Molecular dynamics simulation of melittin in a dimyristoylphosphatidylcholine bilayer membrane.

Molecular dynamics trajectories of melittin in an explicit dimyristoyl phosphatidylcholine (DMPC) bilayer are generated to study the details of lipid-protein interactions at the microscopic level. Melittin, a small amphipathic peptide found in bee venom, is known to have a pronounced effect on the lysis of membranes. The peptide is initially set parallel to the membrane-solution interfacial region in an alpha-helical conformation with unprotonated N-terminus. Solid-state nuclear magnetic resonance (NMR) and polarized attenuated total internal reflectance Fourier transform infrared (PATIR-FTIR) properties of melittin are calculated from the trajectory to characterize the orientation of the peptide relative to the bilayer. The residue Lys7 located in the hydrophobic moiety of the helix and residues Lys23, Arg24, Gln25, and Gln26 at the C-terminus hydrophilic form hydrogen bonds with water molecules and with the ester carbonyl groups of the lipids, suggesting their important contribution to the stability of the helix in the bilayer. Lipid acyl chains are closely packed around melittin, contributing to the stable association with the membrane. Calculated density profiles and order parameters of the lipid acyl chains averaged over the molecular dynamics trajectory indicate that melittin has effects on both layers of the membrane. The presence of melittin in the upper layer causes a local thinning of the bilayer that favors the penetration of water through the lower layer. The energetic factors involved in the association of melittin at the membrane surface are characterized using an implicit mean-field model in which the membrane and the surrounding solvent are represented as structureless continuum dielectric material. The results obtained by solving the Poisson-Bolztmann equation numerically are in qualitative agreement with the detailed dynamics. The influence of the protonation state of the N-terminus of melittin is examined. After 600 ps, the N-terminus of melittin is protonated and the trajectory is continued for 400 ps, which leads to an important penetration of water molecules into the bilayer. These observations provide insights into how melittin interacts with membranes and the mechanism by which it enhances their lysis.     

pH-dependence of interaction between melittin and bacteriorhodopsin

Melittin differentially slowed down the fast (M412f) and the slow (M412s) decay components of the photocyde intermediate M of trimeric bacteriorhodopsin in purple membrane while it accelerated the M412s of Triton X-100-solubilized bacteriorhodopsin monomers. Raising the bulk pH could enhance the effect of melittin on the M412s of bacteriorhodopsin in these two states. From pH 5.5 to 8.8, melittin slightly influenced the yield of intermediate M in purple membrane, whereas the yield of M412s decreased and subsequently reversed with the addition of melittin. Moreover, the monomeric bacteriorhodopsin bleached more readily in the presence of melittin and the higher pH made the bleaching effect of melittin more intensive as well. These results re-certify our former suggestions that there was electrostatic interaction between melittin and bacteriorhodopsin, and indicate that the biphasic M decay may not result from the well-known linear kinetic scheme (M→N →BR). At last the mechanisms underlying the interact     

Attenuated total reflectance Fourier transform infrared studies of the interaction of melittin, two fragments of melittin, and delta-hemolysin with phosphatidylcholines

Attenuated total reflectance Fourier transform infrared spectroscopy (ATR FT-IR) has been used to monitor alterations in phospholipid organization in thin layers of 1,2-dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), induced by the membrane lytic peptide melittin, its fragments 1-15 (hydrophobic fragment) and 16-26 (hydrophilic fragment), and delta-hemolysin. In addition, the secondary structures of the peptides and the orientation of helical fragments were determined with respect to the bilayer. The insertion of melittin into POPC caused large perturbations in the order and increased rates of motion of the acyl chains, as monitored by the frequency and half-width of the symmetric CH2 stretching vibration near 2850 cm-1, as well as by the ATR dichroic ratio for this mode. Changes in DPPC organization were less and were consistent with peptide-induced static disordering (gauche rotamer formation) in the acyl chains. Melittin adopted primarily an alpha-helical secondary structure, although varying small proportions of beta and/or aggregated forms were noted. The helical segments were preferentially oriented perpendicular to the bilayer plane. Several modes of melittin/lipid interaction were considered in an attempt to semiquantitatively understand the observed dichroic ratios. By considering the peptide as a bent rigid rod, a plausible model for its lytic properties has been developed. The hydrophilic fragment in DPPC showed a secondary structure with little alpha-helix present. As judged by its effect on phospholipid acyl chain organizational parameters, the fragment did not penetrate the bilayer substantially. The hydrophobic fragment in DPPC gave amide I spectral patterns consistent with a mixture of predominantly beta-antiparallel pleated sheet with a smaller fraction of alpha-helix.(ABSTRACT TRUNCATED AT 250 WORDS)     

A deuterium and phosphorus-31 nuclear magnetic resonance study of the interaction of melittin with dimyristoylphosphatidylcholine bilayers and the effects of contaminating phospholipase A2

The interaction of bee venom melittin with dimyristolphosphatidylcholine (DMPC) selectively deuteriated in the choline head group has been studied by deuterium and phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy. The action of residual phospholipase A2 in melittin samples resulted in mixtures of DMPC and its hydrolytic products that underwent reversible transitions at temperatures between 30 and 35 degrees C from extended bilayers to micellar particles which gave narrow single-line deuterium and phosphorus-31 NMR spectra. Similar transitions were observed in DMPC-myristoyllysophosphatidylcholine (lysoPC)-myristic acid mixtures containing melittin but not in melittin-free mixtures, indicating that melittin is able to stabilize extended bilayers containing DMPC and its hydrolytic products in the liquid-crystalline phase. Melittin, free of phospholipase A2 activity, and at 3-5 mol% relative to DMPC, induced reversible transitions between extended bilayers and micellar particles on passing through the liquid-crystalline to gel phase transition temperature of the lipid, effects similar to those observed in melittin-acyl chain deuterated dipalmitoylphosphatidylcholine (DPPC) mixtures [Dufourc, E. J., Smith, I. C. P., & Dufourcq, J. (1986) Biochemistry 25, 6448-6455]. LysoPC at concentrations of 20 mol% or greater relative to DMPC induced transitions between extended bilayers and micellar particles with characteristics similar to those induced by melittin. It is proposed that these melittin- and lysoPC-induced transitions share similar mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural and thermodynamic analyses of the interaction between melittin and lipopolysaccharide

Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, is the very first site of interactions with the antimicrobial peptides. In this work, we have determined a solution conformation of melittin, a well-known membrane active amphiphilic peptide from honey bee venom, by transferred nuclear Overhauser effect (Tr-NOE) spectroscopy in its bound state with lipopolysaccharide. The LPS bound conformation of melittin is characterized by a helical structure restricted only to the C-terminus region (residues A15-R24) of the molecule. Saturation transfer difference (STD) NMR studies reveal that several C-terminal residues of melittin including Trp19 are in close proximity with LPS. Isothermal titration calorimetry (ITC) data demonstrates that melittin binding to LPS or lipid A is an endothermic process. The interaction between melittin and lipid A is further characterized by an equilibrium association constant (Ka) of 2.85 x 10(6) M(-1) and a stoichiometry of 0.80, melittin/lipid A. The estimated free energy of binding (delta G0), -8.8 kcal mol(-1), obtained from ITC experiments correlates well with a partial helical structure of melittin in complex with LPS. Moreover, a synthetic peptide fragment, residues L13-Q26 or mel-C, derived from the C-terminus of melittin has been found to contain comparable outer membrane permeabilizing activity against Escherichia coli cells. Intrinsic tryptophan fluorescence experiments of melittin and mel-C demonstrate very similar emission maxima and quenching in presence of LPS micelles. The Red Edge Excitation Shift (REES) studies of tryptophan residue indicate that both peptides are located in very similar environment in complex with LPS. Collectively, these results suggest that a helical conformation of melittin, at its C-terminus, could be an important element in recognition of LPS in the outer membrane.     

Structural and thermodynamic analyses of the interaction between melittin and lipopolysaccharide

Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, is the very first site of interactions with the antimicrobial peptides. In this work, we have determined a solution conformation of melittin, a well-known membrane active amphiphilic peptide from honey bee venom, by transferred nuclear Overhauser effect (Tr-NOE) spectroscopy in its bound state with lipopolysaccharide. The LPS bound conformation of melittin is characterized by a helical structure restricted only to the C-terminus region (residues A15-R24) of the molecule. Saturation transfer difference (STD) NMR studies reveal that several C-terminal residues of melittin including Trp19 are in close proximity with LPS. Isothermal titration calorimetry (ITC) data demonstrates that melittin binding to LPS or lipid A is an endothermic process. The interaction between melittin and lipid A is further characterized by an equilibrium association constant (K_a) of 2.85 × 10⁶ M^− 1 and a stoichiometry of 0.80, melittin/lipid A. The estimated free energy of binding (ΔG⁰), − 8.8 kcal mol^− 1, obtained from ITC experiments correlates well with a partial helical structure of melittin in complex with LPS. Moreover, a synthetic peptide fragment, residues L13-Q26 or mel-C, derived from the C-terminus of melittin has been found to contain comparable outer membrane permeabilizing activity against Escherichia coli cells. Intrinsic tryptophan fluorescence experiments of melittin and mel-C demonstrate very similar emission maxima and quenching in presence of LPS micelles. The Red Edge Excitation Shift (REES) studies of tryptophan residue indicate that both peptides are located in very similar environment in complex with LPS. Collectively, these results suggest that a helical conformation of melittin, at its C-terminus, could be an important element in recognition of LPS in the outer membrane.     

Investigation of the interaction between thallous ions and gramicidin A in dimyristoylphosphatidylcholine vesicles: a thallium-205 NMR equilibrium study

This study reports the first direct observation of multiple occupancy of the gramicidin A channel by Tl+ ions. 205Tl NMR has been used to study the equilibrium binding of Tl+ by gramicidin A incorporated in sonicated dimyristoylphosphatidylcholine vesicles. It is shown that only multiple-channel occupancy can account for the 205Tl chemical shifts measured. The data are analyzed to yield the equilibrium association constants of 450-600 and 5-20 M-1 for the binding of the first and the second ions at 34 degrees C, respectively.     

Effect of hydrophobic mismatch and interdigitation on sterol/sphingomyelin interaction in ternary bilayer membranes

Sphingomyelin (SM) is a major phospholipid in most cell membranes. SMs are composed of a long-chain base (often sphingosine, 18:1(Δ4t)), and N-linked acyl chains (often 16:0, 18:0 or 24:1(Δ15c)). Cholesterol interacts with SM in cell membranes, but the acyl chain preference of this interaction is not fully elucidated. In this study we have examined the effects of hydrophobic mismatch and interdigitation on cholesterol/sphingomyelin interaction in complex bilayer membranes. We measured the capacity of cholestatrienol (CTL) and cholesterol to form sterol-enriched ordered domains with saturated SM species having different chain lengths (14 to 24 carbons) in ternary bilayer membranes. We also determined the equilibrium bilayer partitioning coefficient of CTL with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes containing 20mol% of saturated SM analogs. Ours results show that while CTL and cholesterol formed sterol-enriched domains with both short and long-chain SM species, the sterols preferred interaction with 16:0-SM over any other saturated chain length SM analog. When CTL membrane partitioning was determined with fluid POPC bilayers containing 20mol% of a saturated chain length SM analog, the highest affinity was seen with 16:0-SM (both at 23 and 37°C). These results indicate that hydrophobic mismatch and/or interdigitation attenuate sterol/SM association and thus affect lateral distribution of sterols in the bilayer membrane.     

Direct interaction between cholesterol and phosphatidylcholines in hydrated membranes revealed by ATR-FTIR spectroscopy

By using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and curve fitting we have examined temperature dependence and composition dependence of the shape of the carbonyl band in phosphatidylcholine/cholesterol model membranes. Membranes were hydrated either in excess water or in excess deuterated water. The studied binary mixtures exhibit different lipid phases at appropriate temperature and amount of cholesterol, among them also the so-called liquid-ordered phase. The results confirm that cholesterol has a significant indirect influence on the carbonyl band through conformational and hydration effects. This influence was interpreted in view of the known temperature composition phase diagrams for inspected binary mixtures. In addition, direct interaction was observed, which could point to the presence of hydrogen bond between cholesterol and carbonyl group. This direct interaction, though weak, might play at least a partial role in the stabilization of cholesterol-rich lipid domains in model and biological membranes.     

Effects of bee venom melittin on the order and dynamics of dimyristoylphosphatidylcholine unilamellar and multilamellar vesicles

The effects of bee venom melittin on the order and dynamics of dimyristoylphosphatidylcholine unilamellar and multilamellar vesicles at a protein-to-lipid molar ratio of 1:60 have been investigated by employing the techniques of nanosecond emission anisotropy with 1,6-diphenyl-1,3,5-hexatriene as the fluorescent probe, enhancement by polar groups of the weakly allowed 0-0 vibronic transition in the fluorescence spectrum of pyrene, and Raman spectroscopy. The emission anisotropy results, which are found to be consistent with the wobble-in-cone model, show that the protein induces an increase in the order parameter, S, of the acyl chains of unilamellar vesicles below, at, and above their phase transition temperature, T_t, and it decreases strongly the diffusion rate, D_w, only below T_t. On the other hand, for multilamellar vesicles, the protein induces a decrease in S only at T_t and does not affect D_w. These effects are consistent with the observed changes in the degree of enhancement of the 0-0 vibronic transition of pyrene. Moreover, the protein broadens the thermal transition profile of multilamellar vesicles but sharpens dramatically that of unilamellar vesicles and fuses them without changing significantly the T_t in either case. On the other hand, the Raman data detect a decrease in the inter- and intramolecular order of the acyl chains of multilamellar vesicles below T_t and a decrease of only the former above T_t. This disparity between the Raman and the nanosecond emission anisotropy data is discussed in terms of differences in the time scales of the two techniques and in the state of aggregation of the lipid-bound melittin. The data for the enhancement of the 0-0 vibronic transition of pyrene suggest that, for a melittin-to-lipid ratio of 1:60, the size or structure of channels formed in the bilayer by melittin does not allow the penetration of a neutral molecule the size of pyrene deeply into the bilayer.     

The interaction of calmodulin with melittin

Studies utilizing the interaction of melittin with the 1-106 fragment of calmodulin, the protection of calmodulin from tryptic digestion by melittin, and the interaction of the carbocyanine dye Stains-all with the calmodulin-melittin complex have indicated that complex formation of calmodulin with melittin involves the alpha-helical connecting bridge joining the N- and C-terminal lobes of calmodulin.     

Investigation of the interaction between melittin and dipalmitoylphosphatidylglycerol bilayers by vibrational spectroscopy.

Melittin is shown to affect the structure of the charged phospholipid dipalmitoylphosphatidylglycerol (DPPG). In the gel phase, the presence of melittin leads to (i) an increased lipid interchain vibrational coupling, (ii) a shift of the rectangular to hexagonal lipid packing transition toward low temperatures, (iii) a very small conformational disordering effect, (iv) a decrease of the polarity or hydrogen bonding capability of the lipid ester group surrounding, (v) an important decrease of the water content in the complexes where the remaining water has a more disordered structure than bulk water, and (vi) an interlamellar repeat distance of 79 A. All these observations are rationalized by the following model: adjacent bilayers of DPPG are bridged by tetramers of melittin through electrostatic interactions inducing surface charge neutralization and partial dehydration of the complexes. Melittin also affects the thermotropic behavior of DPPG. When a small amount of the toxin is present, its affinity for charged lipids is such that a phase separation occurs, the domains being stable enough to have their own gel to liquid-crystalline phase transition. In the fluid state, a deeper penetration into the lipid matrix is proposed based on the downshift of the phase transition and the low vibrational interchain coupling. This study brings out general features of cationic species/anionic lipid complexes. The charge neutralization leads to stronger interchain coupling, and electrostatic bridging of adjacent bilayers seems to be common. The hydrophobicity of the peptide is a key factor in the modulation of the gel to liquid-crystalline phase transition and in its insertion in the fluid lipid matrix.     

Structural and thermodynamic aspects of the interaction between heparan sulfate and analogues of melittin

Melittin is an amphipathic cationic peptide derived from honeybee venom with well-known cytolytic and antimicrobial properties. When coupled to cationic polymers or lipid molecules, it forms conjugates with high transfection efficiency and low toxicity with promising applications in gene therapy. A first step in the internalization of melittin and its conjugates is their binding to the cell surface, a reaction likely to involve heparan sulfate proteoglycans (HSPG). In the present work, we characterize the binding equilibrium of heparan sulfate (HS) with two melittin analogues, [Cys(1)]melittin (mel-SH) and retro-inverso [Cys(1)]melittin (ri-mel-SH). The terminal cysteine found in these peptides replaces the N-terminal glycine present in native melittin and allows covalent binding to other molecules. Isothermal titration calorimetry (ITC) reveals a high affinity of each melittin analogue to HS. Association constants of 4.7 x 10(4) and 3.5 x 10(5) M(-)(1) are found at physiological ionic strength and 15 degrees C for ri-mel-SH and mel-SH, respectively. The reaction enthalpy measured under these conditions is DeltaH(degrees)pep= 4.2 kcal/mol for ri-mel-SH and DeltaH(degrees)pep= 1.1 kcal/mol for mel-SH. The peptide-to-HS stoichiometry is approximately 20 for ri-mel-SH and approximately 14 for mel-SH under the same conditions. Temperature dependence studies using ri-mel-SH (mel-SH) show that DeltaH(degrees)pep decreases in magnitude upon increase in temperature, which results in a molar heat capacity of DeltaH(degrees)pep= -322 cal mol(-)(1) K(-)(1) (-45 cal mol(-)(1) K(-)(1)). Such a negative heat capacity change is not expected for a purely electrostatic interaction and indicates that hydrophobic and other interactions are also involved in the binding equilibrium. Salt dependence studies of the binding constants confirm that nonelectrostatic forces are an important component of the HS-melittin interaction. Binding to HS induces conformational changes in both peptides, with ri-mel-SH showing a 6-fold increase of the alpha-helix content when incubated with HS under saturation conditions.     

Potential hydrophobic interaction between two cysteines in interior hydrophobic region improves thermostability of a family 11 xylanase from Neocallimastix Patriciarum

In this study, we employed directed evolution and site‐directed mutagenesis to screen thermostable mutants of a family 11 xylanase from Neocallimastix patriciarum, and found that the thermostability and specific activity are both enhanced when mutations (G201C and C60A) take place in the interior hydrophobic region of the enzyme. Far‐ultraviolet circular dichroism analysis showed that the melting temperatures (T_m) of the G201C and C60A–G201C mutants are higher than that of the wild type by about 10 and 12°C, respectively. At 72°C, their specific activities are about 4 and 6 times as that of the wild type, respectively. Homology modeling and site‐directed mutagenesis demonstrated that the enhanced thermostability of the G201C and C60A–G201C mutants may be mainly attributed to a potential stronger hydrophobic interaction between the two well‐packed cysteines at sites 50 and 201, rather than the disulfide bond formation which was ruled out by thiol titration with dithionitrobenzoic acid (DTNB). And the strength of such interaction depends on the packing of the side‐chain and hydrophobicity of residues at these two sites. This suggests that cysteine could stabilize a protein not only by forming a disulfide bond, but also by the strong hydrophobicity itself. Biotechnol. Bioeng. 2010;105: 861–870. © 2009 Wiley Periodicals, Inc.     

A hydrophilic and hydrophobic organic solvent mixture enhances enzyme stability in organic media

Binary mixtures of hydrophilic and hydrophobic solvents were assessed for their ability to balance enzyme activity with the conservation of enzyme stability in organic media. Acetone, dioxane and dodecane were chosen as model organic solvents, and subtilisin Carlsberg and horseradish peroxidase (HRP) were chosen as model enzymes. Residual enzyme activities were measured to monitor enzyme stability, and the fluorescence intensity of HRP was monitored to investigate structural changes due to the presence of an organic solvent. Enzyme stability increased with the increasing hydrophobicity of the solvent mixture used, and a solvent mixture with a high log P value (~ >4) was capable of conserving enzyme stability. Enzyme stability in organic media can be conserved therefore with a mixture of hydrophilic and hydrophobic solvents: this approach might be used as a general and practical strategy for optimizing enzyme activity and stability for industrial applications.     

The interaction of melittin with calmodulin and its tryptic fragments

Melittin has been found to interact with both the N- and C-terminal half-molecules of calmodulin, as well as the intact molecule, in the presence of Ca2+. The interaction results in a major change in the microenvironment of Trp-19, which is in a more nonpolar, solvent-shielded, and immobilized microenvironment in the complex. The properties of Tyr-99 and Tyr-138 of calmodulin are altered by complex formation. From measurements of the efficiencies of radiationless energy transfer from Trp-19 to the nitro derivatives of Tyr-99 and/or Tyr-138, it is concluded that Trp-19 is located in proximity to the C-terminal lobe of calmodulin in the complex.