Conformations of a synthetic peptide which facilitates the cellular delivery of nucleic acids

Summary We describe the synthesis of an amphipathic vector peptide which is able to form complexes with nucleic acids. Based on circular dichroism investigations, the nature of the structure obtained in water is questioned. The peptide adopts an α-helical structure in TFE and is partially in a β-sheet conformation in phosphate buffer at low peptide concentrations.     

Conformational Analysis of a Synthetic Antimicrobial Peptide in Water and Membrane-Mimicking Solvents: A Molecular Dynamics Simulation Study

We have investigated structural and dynamic properties of the synthetic peptide hlF1-11 (GRRRSVQWCA, i.e., the first 11 N-terminal amino acids of the human lactoferrin protein) in water, 250 mM NaCl solution, 50% (V/V) water–trifluoroethanol mixture, and in the membrane mimetic 4:4:1 methanol–chloroform–water mixture. For comparison, we have also performed analogous simulations for the biologically inactive control peptide featuring Ala substitutions in the 2, 3, 6 and 9 positions of the hlF1-11 sequence. Statistical analyses of the trajectories indicate that only in the membrane-mimicking medium hlF1-11 adopts preferentially a conformation suitable to interact effectively with the membrane. In this conformation the peptide cationic region is rather flexible and elongated, while the C-terminal hydrophobic moiety appears as a more rigid hairpin-shaped loop approximately perpendicular to the cationic region. No such conformation is statistically relevant for the control peptide.     

Production and physicochemical characterization of acidocin D20079, a bacteriocin produced by Lactobacillus acidophilus DSM 20079

Lactobacillus acidophilus DSM 20079 is the producer of a novel bacteriocin termed acidocin D20079. In this paper, a partial sequence of this peptide is determined, together with data on its secondary structure. A modification of the MRS-growth medium (replacing the detergent Tween 80 with oleic acid), was shown to improve the production level of the peptide by one order of magnitude, as well as to stabilize the activity level. Addition of a detergent (Tween 20, less interfering in mass spectrometric analysis), was however necessary for solubilization of the purified acidocin D20079. Digestion of the peptide followed by de-novo sequencing of generated fragments, allowed determination of a partial sequence consisting of 39 of the totally estimated 65 residues. Acidocin D20079 has a high content of glycine residues, hydrophobic residues, and acidic residues. No modified amino acids were found. Edman degradation, and C-terminal sequencing failed, suggesting that the peptide may be cyclic, and a novel member of class IIc bacteriocins. Circular dichroism spectroscopy and secondary structure prediction showed random coil conformation in aqueous solution, but secondary structure was induced in the presence of sodium-dodecyl sulfate. The data could be fitted assuming 2–13% of the residues to be in α-helix and 23–27% of the residues to be in β-strand conformation. This indicates that a membrane/membrane-mimicking hydrocarbon–water interface induces an active conformation.     

Probing the importance of lateral hydrophobic association in self-assembling peptide hydrogelators

A class of peptides has been designed whose ability to self-assemble into hydrogel is dependent on their conformationally folded state. Under unfolding conditions aqueous peptide solutions are freely flowing having the viscosity of water. When folding is triggered by external stimuli, peptides adopt a β-hairpin conformation that self-assembles into a highly crosslinked network of fibrils affording mechanically rigid hydrogels. MAX 1, a 20 residue, amphiphilic hairpin self-assembles via a mechanism which entails both lateral and facial self-assembly events to form a network of fibrils whose local structure consists of a bilayer of hairpins hydrogen bonded in the direction of fibril growth. Lateral self-assembly along the long axis of the fibril is mainly facilitated by intermolecular hydrogen bonding between the strands of distinct hairpins and the formation of hydrophobic contacts between residue side chains of laterally associating hairpins. Facial assembly is driven by the hydrophobic collapse of the valine-rich faces of the amphiphilic hairpins affording a bilayer laminate. The importance of forming lateral hydrophobic contacts during hairpin self-assembly and the relative contribution these interactions have towards nano-scale morphology and material rigidity is probed via the study of: MAX1, a hairpin designed to exploit lateral hydrophobic interactions; MAX 4, a peptide with reduced ability to form these interactions; and MAX5, a control peptide. CD spectroscopy and rheological experiments suggest that the formation of lateral hydrophobic interactions aids the kinetics of assembly and contributes to the mechanical rigidity of the hydrogel. Transmission electron microscopy (TEM) shows that these interactions play an essential role in the self-assembly process leading to distinct nano-scale morphologies. Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Incorporation of a β-turn mimic in the N-terminal fragment of the B1 domain of streptococcal protein G

Summary A dibenzofuran-based β-turn mimic has been incorporated in the B1_2–29 fragment of the B1 domain of streptococcal protein G. This amino acid sequence adopts a β-hairpin structure in the complete B1 domain (B1_2–56). The modified peptide was studied by CD and NMR spectroscopy and its solution behavior was compared with the conformation adopted by the same sequence in the modified B1 domain.     

Molecular mechanism of action of β-hairpin antimicrobial peptide arenicin: oligomeric structure in dodecylphosphocholine micelles and pore formation in planar lipid bilayers

The membrane-active, cationic, β-hairpin peptide, arenicin, isolated from marine polychaeta Arenicola marina exhibits a broad spectrum of antimicrobial activity. The peptide in aqueous solution adopts the significantly twisted β-hairpin conformation without pronounced amphipathicity. To assess the mechanism of arenicin action, the spatial structure and backbone dynamics of the peptide in membrane-mimicking media and its pore-forming activity in planar lipid bilayers were studied. The spatial structure of the asymmetric arenicin dimer stabilized by parallel association of N-terminal strands of two β-hairpins was determined using triple-resonance nuclear magnetic resonance (NMR) spectroscopy in dodecylphosphocholine (DPC) micelles. Interaction of arenicin with micelles and its oligomerization significantly decreased the right-handed twist of the β-hairpin, increased its amphipathicity, and led to stabilization of the peptide backbone on a picosecond to nanosecond time scale. Relaxation enhancement induced by water-soluble (Mn(2+)) and lipid-soluble (16-doxylstearate) paramagnetic probes pointed to the dimer transmembrane arrangement. Qualitative NMR and circular dichroism study of arenicin-2 in mixed DPC/1,2-dioleoyl-sn-glycero-3-phosphoglycerol bicelles, sodium dodecyl sulfate micelles, and lipid vesicles confirmed that a similar dimeric assembly of the peptide was retained in membrane-mimicking systems containing negatively charged lipids and detergents. Arenicin-induced conductance was dependent on the lipid composition of the membrane. Arenicin low-conductivity pores were detected in the phosphatidylethanolamine-containing lipid mixture, whereas the high-conductivity pores were observed in an exclusively anionic lipid system. The measured conductivity levels agreed with the model in which arenicin antimicrobial activity was mediated by the formation of toroidal pores assembled of two, three, or four β-structural peptide dimers and lipid molecules. The structural transitions involved in arenicin membrane-disruptive action are discussed.     

Structural studies and model membrane interactions of two peptides derived from bovine lactoferricin.

The powerful antimicrobial properties of bovine lactoferricin (LfcinB) make it attractive for the development of new antimicrobial agents. An 11-residue linear peptide portion of LfcinB has been reported to have similar antimicrobial activity to lactoferricin itself, but with lower hemolytic activity. The membrane-binding and membrane-perturbing properties of this peptide were studied together with an amidated synthetic version with an added disulfide bond, which was designed to confer increased stability and possibly activity. The antimicrobial and cytotoxic properties of the peptides were measured against Staphylococcus aureus and Escherichia coli and by hemolysis assays. The peptides were also tested in an anti-cancer assay against neuroblastoma cell lines. Vesicle disruption caused by these LfcinB derivatives was studied using the fluorescent reporter molecule calcein. The extent of burial of the two Trp residues in membrane mimetic environments were quantitated by fluorescence. Finally, the solution NMR structures of the peptides bound to SDS micelles were determined to provide insight into their membrane bound state. The cyclic peptide was found to have greater antimicrobial potency than its linear counterpart. Consistent with this property, the two Trp residues of the modified peptide were suggested to be embedded deeper into the membrane. Although both peptides adopt an amphipathic structure without any regular alpha-helical or beta-sheet conformation, the 3D-structures revealed a clearer partitioning of the cationic and hydrophobic faces for the cyclic peptide.     

Role of β-Hairpin Formation in Aggregation: The Self-Assembly of the Amyloid-β(25-35) Peptide

The amyloid-β(25-35) peptide plays a key role in the etiology of Alzheimer's disease due to its extreme toxicity even in the absence of aging. Because of its high tendency to aggregate and its low solubility in water, the structure of this peptide is still unknown. In this work, we sought to understand the early stages of aggregation of the amyloid-β(25-35) peptide by conducting simulations of oligomers ranging from monomers to tetramers. Our simulations show that although the monomer preferentially adopts a β-hairpin conformation, larger aggregates have extended structures, and a clear transition from compact β-hairpin conformations to extended β-strand structures occurs between dimers and trimers. Even though β-hairpins are not present in the final architecture of the fibril, our simulations indicate that they play a critical role in fibril growth. Our simulations also show that β-sheet structures are stabilized when a β-hairpin is present at the edge of the sheet. The binding of the hairpin to the sheet leads to a subsequent destabilization of the hairpin, with part of the hairpin backbone dangling in solution. This free section of the peptide can then recruit an extra monomer from solution, leading to further sheet extension. Our simulations indicate that the peptide must possess sufficient conformational flexibility to switch between a hairpin and an extended conformation in order for β-sheet extension to occur, and offer a rationalization for the experimental observation that overstabilizing a hairpin conformation in the monomeric state (for example, through chemical cross-linking) significantly hampers the fibrillization process.     

Selective emergence and survival of early polypeptides in water

Oligopeptides essential to primitive cells could not be obtained just by raising the background noise of organic compounds produced by a prebiotic chemistry working at random. Selection pathways were required. Experimental evidence is given for selective condensation of amino acids in water as well as for selective resistance to degradation. It is shown that N-carboxyanhydrides are good candidates for chemical selection in water. They are formed when active esters of amino acids are left in the presence of bicarbonate ions or when N,N'-carbonyldiimidazole is used as condensing agent. Polymerization of a mixture of proteinaceous and non-proteinaceous amino acids leads to an enrichment in the proteinaceous ones plusα-aminobutyric acid. Selective resistance toward degradation ofβ-pleated sheet conformation is used to exemplify a possible accumulation of homochiral sequences made of hydrophilic and strong hydrophobic residues. Amino acids with branched aliphatic side-chains are selected but those having short linear aliphatic side-chains such asα-aminobutyric acid or norvaline are not.     

Human Antimicrobial Peptides: Defensins, Cathelicidins and Histatins

Antimicrobial peptides, which have been isolated from many bacteria, fungi, plants, invertebrates and vertebrates, are an important component of the natural defenses of most living organisms. The isolated peptides are very heterogeneous in length, sequence and structure, but most of them are small, cationic and amphipathic. These peptides exhibit broad-spectrum activity against Gram-positive and Gram-negative bacteria, yeasts, fungi and enveloped viruses. A wide variety of human proteins and peptides also have antimicrobial activity and play important roles in innate immunity. In this review we discuss three important groups of human antimicrobial peptides. The defensins are cationic non-glycosylated peptides containing six cysteine residues that form three intramolecular disulfide bridges, resulting in a triple-stranded β-sheet structure. In humans, two classes of defensins can be found: α-defensins and β-defensins. The defensin-related HE2 isoforms will also be discussed. The second group is the family of histatins, which are small, cationic, histidine-rich peptides present in human saliva. Histatins adopt a random coil conformation in aqueous solvents and form α-helices in non-aqueous solvents. The third group comprises only one antimicrobial peptide, the cathelicidin LL−37. This peptide is derived proteolytically from the C-terminal end of the human CAP18 protein. Just like the histatins, it adopts a largely random coil conformation in a hydrophilic environment, and forms an α-helical structure in a hydrophobic environment.     

Bilayer conformation of fusion peptide of influenza virus hemagglutinin: a molecular dynamics simulation study

Unraveling the conformation of membrane-bound viral fusion peptides is essential for understanding how those peptides destabilize the bilayer topology of lipids that is important for virus-cell membrane fusion. Here, molecular dynamics (MD) simulations were performed to investigate the conformation of the 20 amino acids long fusion peptide of influenza hemagglutinin of strain X31 bound to a dimyristoyl phosphatidylcholine (DMPC) bilayer. The simulations revealed that the peptide adopts a kinked conformation, in agreement with the NMR structures of a related peptide in detergent micelles. The peptide is located at the amphipathic interface between the headgroups and hydrocarbon chains of the lipid by an energetically favorable arrangement: The hydrophobic side chains of the peptides are embedded into the hydrophobic region and the hydrophilic side chains are in the headgroup region. The N-terminus of the peptide is localized close to the amphipathic interface. The molecular dynamics simulations also revealed that the peptide affects the surrounding bilayer structure. The average hydrophobic thickness of the lipid phase close to the N-terminus is reduced in comparison with the average hydrophobic thickness of a pure dimyristoyl phosphatidylcholine bilayer.     

Computational studies of colicin insertion into membranes: the closed state

Colicins are water-soluble toxins that, upon interaction with membranes, undergo a conformational change, insert, and form pores in them. Pore formation activity is localized in a bundle of 10 α-helices named the pore-forming domain (PFD). There is evidence that colicins attach to the membrane via a hydrophobic hairpin embedded in the core of the PFD. Two main models have been suggested for the membrane-bound state: penknife and umbrella, differing in regard to the orientation of the hydrophobic hairpin with respect to the membrane. The arrangement of the amphipathic helices has been described as either a compact three-dimensional structure or a two-dimensional array of loosely interacting helices on the membrane surface. Using molecular dynamics simulations with an implicit membrane model, we studied the structure and stability of the conformations proposed earlier for four colicins. We find that colicins are initially driven towards the membrane by electrostatic interactions between basic residues and the negatively charged membrane surface. They do not have a unique binding orientation, but in the predominant orientations the central hydrophobic hairpin is parallel to the membrane. In the inserted state, the estimated free energy tends to be lower for the compact arrangements of the amphipathic helix, but the more expanded ones are in better agreement with experimental distance distributions. The difference in energy between penknife and umbrella conformations is small enough for equilibrium to exist between them. Elongation of the hydrophobic hairpin helices and membrane thinning were found unable to produce stabilization of the transmembrane configuration of the hydrophobic hairpin.     

Structural basis for poor uracil excision from hairpin DNA. An NMR study.

Two-dimensional NMR and molecular dynamics simulations have been used to determine the three-dimensional structures of two hairpin DNA structures: d-CTAGAG GATCCUTTTGGATCCT (abbreviated as U1-hairpin) and d-CTAGAGGATCCTTUTGGATCCT (abbreviated as U3-hairpin). The 1H resonances of both of these hairpin structures have been assigned almost completely. NMR restrained molecular dynamics and energy minimization procedures have been used to describe the three-dimensional structures of these hairpins. This study and concurrent NMR structural studies on two other d-CTAGAGGA TCCTUTTGGATCCT (abbreviated as U2-hairpin) and d-CTAGAGGATCCTTTUGGATCCT (abbreviated as U4-hairpin) have shed light upon various interactions reported between Echerichia coli uracil DNA glycosylase (UDG) and uracil-containing DNA. The backbone torsion angles, which partially influence the local conformation of U12 and U14 in U1 and U3-hairpins, respectively, are probably locked in the trans conformation as in the case of U13 in the U2-hairpin. Such a stretched-out backbone conformation in the vicinity of U12 and U14 is thought to be the reason why the Km value is poor for U1- and U3-hairpins as it is for the U2-hairpin. Furthermore, the bases U12 and U14 in both U1- and U3-hairpins adopt an anti conformation, in contrast with the base conformation of U13 in the U2-hairpin, which adopts a syn conformation. The clear discrepancy observed in the U-base orientation with respect to the sugar moieties could explain why the Vmax value is 10- to 20-fold higher for the U1- and U3-hairpins compared with the U2-hairpin. Taken together, these observations support our interpretation that the unfavourable backbone results in a poor Km value, whereas the unfavourable nucleotide conformation results in a poor Vmax value. These two parameters therefore make the U1- and U3-hairpins better substrates for UDG compared with the U2-hairpin, as reported earlier [Kumar, N. V. & Varshney, U. (1997) Nucleic Acids Res. 25, 2336-2343.].     

Membrane destabilization induced by the human immunodeficiency virus type-1 fusion peptide

Summary The human immunodeficiency virus type-1 (HIV-1) fusion peptide, corresponding to a sequence of 23 amino acid residues at the N-terminus of the spike transmembrane subunit gp41, has the capacity to destabilize negatively charged and neutral large unilamellar vesicles, representing, respectively, the acidic and the neutral fraction of the plasma membrane lipids of viral target cells. As revealed by infrared spectroscopy, the peptide associated with the vesicles may exist in different conformations. In negatively charged membranes the structure is mainly an α-helix, while in Ca²⁺-neutralized negatively charged membranes the conformation switches to a predominantly extended conformation. In membranes composed of zwitterionic phospholipids and cholesterol, the peptide also adopts a predominant extended structure. The α-helical structure permeabilizes negatively charged vesicles but does not induce membrane fusion. The peptide in β-type conformation, on the other hand, permeabilizes neutral membranes and triggers fusion. As seen by³¹P NMR, the latter structure also exhibits the capacity to alter the lamellar organization of the membrane.     

The structure of the antimicrobial active center of lactoferricin B bound to sodium dodecyl sulfate micelles

Lactoferricin B (LfcinB) is a 25-residue antimicrobial peptide released from bovine lactoferrin upon pepsin digestion. The antimicrobial center of LfcinB consists of six residues (RRWQWR-NH2), and it possesses similar bactericidal activity to LfcinB. The structure of the six-residue peptide bound to sodium dodecyl sulfate (SDS) micelles has been determined by NMR spectroscopy and molecular dynamics refinement. The peptide adopts a well defined amphipathic structure when bound to SDS micelles with the Trp sidechains separated from the Arg residues. Additional evidence demonstrates that the peptide is oriented in the micelle such that the Trp residues are more deeply buried in the micelle than the Arg and Gln residues.     

Structural characterisation of a uracil containing hairpin DNA by NMR and molecular dynamics.

Three-dimensional (3D) structure of a hairpin DNA d-CTAGAGGATCCTTTUGGATCCT (22mer; abbreviated as U4-hairpin), which has a uracil nucleotide unit at the fourth position from the 5' end of the tetra-loop has been solved by NMR spectroscopy. The(1)H resonances of this hairpin have been assigned almost completely. NMR restrained molecular dynamics and energy minimisation procedures have been used to describe the 3D structure of the U4 hairpin. This study establishes that the stem of the hairpin adopts a right handed B-DNA conformation while the T(12)and U(15)nucleotide stack upon 3' and 5' ends of the stem, respectively. Further, T(14)stacks upon both T(12)and U(15)while T(13)partially stacks upon T(14). Very weak stacking interaction is observed between T(13)and T(12). All the individual nucleotide bases adopt ' anti ' conformation with respect to their sugar moiety. The turning phosphate in the loop is located between T(13)and T(14). The stereochemistry of U(15)mimics the situation where uracil would stack in a B-DNA conformation. This could be the reason as to why the U4-hairpin is found to be the best substrate for its interaction with uracil DNA glycosylase (UDG) compared to the other substrates in which the uracil is at the first, second and third positions of the tetra-loop from its 5' end, as reported previously.     

Role of hydrophobic interactions and salt-bridges in β-hairpin folding

β-Hairpins are the simplest form of β-sheets which, due to the presence of long-range interactions, can be considered as tertiary structures. Molecular dynamics simulation is a powerful tool that can unravel whole pathways of protein folding/unfolding at atomic resolution. We have performed several molecular dynamics simulations, to a total of over 250 ns, of a β-hairpin peptide in water using GROMACS. We show that hydrophobic interactions are necessary for initiating the folding of the peptide. Once formed, the peptide is stabilized by hydrogen bonds and disruption of hydrophobic interactions in the folded peptide does not denature the structure. In the absence of hydrophobic interactions, the peptide fails to fold. However, the introduction of a salt-bridge compensates for the loss of hydrophobic interactions to a certain extent. Figure Model of b-hairpin folding: Folding is initiated by hydrophobic interactions (Brown circles). The folded structure, once formed, is stabilized by hydrogen bonds (red lines) and is unaffected by loss of hydrophobic contacts     

Conformations of a synthetic peptide which facilitates the cellular delivery of nucleic acids

We describe the synthesis of an amphipathic vectorpeptide which is able to form complexes with nucleicacids. Based on circular dichroism investigations, the nature of thestructure obtained in water is questioned. Thepeptide adopts an -helical structure in TFEand is partially in a -sheet conformation inphosphate buffer at low peptide concentrations.     

Membrane-bound dimer structure of a beta-hairpin antimicrobial peptide from rotational-echo double-resonance solid-state NMR

The intermolecular packing of a beta-hairpin antimicrobial peptide, PG-1, in lipid bilayers is determined using solid-state NMR distance measurements. Previous spin counting experiments showed that PG-1 associates as dimers in POPC bilayers; however, the detailed dimer structure was unknown. We have now measured several intermolecular 13C-19F, 1H-13C, and 15N-13C distances in site-specifically labeled PG-1 to constrain the structure of the intermolecular interface. The distances are measured using the rotational-echo double-resonance (REDOR) technique under magic-angle spinning. The results indicate that two PG-1 molecules align in a parallel fashion with the C-terminal strand of the hairpin forming the dimer interface. Six hydrogen bonds stabilize this interface, and the Phe12 side chain adopts the g- conformation in the membrane as in solution. The parallel packing of the peptide in the lipid bilayer differs from the antiparallel dimer found in DPC micelles and may be stabilized by its strong amphipathic character, which should facilitate its insertion into the amphipathic lipid bilayer. This study demonstrates the utility of the REDOR NMR technique for the elucidation of the oligomeric structure of membrane proteins.     

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.