Solution structures and model membrane interactions of lactoferrampin, an antimicrobial peptide derived from bovine lactoferrin

Bovine lactoferrampin (LFampinB) has been identified as a novel antimicrobial peptide, which is derived from the N-terminal lobe of bovine lactoferrin. In this study, the solution structure of LFampinB bound to negatively charged sodium dodecyl sulphate micelles and zwitterionic dodecyl phosphocholine micelles was determined using 2-dimensional nuclear magnetic resonance (NMR) spectroscopy. The interaction between LFampinB and multilamellar phospholipid vesicles, containing choline and glycerol head groups, was examined using differential scanning calorimetry (DSC). In addition, the interaction between the N-terminal tryptophan residue and model membranes of varying composition was analyzed by fluorescence spectroscopy. LFampinB adopts an amphipathic alpha-helical conformation across the first 11 residues of the peptide but remains relatively unstructured at the C-terminus. The hydrophobic surface of the amphipathic helix is bordered by the side chains of Trp1 and Phe11, and is seen in both micelle-bound structures. The fluorescence results suggest that Trp1 inserts into the membrane at the lipid/water interface. The phenyl side chain of Phe11 is oriented in the same direction as the indole ring of Trp1, allowing these two residues to serve as anchors for the lipid bilayer. The DSC results also indicate that LFampinB interacts with glycerol head groups in multilamellar vesicles but has little effect on acyl chain packing. Our results support a two step model of antimicrobial activity where the initial attraction of LFampinB is mediated by the cluster of positive charges on the C-terminus followed by the formation of the N-terminal helix which binds to the surface of the bacterial lipid bilayer.     

Novel lactoferrampin antimicrobial peptides derived from human lactoferrin

Human lactoferrampin is a novel antimicrobial peptide found in the cationic N-terminal lobe of the iron-binding human lactoferrin protein. The amino acid sequence that directly corresponds to the previously characterized bovine lactoferrin-derived lactoferrampin peptide is inactive on its own (WNLLRQAQEKFGKDKSP, residues 269-285). However, by increasing the net positive charge near the C-terminal end of human lactoferrampin, a significant increase in its antibacterial and Candidacidal activity was obtained. Conversely, the addition of an N-terminal helix cap (sequence DAI) did not have any appreciable effect on the antibacterial or antifungal activity of human lactoferrampin peptides, even though it markedly influenced that of bovine lactoferrampin. The solution structure of five human lactoferrampin variants was determined in SDS micelles and all of the structures display a well-defined amphipathic N-terminal helix and a flexible cationic C-terminus. Differential scanning calorimetry studies indicate that this peptide is capable of inserting into the hydrophobic core of a membrane, while fluorescence spectroscopy results suggest that a hydrophobic patch encompassing the single Trp and Phe residues as well as Leu, Ile and Ala side chains mediates the interaction between the peptide and the hydrophobic core of a phospholipid bilayer.     

Interactions of the antimicrobial peptide Ac-FRWWHR-NH(2) with model membrane systems and bacterial cells.

The acetylated and amidated hexapeptide FRWWHR (combi-2), previously identified by combinatorial chemistry methods, shows strong antimicrobial activity. The binding of the peptide to 1-palmitoyl-2-oleoyl-sn-glycero-3-[(phospho-rac-(1-glycerol)] (POPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles was studied using fluorescence spectroscopy and isothermal titration calorimetry (ITC). Differential scanning calorimetry (DSC) with dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) multilamellar vesicles was performed to determine changes in the lipid phase behaviour upon binding the peptide. Two-dimensional proton nuclear magnetic resonance (NMR) spectroscopy, to solve the bound peptide structure, was performed in the presence of dodecylphosphatidylcholine (DPC) and sodium dodecyl sulphate (SDS) micelles. The fluorescence, ITC and DSC studies indicate that the peptide interacts preferentially with lipid vesicles containing negatively charged head groups. Conformational information determined using NMR indicate that the combi-2 peptide adopts a coiled amphipathic conformation when bound to SDS and DPC micelles. Leakage assays indicate that the peptide is not very efficient at causing leakage from calcein-filled large unilamellar vesicles comprised of POPG/POPC (1 : 1). The rapid passage of either the fluorescent-tagged peptides combi-2 or the previously studied peptide Ac-RRWWRF-NH(2) (combi-1) into Escherichia coli and Staphylococcus aureus suggests that instead of membrane disruption, the main bactericidal site of action of these peptides might be located inside bacteria.     

Structural studies of porcine myeloid antibacterial peptide PMAP-23 and its analogues in DPC micelles by NMR spectroscopy.

PMAP-23 is a cathelicidin-derived antimicrobial peptide identified from porcine leukocytes. PMAP-23 was reported to show potent antimicrobial activity against Gram-negative and Gram-positive bacteria without hemolytic activity. To study the structure-antibiotic activity relationships of PMAP-23, two analogues by replacing Trp with Ala were synthesized and their tertiary structures bound to DPC micelles have been studied by NMR spectroscopy. PMAP-23 has two alpha-helices, one from Arg1 to Arg10 in the N-terminal region and the other from Phe18 to Arg23 in the C-terminal region. PMAP-1 (Trp(7)-->Ala) shows similar structure to PMAP-23, while PMAP-2 (Trp(21)-->Ala) has a random structure in the C-terminus. PMAP-2 was found to show less antibacterial and vesicle-disrupting activities than PMAP-23 and PMAP-1 [J. H. Kang, S. Y. Shin, S. Y. Jang, K. L. Kim, and K.-S. Hahm (1999) Biochem. Biophys. Res. Commun. 264, 281-286]. Trp(21) in PMAP-23 which induces an alpha-helical structure in the second alpha-helix is essential for the antibacterial activity of PMAP-23. Also, the fluorescence data proved that Trp(21) at the second alpha-helix is buried deep into the phospholipid in the membrane. Therefore, it implies that Trp(21) in the second alpha-helix at the C-terminus of PMAP-23 may play an important role on the interactions with the membrane and the flexible region including two proline residues may allow this alpha-helix to span the lipid bilayer.     

Influence of specific amino acid side-chains on the antimicrobial activity and structure of bovine lactoferrampin

Lactoferrin is an 80 kDa iron binding protein found in the secretory fluids of mammals and it plays a major role in host defence. An antimicrobial peptide, lactoferrampin, was identified through sequence analysis of bovine lactoferrin and its antimicrobial activity against a wide range of bacteria and yeast species is well documented. In the present work, the contribution of specific amino acid residues of lactoferrampin was examined to evaluate the role that they play in membrane binding and bilayer disruption. The structures of all the bovine lactoferrampin derivatives were examined with circular dichroism and nuclear magnetic resonance spectroscopy, and their interactions with phospholipids were evaluated with differential scanning calorimetry and isothermal titration calorimetry techniques. From our results it is apparent that the amphipathic N-terminal helix anchors the peptide to membranes with Trp 268 and Phe 278 playing important roles in determining the strength of the interaction and for inducing peptide folding. In addition, the N-terminal helix capping residues (DLI) increase the affinity for negatively charged vesicles and they mediate the depth of membrane insertion. Finally, the unique flexibility in the cationic C-terminal region of bovine lactoferrampin does not appear to be essential for the antimicrobial activity of the peptide.     

Interactions of tryptophan-rich cathelicidin antimicrobial peptides with model membranes studied by differential scanning calorimetry

The 13-residue cathelicidins indolicidin and tritrpticin are part of a group of relatively short tryptophan-rich antimicrobial peptides that hold potential as future substitutes for antibiotics. Differential scanning calorimetry (DSC) has been applied here to study the effect of indolicidin and tritrpticin as well as five tritrpticin analogs on the phase transition behaviour of model membranes made up of zwitterionic dimyristoylphosphatidylcholine (DMPC, DMPC/cholesterol) and anionic dimyristoylphosphatidyl glycerol (DMPG) phospholipids. Most of the peptides studied significantly modified the phase transition profile, suggesting the importance of hydrophobic forces for the peptide interactions with the lipid bilayers and their insertion into the bilayer. Indolicidin and tritrpticin are both known to be flexible in aqueous solution, but they adopt turn-turn structures when they bind to and insert in a membrane surface. Pro-to-Ala substitutions in tritrpticin, which result in the formation of a stable α-helix in this peptide, lead to a substantial increase in the peptide interactions with both zwitterionic and anionic phospholipid vesicles. In contrast, the substitution of the three Trp residues by Tyr or Phe resulted in a significant decrease of the peptide's interaction with anionic vesicles and virtually eliminated binding of these peptides to the zwitterionic vesicles. An increase of the cationic charge of the peptide induced much smaller changes to the peptide interaction with all lipid systems than substitution of particular amino acids or modification of the peptide conformation. The presence of multiple lipid domains with a non-uniform peptide distribution was noticed. Slow equilibration of the lipid-peptide systems due to peptide redistribution was observed in some cases. Generally good agreement between the present DSC data and peptide antimicrobial activity data was obtained.     

Solution structure of a novel tryptophan-rich peptide with bidirectional antimicrobial activity

Trp-rich antimicrobial peptides play important roles in the host innate defense mechanisms of many plants, insects, and mammals. A new type of Trp-rich peptide, Ac-KWRRWVRWI-NH(2), designated Pac-525, was found to possess improved activity against both gram-positive and -negative bacteria. We have determined that the solution structures of Pac-525 bound to membrane-mimetic sodium dodecyl sulfate (SDS) micelles. The SDS micelle-bound structure of Pac-525 adopts an alpha-helical segment at residues Trp2, Arg3, and Arg4. The positively charged residues are clustered together to form a hydrophilic patch. The three hydrophobic residues Trp2, Val6, and Ile9 form a hydrophobic core. The surface electrostatic potential map indicates the three tryptophan indole rings are packed against the peptide backbone and form an amphipathic structure. Moreover, the reverse sequence of Pac-525, Ac-IWRVWRRWK-NH(2), designated Pac-525(rev), also demonstrates similar antimicrobial activity and structure in membrane-mimetic micelles and vesicles. A variety of biophysical and biochemical methods, including circular dichroism, fluorescence spectroscopy, and microcalorimetry, were used to show that Pac-525 interacted strongly with negatively charged phospholipid vesicles and induced efficient dye release from these vesicles, suggesting that the antimicrobial activity of Pac-525 may be due to interactions with bacterial membranes.     

MSI-78, an analogue of the magainin antimicrobial peptides,disrupts lipid bilayer structure via positive curvature strain

In this work, we present the first characterization of the cell lysing mechanism of MSI-78, an antimicrobial peptide. MSI-78 is an amphipathic alpha-helical peptide designed by Genaera Corporation as a synthetic analog to peptides from the magainin family. (31)P-NMR of mechanically aligned samples and differential scanning calorimetry (DSC) were used to study peptide-containing lipid bilayers. DSC showed that MSI-78 increased the fluid lamellar to inverted hexagonal phase transition temperature of 1,2-dipalmitoleoyl-phosphatidylethanolamine indicating the peptide induces positive curvature strain in lipid bilayers. (31)P-NMR of lipid bilayers composed of MSI-78 and 1-palmitoyl-2-oleoyl-phosphatidylethanolamine demonstrated that the peptide inhibited the fluid lamellar to inverted hexagonal phase transition of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine, supporting the DSC results, and the peptide did not induce the formation of nonlamellar phases, even at very high peptide concentrations (15 mol %). (31)P-NMR of samples containing 1-palmitoyl-2-oleoyl-phosphatidylcholine and MSI-78 revealed that MSI-78 induces significant changes in the bilayer structure, particularly at high peptide concentrations. At lower concentrations (1-5%), the peptide altered the morphology of the bilayer in a way consistent with the formation of a toroidal pore. Higher concentrations of peptide (10-15%) led to the formation of a mixture of normal hexagonal phase and lamellar phase lipids. This work shows that MSI-78 induces significant changes in lipid bilayers via positive curvature strain and presents a model consistent with both the observed spectral changes and previously published work.     

Antimicrobial activity and membrane selective interactions of a synthetic lipopeptide MSI-843

Lipopeptide MSI-843 consisting of the nonstandard amino acid ornithine (Oct-OOLLOOLOOL-NH₂) was designed with an objective towards generating non-lytic short antimicrobial peptides, which can have significant pharmaceutical applications. Octanoic acid was coupled to the N-terminus of the peptide to increase the overall hydrophobicity of the peptide. MSI-843 shows activity against bacteria and fungi at micromolar concentrations. It permeabilizes the outer membrane of Gram-negative bacterium and a model membrane mimicking bacterial inner membrane. Circular dichroism investigations demonstrate that the peptide adopts α-helical conformation upon binding to lipid membranes. Isothermal titration calorimetry studies suggest that the peptide binding to membranes results in exothermic heat of reaction, which arises from helix formation and membrane insertion of the peptide. ²H NMR of deuterated-POPC multilamellar vesicles shows the peptide-induced disorder in the hydrophobic core of bilayers. ³¹P NMR data indicate changes in the lipid head group orientation of POPC, POPG and Escherichia colitotal lipid bilayers upon peptide binding. Results from ³¹P NMR and dye leakage experiments suggest that the peptide selectively interacts with anionic bilayers at low concentrations (up to 5 mol%). Differential scanning calorimetry experiments on DiPOPE bilayers and ³¹P NMR data from E.coli total lipid multilamellar vesicles indicate that MSI-843 increases the fluid lamellar to inverted hexagonal phase transition temperature of bilayers by inducing positive curvature strain. Combination of all these data suggests the formation of a lipid-peptide complex resulting in a transient pore as a plausible mechanism for the membrane permeabilization and antimicrobial activity of the lipopeptide MSI-843.     

Structure and dynamics of helix-0 of the N-BAR domain in lipid micelles and bilayers

Bin/Amphiphysin/Rvs-homology (BAR) domains generate and sense membrane curvature by binding the negatively charged membrane to their positively charged concave surfaces. N-BAR domains contain an N-terminal extension (helix-0) predicted to form an amphipathic helix upon membrane binding. We determined the NMR structure and nano-to-picosecond dynamics of helix-0 of the human Bin1/Amphiphysin II BAR domain in sodium dodecyl sulfate and dodecylphosphocholine micelles. Molecular dynamics simulations of this 34-amino acid peptide revealed electrostatic and hydrophobic interactions with the detergent molecules that induce helical structure formation from residues 8-10 toward the C-terminus. The orientation in the micelles was experimentally confirmed by backbone amide proton exchange. The simulation and the experiment indicated that the N-terminal region is disordered, and the peptide curves to adopted the micelle shape. Deletion of helix-0 reduced tubulation of liposomes by the BAR domain, whereas the helix-0 peptide itself was fusogenic. These findings support models for membrane curving by BAR domains in which helix-0 increases the binding affinity to the membrane and enhances curvature generation.     

Structure and Membrane Interactions of the Antibiotic Peptide Dermadistinctin K by Multidimensional Solution and Oriented N and P Solid-State NMR Spectroscopy

DD K, a peptide first isolated from the skin secretion of the Phyllomedusa distincta frog, has been prepared by solid-phase chemical peptide synthesis and its conformation was studied in trifluoroethanol/water as well as in the presence of sodium dodecyl sulfate and dodecylphosphocholine micelles or small unilamellar vesicles. Multidimensional solution NMR spectroscopy indicates an α-helical conformation in membrane environments starting at residue 7 and extending to the C-terminal carboxyamide. Furthermore, DD K has been labeled with ¹⁵N at a single alanine position that is located within the helical core region of the sequence. When reconstituted into oriented phosphatidylcholine membranes the resulting ¹⁵N solid-state NMR spectrum shows a well-defined helix alignment parallel to the membrane surface in excellent agreement with the amphipathic character of DD K. Proton-decoupled ³¹P solid-state NMR spectroscopy indicates that the peptide creates a high level of disorder at the level of the phospholipid headgroup suggesting that DD K partitions into the bilayer where it severely disrupts membrane packing.     

Conditions affecting the re-alignment of the antimicrobial peptide PGLa in membranes as monitored by solid state H-NMR

The cationic antimicrobial peptide PGLa is electrostatically attracted to bacterial membranes, binds as an amphiphilic α-helix, and is thus able to permeabilize the lipid bilayer. Using solid state ²H-NMR of non-perturbing Ala-d₃ labels on the peptide, we have characterized the helix alignment under a range of different conditions. Even at a very high peptide-to-lipid ratio (1:20) and in the presence of negatively charged lipids, there was no indication of a toroidal wormhole structure. Instead, PGLa re-aligns from a surface-bound S-state to an obliquely tilted T-state, which is presumably dimeric. An intermediate structure half-way between the S- and T-state was observed in fully hydrated multilamellar DMPC vesicles at 1:50, suggesting a fast exchange between the two states on the time scale of >50 kHz. We demonstrate that this equilibrium is shifted from the S- towards the T-state either upon (i) increasing the peptide concentration, (ii) adding negatively charged DMPG, or (iii) decreasing the level of hydration. The threshold concentration for re-alignment in DMPC is found to be between 1:200 and 1:100 in oriented samples at 96% humidity. In fully hydrated multilamellar DMPC vesicles, it shifts to an effective peptide-to-lipid ratio of 1:50 as some peptides are able to escape into the bulk water phase.     

Structure and Dynamics of the Membrane-Bound Form of Pf1 Coat Protein: Implications of Structural Rearrangement for Virus Assembly

The three-dimensional structure of the membrane-bound form of the major coat protein of Pf1 bacteriophage was determined in phospholipid bilayers using orientation restraints derived from both solid-state and solution NMR experiments. In contrast to previous structures determined solely in detergent micelles, the structure in bilayers contains information about the spatial arrangement of the protein within the membrane, and thus provides insights to the bacteriophage assembly process from membrane-inserted to bacteriophage-associated protein. Comparisons between the membrane-bound form of the coat protein and the previously determined structural form found in filamentous bacteriophage particles demonstrate that it undergoes a significant structural rearrangement during the membrane-mediated virus assembly process. The rotation of the transmembrane helix (Q16-A46) around its long axis changes dramatically (by 160°) to obtain the proper alignment for packing in the virus particles. Furthermore, the N-terminal amphipathic helix (V2-G17) tilts away from the membrane surface and becomes parallel with the transmembrane helix to form one nearly continuous long helix. The spectra obtained in glass-aligned planar lipid bilayers, magnetically aligned lipid bilayers (bicelles), and isotropic lipid bicelles reflect the effects of backbone motions and enable the backbone dynamics of the N-terminal helix to be characterized. Only resonances from the mobile N-terminal helix and the C-terminus (A46) are observed in the solution NMR spectra of the protein in isotropic q > 1 bicelles, whereas only resonances from the immobile transmembrane helix are observed in the solid-state ¹H/¹⁵N-separated local field spectra in magnetically aligned bicelles. The N-terminal helix and the hinge that connects it to the transmembrane helix are significantly more dynamic than the rest of the protein, thus facilitating structural rearrangement during bacteriophage assembly.     

Possible role of a PXXP central hinge in the antibacterial activity and membrane interaction of PMAP-23, a member of cathelicidin family

Cathelicidins are essential components of the innate immune system of mammals, providing them a weapon against microbial invasion. PMAP-23 adopting a helix-hinge-helix structure with a central PXXP motif is a member of the cathelicidin family and has potent killing activities against a broad spectrum of microbial organisms. Although the antimicrobial effect of PMAP-23 is believed to be mediated by membrane disruption, many details of this event remain unclear. Here, we try to characterize the interaction between PMAP-23 and membrane phospholipids, focusing on the function of the central PXXP motif. PMAP-PA, in which the Pro residues were substituted by Ala, had significantly more alpha-helical content than PMAP-23, but was less amphipathic and more damaging to human erythrocytes and zwitterionic liposomes. The observed differences in the structures and biological activities of PMAP-23 and PMAP-PA confirmed the functional importance of the central hinge PXXP motif, which enables PMAP-23 to adopt a well-defined amphipathic conformation along its entire length and to have selective antimicrobial activity. CD and Trp fluorescence studies using fragments corresponding to the two helical halves of PMAP-23 revealed that the N-terminal half binds to anionic phospholipids and is more stable than the C-terminal half. In addition, Trp fluorescence quench analyses revealed that the C-terminal helix inserts more deeply into the hydrophobic region of the membrane than the N-terminal helix. Finally, observations made using biosensor technology enabled us to distinguish between the membrane binding and insertion steps, substantiating a proposed kinetic mode of the peptide-membrane interaction in which PMAP-23 first attaches to the membrane via the N-terminal amphipathic helix, after which bending and/or swiveling of the PXXP motif enables insertion of the C-terminal helix into the lipid bilayer.     

Interactions of tryptophan-rich cathelicidin antimicrobial peptides with model membranes studied by differential scanning calorimetry

The 13-residue cathelicidins indolicidin and tritrpticin are part of a group of relatively short tryptophan-rich antimicrobial peptides that hold potential as future substitutes for antibiotics. Differential scanning calorimetry (DSC) has been applied here to study the effect of indolicidin and tritrpticin as well as five tritrpticin analogs on the phase transition behaviour of model membranes made up of zwitterionic dimyristoylphosphatidylcholine (DMPC, DMPC/cholesterol) and anionic dimyristoylphosphatidyl glycerol (DMPG) phospholipids. Most of the peptides studied significantly modified the phase transition profile, suggesting the importance of hydrophobic forces for the peptide interactions with the lipid bilayers and their insertion into the bilayer. Indolicidin and tritrpticin are both known to be flexible in aqueous solution, but they adopt turn-turn structures when they bind to and insert in a membrane surface. Pro-to-Ala substitutions in tritrpticin, which result in the formation of a stable alpha-helix in this peptide, lead to a substantial increase in the peptide interactions with both zwitterionic and anionic phospholipid vesicles. In contrast, the substitution of the three Trp residues by Tyr or Phe resulted in a significant decrease of the peptide's interaction with anionic vesicles and virtually eliminated binding of these peptides to the zwitterionic vesicles. An increase of the cationic charge of the peptide induced much smaller changes to the peptide interaction with all lipid systems than substitution of particular amino acids or modification of the peptide conformation. The presence of multiple lipid domains with a non-uniform peptide distribution was noticed. Slow equilibration of the lipid-peptide systems due to peptide redistribution was observed in some cases. Generally good agreement between the present DSC data and peptide antimicrobial activity data was obtained.     

Interaction of staphylococcal delta-toxin and synthetic analogues with erythrocytes and phospholipid vesicles. Biological and physical properties of the amphipathic peptides

Staphylococcal delta-toxin, a 26-residue amphiphilic peptide is lytic for cells and phospholipid vesicles and is assumed to insert as an amphipathic helix and oligomerize in membranes. For the first time, the relationship between these properties and toxin structure is investigated by means of eight synthetic peptides, one identical in sequence to the natural toxin, five 26-residue analogues and two shorter peptides corresponding to residues 1-11 and 11-26. These peptides were designed by the Edmundson wheel axial projection in order to maintain: (a) the hydrophilic/hydrophobic balance while rationalizing the sequence, (b) the alpha-helical configuration and (c) the common epitopic structure. The fluorescence of the single Trp residue was used to monitor the behaviour of the natural toxin and analogues. All 26-residue analogues were hemolytically active although to a lesser extent than natural toxin. The peptide of residues 11-26 bound lipids weakly and was hemolytic at high concentration. The peptide of residues 1-11 did not bind lipids and was hemolytically inactive. All peptides except the latter cross-reacted in immunoprecipitation tests with the natural toxin. The study of a 26-residue analogue by circular dichroism revealed an alpha-helical configuration in both the free and lipid-bound state. Changes in the fluorescence of the peptides in the presence of lipid micelles and bilayers varied according to the position of the reporter group. When bound to lipids, Trp5, Trp16 and the Fmoc-1 positions of the analogues became buried while Trp15 of the natural toxin and its synthetic replicate remained more exposed. All changes are rationalized by the proposal of an amphipathic helix whose hydrophobic face is embedded within the apolar core of bilayers while the hydrophilic and charged face remains more exposed to solvent.     

Anchoring mechanisms of membrane-associated M13 major coat protein

Bacteriophage M13 major coat protein is extensively used as a biophysical, biochemical, and molecular biology reference system for studying membrane proteins. The protein has several elements that control its position and orientation in a lipid bilayer. The N-terminus is dominated by the presence of negatively charged amino acid residues (Glu2, Asp4, and Asp5), which will always try to extend into the aqueous phase and therefore act as a hydrophilic anchor. The amphipathic and the hydrophobic transmembrane part contain the most important hydrophobic anchoring elements. In addition there are specific aromatic and charged amino acid residues in these domains (Phe 11, Tyr21, Tyr24, Trp26, Phe42, Phe45, Lys40, Lys43, and Lys44) that fine-tune the association of the protein to the lipid bilayer. The interfacial Tyr residues are important recognition elements for precise protein positioning, a function that cannot be performed optimally by residues with an aliphatic character. The Trp26 anchor is not very strong: depending on the context, the tryptophan residue may move in or out of the membrane. On the other hand, Lys residues and Phe residues at the C-terminus of the protein act in a unique concerted action to strongly anchor the protein in the lipid bilayer.     

Antimicrobial and Membrane Disrupting Activities of a Peptide Derived from the Human Cathelicidin Antimicrobial Peptide LL37

A 21-residue peptide segment, LL7-27 (RKSKEKIGKEFKRIVQRIKDF), corresponding to residues 7-27 of the only human cathelicidin antimicrobial peptide, LL37, is shown to exhibit potent activity against microbes (particularly Gram-positive bacteria) but not against erythrocytes. The structure, membrane orientation, and target membrane selectivity of LL7-27 are characterized by differential scanning calorimetry, fluorescence, circular dichroism, and NMR experiments. An anilinonaphthalene-8-sulfonic acid uptake assay reveals two distinct modes of Escherichia coli outer membrane perturbation elicited by LL37 and LL7-27. The circular dichroism results show that conformational transitions are mediated by lipid-specific interactions in the case of LL7-27, unlike LL37. It folds into an α-helical conformation upon binding to anionic (but not zwitterionic) vesicles, and also does not induce dye leakage from zwitterionic lipid vesicles. Differential scanning calorimetry thermograms show that LL7-27 is completely integrated with DMPC/DMPG (3:1) liposomes, but induces peptide-rich and peptide-poor domains in DMPC liposomes. ¹⁵N NMR experiments on mechanically aligned lipid bilayers suggest that, like the full-length peptide LL37, the peptide LL7-27 is oriented close to the bilayer surface, indicating a carpet-type mechanism of action for the peptide. ³¹P NMR spectra obtained from POPC/POPG (3:1) bilayers containing LL7-27 show substantial disruption of the lipid bilayer structure and agree with the peptide's ability to induce dye leakage from POPC/POPG (3:1) vesicles. Cholesterol is shown to suppress peptide-induced disorder in the lipid bilayer structure. These results explain the susceptibility of bacteria and the resistance of erythrocytes to LL7-27, and may have implications for the design of membrane-selective therapeutic agents.     

Interaction of a pseudosubstrate peptide of protein kinase C and its myristoylated form with lipid vesicles: Only the myristoylated form translocates into the lipid bilayer

Lipopeptides derived from protein kinase C (PKC) pseudosubstrates have the ability to cross the plasma membrane in cells and modulate the activity of PKC in the cytoplasm. Myristoylation or palmitoylation appears to promote translocation across membranes, as the non-acylated peptides are membrane impermeant. We have investigated, by fluorescence spectroscopy, how myristoylation modulates the interaction of the PKC pseudosubstrate peptide KSIYRRGARRWRKL with lipid vesicles and translocation across the lipid bilayer. Our results indicate that myristoylated peptides are intimately associated with lipid vesicles and are not peripherally bound. When visualized under a microscope, myristoylation does appear to facilitate translocation across the lipid bilayer in multilamellar lipid vesicles. Translocation does not involve large-scale destabilization of the bilayer structure. Myristoylation promotes translocation into the hydrophobic interior of the lipid bilayer even when the non-acylated peptide has only weak affinity for membranes and is also only peripherally associated with lipid vesicles.     

Tryptophan Fluorescence Reveals Structural Features of α-Synuclein Oligomers

Oligomeric α-synuclein (αS) is considered to be the potential toxic species responsible for the onset and progression of Parkinson's disease, possibly through the disruption of lipid membranes. Although there is evidence that oligomers contain considerable amounts of secondary structure, more detailed data on the structural characteristics and how these mediate oligomer-lipid binding are critically lacking. This report is, to our knowledge, the first study that aimed to address the structure of oligomeric αS on a more detailed level. We have used tryptophan (Trp) fluorescence spectroscopy to gain insight into the structural features of oligomeric αS and the structural basis for oligomer-lipid interactions. Several single Trp mutants of αS were used to gain site-specific information about the microenvironments of monomeric αS, oligomeric αS and lipid-bound oligomeric αS. Acrylamide quenching and spectral analyses indicate that the Trp residues are considerably more solvent protected in the oligomeric form compared with the monomeric protein. In the oligomers, the negatively charged C-terminus was the most solvent exposed part of the protein. Upon lipid binding, a blue shift in fluorescence was observed for αS mutants where the Trp is located within the N-terminal region. These results suggest that, as in the case of monomeric αS, the N-terminus is critical in determining oligomer-lipid binding.