC headgroup protons. These NOEs are absent in the 2F.DMPC complex. In addition, the conformation of the DMPC sn-3 chain in 4F.DMPC complex is different than in the 2F.DMPC complex as evidenced by the NOE between lipid 2.CH and betaCH(2) protons in 4F.DMPC, but not in 2F.DMPC, complex. Based on the results of this study, we infer that the antiatherogenic properties of 4F may result from its preferential interaction with lipid headgroups.

Melectin: A Novel Antimicrobial Peptide from the Venom of the Cleptoparasitic Bee Melecta albifrons

A novel antimicrobial peptide designated melectin was isolated from the venom of the cleptoparasitic bee Melecta albifrons. Its primary sequence was established as H-Gly-Phe-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-Pro-Lys-Val-Met-Ala-His-Met-Lys-NH(2) by Edman degradation and ESI-QTOF mass spectrometry. Synthetic melectin exhibited antimicrobial activity against both gram-positive and -negative bacteria and it degranulated rat peritoneal mast cells, but its hemolytic activity was low. The CD spectra of melectin measured in the presence of trifluoroethanol and sodium dodecyl sulfate showed a high content alpha-helices, which indicates that melectin can adopt an amphipathic alpha-helical secondary structure in an anisotropic environment such as the bacterial cell membrane. To envisage the role of the proline residue located in the middle of the peptide chain on biological activity and secondary structure, we prepared several melectin analogues in which the Pro11 residue was either replaced by other amino acid residues or was omitted. The results of biological testing suggest that a Pro kink in the alpha-helical structure of melectin plays an important role in selectivity for bacterial cells. In addition, a series of N- and C-terminal-shortened analogues was synthesized to examine which region of the peptide is related to antimicrobial activity.

Solution Structure and Model Membrane Interactions of Temporins-SH, Antimicrobial Peptides from Amphibian Skin. A NMR Spectroscopy and Differential Scanning Calorimetry Study

Temporin-SHa and temporin-SHc are 13 residue long antimicrobial peptides from frog skin that have similar sequences but differ markedly in their membrane-damaging properties. Temporin-SHa contains a single basic lysine residue and has a unique antimicrobial spectrum of action among temporins, being very potent against Gram-positive and Gram-negative bacteria, yeasts, fungi, and protozoa. Temporin-SHc, which contains a single basic histidine residue, is inactive against Gram-negative bacteria, has a reduced efficacy against Gram-positive bacteria, but is still active against yeasts and fungi. Temporin-SHb, with no basic residue, has no antimicrobial activity. The three-dimensional structures of the peptides bound to SDS micelles were analyzed by CD and NMR spectroscopy combined with restrained molecular dynamics calculations. The peptides adopt well-defined amphipathic alpha-helical structures extending from residue 3 to residue 12, when bound to SDS micelles. The structures are stabilized by extensive interactions between aliphatic and aromatic side chains on the nonpolar face. Relaxation enhancements caused by paramagnetic probes showed that the peptides adopt nearly parallel orientations to the micelle surface and do not deeply penetrate into the micelle. The interaction of the peptides with model membranes was investigated by differential scanning calorimetry on anionic and zwitterionic multilamellar vesicles and membrane-permeabilization assays on calcein-loaded large unilamellar vesicles. Calorimetric data indicated that both temporin-SHa and -SHc reside at the hydrocarbon core-water interface of the anionic lipid bilayer but interact with anionic bilayers in a very different manner. This suggests that the charge-induced activity of temporins-SH for bacterial cells is due to changes in the membrane-disturbing mechanism of the bound peptides.

Selectivity in the mechanism of action of antimicrobial mastoparan peptide Polybia-MP1

Many potent antimicrobial peptides also present hemolytic activity, an undesired collateral effect for the therapeutic application. Unlike other mastoparan peptides, Polybia-MP1 (IDWKKLLDAAKQIL), obtained from the venom of the social wasp Polybia paulista, is highly selective of bacterial cells. The study of its mechanism of action demonstrated that it permeates vesicles at a greater rate of leakage on the anionic over the zwitterionic, impaired by the presence of cholesterol or cardiolipin; its lytic activity is characterized by a threshold peptide to lipid molar ratio that depends on the phospholipid composition of the vesicles. At these particular threshold concentrations, the apparent average pore number is distinctive between anionic and zwitterionic vesicles, suggesting that pores are similarly formed depending on the ionic character of the bilayer. To prospect the molecular reasons for the strengthened selectivity in Polybia-MP1 and its absence in Mastoparan-X, MD simulations were carried out. Both peptides presented amphipathic alpha-helical structures, as previously observed in Circular Dichroism spectra, with important differences in the extension and stability of the helix; their backbone solvation analysis also indicate a different profile, suggesting that the selectivity of Polybia-MP1 is a consequence of the distribution of the charged and polar residues along the peptide helix, and on how the solvent molecules orient themselves according to these electrostatic interactions. We suggest that the lack of hemolytic activity of Polybia-MP1 is due to the presence and position of Asp residues that enable the equilibrium of electrostatic interactions and favor the preference for the more hydrophilic environment.

Association of a Model Class A (Apolipoprotein) Amphipathic α Helical Peptide with Lipid: HIGH RESOLUTION NMR STUDIES OF PEPTIDE·LIPID DISCOIDAL COMPLEXES

Class A amphipathic helical peptides have been shown to mimic apolipoprotein A-I, the major protein component of high density lipoproteins and have been shown to inhibit atherosclerosis in several dyslipidemic mouse models. Previously we reported the NMR structure of Ac-18A-NH2, the base-line model class A amphipathic helical peptide in a 50% (v/v) trifluoroethanol-d3/water mixture, a membrane-mimic environment (Mishra, V. K., Palgunachari, M. N., Anantharamaiah, G. M., Jones, M. K., Segrest, J. P., and Krishna, N. R. (2001) Peptides 22, 567-573). The peptide Ac-18A-NH2 forms discoidal nascent high density lipoprotein-like particles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine. Because subtle structural changes in the peptide.lipid complexes have been shown to be responsible for their antiatherogenic properties, we undertook high resolution NMR studies to deduce detailed structure of recombinant peptide.1,2-dimyristoyl-sn-glycero-3-phosphocholine complexes. The peptide adopts a well defined amphipathic alpha helical structure in association with the lipid at a 1:1 peptide:lipid weight ratio. Nuclear Overhauser effect spectroscopy revealed a number of intermolecular close contacts between the aromatic residues in the hydrophobic face of the helix and the lipid acyl chain protons. The pattern of observed peptide-lipid nuclear Overhauser effects is consistent with a parallel orientation of the amphipathic alpha helix, with respect to the plane of the lipid bilayer, on the edge of the disc (the belt model). Based on the results of chemical cross-linking and molecular modeling, we propose that peptide helices are arranged in a head to tail fashion to cover the edge of the disc. This arrangement of peptides is also consistent with the pKa values of the Lys residues determined previously. Taken together, these results provide for the first time a high resolution structural view of the peptide.lipid discoidal complexes formed by a class A amphipathic alpha helical peptide.

HFIP‐induced structures and assemblies of the peptides from the transmembrane domain 4 of membrane protein Nramp1

Membrane protein Nramp1 (natural resistance-associated macrophage protein 1) is a pH-dependent divalent metal cation transporter that regulates macrophage activation in infectious and autoimmune diseases. A naturally occurring glycine to aspartic acid substitution at position 169 (G169D) within the transmembrane domain 4 (TM4) of Nramp1 makes mice susceptible to Leishmania donovani, Salmonella typhimurium, and Mycobacterium bovis. Here we present a structural and self-assembling study on two synthetic 24-residue peptides, corresponding to TM4 of mouse Nramp1 and its G169D mutant, respectively, in 1,1,1,3,3,3-hexafluoroisopropanol-d(2) (HFIP-d(2)) aqueous solution by nuclear magnetic resonance (NMR) spectroscopy. The results show that amphipathic alpha-helical structures are formed from residue Ile173 to Tyr187 for the wild-type peptide and from Trp168 to Tyr187 for the G169D mutant, respectively. The segment of the N-terminus from Leu167 to Leu172 is poorly structured for the wild-type peptide, whereas it is well defined for the G169D mutant. Both peptides aggregate to form a tetramer and the monomeric peptides in peptide bundles are structurally and orientationally similar. The intermolecular interactions in assemblies could be stronger in the C-terminal regions related to residues Phe180-Leu184 than those in the central helical segments for both peptides. The G169D mutation may change the size of the opening on the termini of assembly.