Melittin In Aqueous Solution Research Articles

Understanding Antimicrobial Peptide Synergy: Differential Binding Interactions and Their Impact on Membrane Integrity.

Research on antimicrobial peptides (AMPs) has been conducted as a solution to overcome antibiotic resistance. In particular, the synergistic effect that appears when two or more AMPs are used in combination has been observed. To find an effective synergistic combination, it is necessary to understand the underlying mechanism. However, a consistent explanation for this phenomenon has not yet been provided due to limitations in experimentally determining or predicting the structure of the heteroaggregates formed by the interactions between different AMPs and the interaction of the aggregate surface with the lipid membrane surface. In this study, we conducted molecular dynamics simulations for two heterogeneous aggregates of melittin-indolicidin and pexiganan-indolicidin to observe their structures in the solution phase and their interactions with the lipid membrane. We aimed to determine how the surfaces of these aggregates interact with the lipid membrane. Due to the different amino acid residue sequence characteristics of melittin and pexiganan, we found that when the two AMPs bind to indolicidin, they form aggregates with completely different structural characteristics. Accordingly, the sequence characteristics of pexiganan, which exhibits a relatively unstable structure compared to melittin in aqueous solution or on lipid membranes, allow for a more stable interaction with the lipid membrane when forming aggregates with indolicidin, effectively inhibiting the integrity of the lipid membranes. We also found that the amino acid residues forming the surface of the AMP aggregate show differential binding strengths to different lipid species forming the lipid membrane, thereby disrupting the membrane in a way that weakens its integrity. Through this, we provided insight into the basic principle of how the synergistic effect of AMPs occurs.

Topology and dynamics of melittin within the liposome revealed by a combination of mass spectrometry and chemical modification

The topology and dynamics of melittin within the liposome were investigated by a mass spectrometry coupled with acetylation. The MALDI-TOF MS and MALDI-QIT-TOF MS/MS analyses revealed that only N-terminal amine of melittin was dominantly acetylated in the presence of liposome although all of four primary amines were completely and rapidly acetylated in aqueous solution. This result indicates that melittin adopts the N-terminal-outside transmembrane topology within the liposome. The time course of acetylation followed the first-order kinetics at any examined temperatures (6–30°C). The rate constant was less than that of the acetylation of melittin in aqueous solution. The activation energy for acetylation (74kJmol−1) was comparable to that for dissociation of a lipid monomer from the membrane, suggesting a float-like longitudinal motion of melittin within the liposome. These results demonstrate that a mass spectrometry combined with chemical modification is very efficient way for clarifying the topology and dynamics of peptides bound to the membrane.

質量分析と化学修飾を用いた膜貫通ヘリックスの膜内ダイナミクス解析

We investigated the topology and dynamics of melittin within the liposomes using mass spectrometry combined with acetylation. According to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry/mass spectrometry (MALDI-QIT-TOF MS/MS) analyses, melittin within the liposomes was mono acetylated; with the acetylated position being the N-terminal of melittin. This acetylation followed first-order kinetics. The rate constant was less than that of the acetylation of melittin in aqueous solution. Thus, it is suggested that the observed rate constant is that for the release of the N-terminal region of melittin to a water phase from the hydrophobic core of the liposomes. This approach has opened a new method in the study of dynamics of transmembrane helices within the membranes using mass spectrometry.

Peptide Imprinted Polymer Nanoparticles: A Plastic Antibody

A novel method for preparation of biomacromolecular imprinted nanoparticles is described. Combinations of functional monomers were polymerized in the presence of the imprinting peptide melittin in aqueous solution at room temperature to produce a small library of polymer nanoparticles. The template peptide and unreacted monomers are subsequently removed by dialysis. Nanoparticles (NPs) from the library were evaluated for their binding to melittin by 27 MHz QCM analysis. NPs prepared with optimized functional monomer combinations bind strongly to the target molecule. Nanoparticles that were polymerized in the absence of template peptide were found to have little affinity to the peptide. Binding affinity and the size of imprinted particles are comparable to those of natural antibodies. They interact specifically with the target peptide and show little affinity for other proteins. These NPs are of interest as inert and stable substitutes for antibodies. Extension of this approach to other targets of biological importance and the applications of these materials are currently being evaluated.

Effect of ionic strength on folding and aggregation of the hemolytic peptide melittin in solution

Melittin is a cationic, amphipathic, hemolytic peptide composed of 26 amino acid residues. It is intrinsically fluorescent due to the presence of a single tryptophan residue, which has been shown to be crucial for its hemolytic activity. It undergoes a structural transition from a random coil monomer to an alpha-helical tetramer at high ionic strength. Although the aggregation behavior of melittin in solution is well characterized, dynamic information associated with the aggregation of melittin is lacking. In this paper, we have monitored the effect of ionic strength on the dynamics and aggregation behavior of melittin in aqueous solution by utilizing sensitive fluorescence approaches, which include the red edge excitation shift (REES) approach. Importantly, we demonstrate that REES is sensitive to the self-association of melittin induced by ionic strength. The change in environment experienced by melittin tryptophan(s) is supported by changes in fluorescence emission maximum, polarization, and lifetime. In addition, the accessibility of the tryptophan residue was probed by fluorescence quenching experiments using acrylamide and trichloroethanol as soluble and hydrophobic quenchers, respectively. Circular dichroism studies confirm the ionic strength-induced change in the secondary structure of melittin. Taken together, these results constitute the first report showing that REES could be used as a sensitive tool to monitor the aggregation behavior of melittin in particular and other proteins and peptides in general.

A molecular dynamics study of the bee venom melittin in aqueous solution, in methanol, and inserted in a phospholipid bilayer

The structural properties of melittin, a small amphipathic peptide found in the bee venom, are investigated in three different environments by molecular dynamics simulation. Long simulations have been performed for monomeric melittin solvated in water, in methanol, and shorter ones for melittin inserted in a dimyristoylphosphatidylcholine bilayer. The resulting trajectories were analysed in terms of structural properties of the peptide and compared to the available NMR data. While in water and methanol solution melittin is observed to partly unfold, the peptide retains its structure when embedded in a lipid bilayer. The latter simulation shows good agreement with the experimentally derived (3)J-coupling constants. Generally, it appears that higher the stability of the helical conformation of melittin, lower is the dielectric permittivity of the environment. In addition, peptide-lipid interactions were investigated showing that the C-terminus of the peptide provides an anchor to the lipid bilayer by forming hydrogen bonds with the lipid head groups.

Kinetics of conformational changes in melittin. A circular-dichroic stopped-flow study.

The kinetics of the conformational changes undergone by melittin in aqueous solution upon interaction with ions and/or detergents were studied by following the variations of intrinsic ellipticity of the peptide with a circular-dichroic stopped-flow apparatus. The results were consistent with a simplified model in which salt induces a modification of the structure of melittin monomer, which may then aggregate into a polymeric assembly. Interaction with detergent micelles followed more complex kinetics.

High-resolution 1H-NMR studies of self-aggregation of melittin in aqueous solution

4 mM melittin solution in 0.05 M sodium phosphate buffer at p 2H 7.0 and 30°C was shown by ultracentrifugation to contain tetrameric melittin. Using the spectra of this species and the previously characterized monomeric melittin as references, high-resolution 1H-NMR at 360 MHz was used to investigate self-aggregation of melittin at variable temperatures, pH and ionic strength. The NMR parameters show that the spatial structure of aggregated melittin is different from monomeric melittin in aqueous solution but resembles closely the conformation adopted by melittin bound to detergent micelles. Comparison of melittin bound to different detergent micelles and self-aggregated melittin in different aqueous media indicates that the melittin monomers adopt similar conformations in all these systems. The present data suggest that melittin assumes an amphiphilic spatial structure which is stabilized both by the formation of mixed micelles with detergents or by self-aggregation.

High-resolution 1H-NMR studies of monomeric melittin in aqueous solution

High resolution 1H-NMR at 360 MHz was used to characterize monomeric melittin in aqueous solution. The monomeric form of melittin was found to prevail at 3 mM concentration, pH 3.0, and temperatures between 30 and 90°C, both in the absence of salt and with 6 M guanidium chloride. From comparison with model peptides and studies of the effects of 6 M guanidium chloride and variable temperature on the NMR parameters it was concluded that monomeric melittin is predominantly in an extended flexible form, with the fragments 5–9 and 14–20 more highly structured than the rest of the amino acid sequence. The appearance of a second, low abundant form of monomeric melittin, which is in slow exchange on the NMR time scale with both the more abundant monomeric conformation and aggregated melittin, was attributed to cis-trans isomerism of the peptide bond Leu-13–Pro-14.

Physicochemical studies of the protein-lipid interactions in melittin-containing micelles

Complexes of melittin with detergents and phospholipids have been characterized by fluorescence, circular dichroism, ultracentrifugation, quasi-elastic light scattering and 1H nuclear magnetic resonance (NMR) experiments. By ultracentrifugation and quasi-elastic light-scattering measurements it is shown that melittin forms stoichiometrically well-defined complexes with dodecylphosphocholine micelles consisting of one melittin molecule and approximately forty detergent molecules. Evidence from fluorescence, circular dichroism and 1H nuclear magnetic resonance experiments indicates that the conformation of melittin bound to micelles of various detergents or of diheptanoyl phosphatidylcholine is largely independent of the type of lipid and furthermore appears to be quite closely related to the conformation of melittin bound to phosphatidylcholine bilayers. 1H NMR is used to investigate the conformation of micelle-bound melittin in more detail and to compare certain aspects of the melittin conformation in the micelles with the spatial structures of monomeric and self-aggregated tetrameric melittin in aqueous solution. The experience gained with this system demonstrates that high resolution NMR of complexes of membrane proteins with micelles provides a viable method for conformational studies of membrane proteins.

The structure of melittin in lipid bilayer membranes

0.15 M inorganic phosphate dramatically increased the α-helix content of melittin in aqueous solution. When melittin interacted with egg yolk phosphatidylcholine liposomes in the absence of inorganic phosphate, it was converted to an α-helix rich form, as postulated by Dawson et al. (Dawson, C.R., Drake, A.F. Helliwell, J. and Hider, R.C. (1978) Biochim. Biophys. Acta 510, 75–86).

Weitere Untersuchungen zur Aminosäuresequenz des Melittins

Melittin is the (main) toxic peptide of bee venom having a molecular weight of 2840, with a known sequence (s. fig. 1). Optical rotatory dispersion of non-crystalline melittin in aqueous solution suggests that the polypeptide chain is random, although 7% α-helix has been determined. These results are in agreement with the amino acid sequence of melittin and the assumption that the biological activity is attributable to its surface active character.