Effect of lipid headgroup composition on the interaction between melittin and lipid bilayers

Strömstedt, Adam A.; Wessman, Per; Ringstad, Lovisa; Edwards, Katarina; Malmsten, Martin

doi:10.1016/j.jcis.2007.02.070

Cited by 63 publications

(78 citation statements)

References 51 publications

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“…Thus, for DOPG, melittin did not cause leakage in freshly added liposomes, presumably because of the extremely high binding coefficient (e.g. ϳ10-fold higher than for PC (14,16)), which dropped the free concentration of melittin to below that needed for leakage. Indeed, binding experiments indicated that melittin bound much more strongly to DOPG than to DOPC liposomes because virtually all melittin was absorbed to the membrane for melittin/lipid molecule ratios up to 0.1.…”

Section: Resultsmentioning

confidence: 97%

“…1A, step 1) and at higher concentrations shifts toward the perpendicular orientation (step 2), causing pore formation (10 -12). The transition from parallel to perpendicular is still poorly understood, especially because the cationic (5ϩ at neutral pH) melittin interacts strongly with the lipid headgroups (partitioning coefficient of 10 (12)(13)(14)(15)(16)(17)), and the energy of the transition must be very high (3,11). Based on the high affinity of melittin for PC headgroups, it has been widely accepted that the concentration of melittin needed for pore formation is not dependent on the absolute melittin concentration but rather on the ratio of melittin to lipid molecules.…”

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confidence: 99%

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On the Mechanism of Pore Formation by Melittin

Bogaart

Guzman

Mika

et al. 2008

Journal of Biological Chemistry

180

138

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The mechanism of pore formation of lytic peptides, such as melittin from bee venom, is thought to involve binding to the membrane surface, followed by insertion at threshold levels of bound peptide. We show that in membranes composed of zwitterionic lipids, i.e. phosphatidylcholine, melittin not only forms pores but also inhibits pore formation. We propose that these two modes of action are the result of two competing reactions: direct insertion into the membrane and binding parallel to the membrane surface. The direct insertion of melittin leads to pore formation, whereas the parallel conformation is inactive and prevents other melittin molecules from inserting, hence preventing pore formation.Lytic or antimicrobial peptides are small proteins (12-50 amino acids) that affect cells by disrupting the barrier function of lipid membranes (1). There is much interest in these peptides because of their potential pharmaceutical applications, e.g. as cancer drugs and antibiotics (2). Because of their small size and high stability, they can be obtained in large quantities. Of all lytic peptides, the 26-residue melittin is the best studied to date (for review see Ref.3). Melittin is the major constituent of the venom of the European honeybee Apis mellifera. Melittin has been reported to have anticancer effects and is already being used to treat pain and arthritis in Asia (4).Melittin can bind within milliseconds to lipid membranes and adopts an amphipatic ␣-helical conformation, oriented either parallel or perpendicular to the plane of the membrane. The perpendicular conformation is embedded in the membrane and is needed for pore formation, whereas the parallel conformation is inactive (5-10). These observations led to the proposal of a two-step model for pore formation, where melittin at low concentrations binds parallel to the membrane (Fig. 1A, step 1) and at higher concentrations shifts toward the perpendicular orientation (step 2), causing pore formation (10 -12). The transition from parallel to perpendicular is still poorly understood, especially because the cationic (5ϩ at neutral pH) melittin interacts strongly with the lipid headgroups (partitioning coefficient of 10 4 -10 5 M Ϫ1 for phosphatidylcholine (PC) 2 (12-17)), and the energy of the transition must be very high (3,11). Based on the high affinity of melittin for PC headgroups, it has been widely accepted that the concentration of melittin needed for pore formation is not dependent on the absolute melittin concentration but rather on the ratio of melittin to lipid molecules. However, this important implication has never been directly tested by varying the lipid concentration and determining the fractions of bound and free melittin. In this study we analyzed the lipid concentration dependence of melittin action and studied the reversibility of melittin binding, thereby resolving pore formation from binding events. MATERIALS AND METHODSMelittin was purchased from Genscript (Piscataway, NJ), and lipids were from Avanti Polar Lipids (Albaster, AL). Liposomes ...

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Section: Resultsmentioning

confidence: 97%

mentioning

confidence: 99%

On the Mechanism of Pore Formation by Melittin

Bogaart

Guzman

Mika

et al. 2008

Journal of Biological Chemistry

180

138

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“…A J810 spectropolarimeter (Jasco Corporation, Easton, MD) was used in the range of 190 to 260 nm at 37°C in a quartz cuvette at a peptide concentration of 10 M. The helix content was quantified at 222 nm (17) to 225 VOL. 53,2009 METHODS FOR INCREASING PROTEOLYTIC STABILITY 595 nm (49) using reference spectra for samples containing 100% ␣-helix and 100% random coil. As a reference, samples of 0.133 mM (monomer concentration) poly-L-lysine in 0.1 M NaOH or in 0.1 M HCl, respectively, were used (49).…”

Section: Methodsmentioning

confidence: 99%

“…on May 11, 2018 by guest http://aac.asm.org/ interferes with electrostatic arrest at the interface of anionic membranes (in analogy with melittin and phosphatidylinositol membranes) (53), resulting in a more profound effect of W substitutions on bacteria. Furthermore, since the W substitutions may in our case have a reducing effect on peptide hydrophobicity depending on the hydrophobicity scale employed, it is of interest in the present context since it has been found that an increased level of hydrophobicity for helical amphiphilic AMP may specifically increase the lytic property on erythrocytes rather than bacteria (67).…”

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confidence: 99%

Evaluation of Strategies for Improving Proteolytic Resistance of Antimicrobial Peptides by Using Variants of EFK17, an Internal Segment of LL-37

Strömstedt

Pasupuleti

Schmidtchen

et al. 2009

Antimicrob Agents Chemother

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177

143

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Methods for increasing the proteolytic stability of EFK17 (EFKRIVQRIKDFLRNLV), a new peptide sequence with antimicrobial properties derived from LL-37, were evaluated. EFK17 was modified by four d-enantiomer or tryptophan (W) substitutions at known protease cleavage sites as well as by terminal amidation and acetylation. The peptide variants were studied in terms of proteolytic resistance, antibacterial potency, and cytotoxicity but also in terms their adsorption at model lipid membranes, liposomal leakage generation, and secondary-structure behavior. The W substitutions resulted in a marked reduction in the proteolytic degradation caused by human neutrophil elastase, Staphylococcus aureus aureolysin, and V8 protease but not in the degradation caused by Pseudomonas aeruginosa elastase. For the former two endoproteases, amidation and acetylation of the terminals also reduced proteolytic degradation but only when used in combination with W substitutions. The d-enantiomer substitutions rendered the peptides indigestible by all four proteases; however, those peptides displayed little antimicrobial potency. The W-and end-modified peptides, on the other hand, showed an increased bactericidal potency compared to that of the native peptide sequence, coupled with a moderate cytotoxicity that was largely absent in serum. The bactericidal, cytotoxic, and liposome lytic properties correlated with each other as well as with the amount of peptide adsorbed at the lipid membrane and the extent of helix formation associated with the adsorption. The lytic properties of the W-substituted peptides were less impaired by increased ionic strength, presumably by a combination of W-mediated stabilization of the largely amphiphilic helix conformation and a nonelectrostatic W affinity for the bilayer interface. Overall, W substitutions constitute an interesting means to reduce the proteolytic susceptibility of EFK17 while also improving antimicrobial performance.The cathelicidins are a major family of antibacterial peptides in mammals (66). The human cathelicidin hCAP-18 is released by several types of epithelial and immune cells, notably neutrophils, in response to inflammation and subsequently cleaved to generate the active C-terminal LL-37 peptide (18, 52). LL-37 is an extensively investigated peptide displaying strong antibacterial effects also at a high ionic strength (57). It also displays a range of other interesting properties, e.g., relating to lipopolysaccharide neutralization, immune cell stimulation and attraction, reepithelization, and wound closure (13). Although LL-37 displays some cytotoxicity against human cells, possibly originating from a hydrophobic interaction with cell membranes (31), this is largely absent in human serum due to interactions with apolipoproteins and other serum factors (61). It was previously found that reducing the number of residues from the N terminus of LL-37 decreases its cytotoxicity while retaining the bactericidal properties, although such truncations may also result in an increased proteolyt...

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“…A wide range of techniques is available to characterize protein-membrane interactions. To name but a few, ellipsometry may be used to determine the amount of adsorbed dry mass [17,18] while surface plasmon resonance follows the kinetics of adsorption and release [19,20] and solid state 31 P, 14 N and 2 H-NMR can follow the changes in the chemical environment of the lipid headgroups [2,21]. We describe the orientation of the a-helical structure in lipid membranes using oriented circular dichroism (OCD) and describe the membrane perturbation using quartz crystal microbalance with dissipation (QCM-D) to measure the mass and determine the reversibility of peptide binding to supported lipid bilayers.…”

Section: Introductionmentioning

confidence: 99%