Melittin Tetramer Research Articles

Inhibition of Melittin Activity Using a Small Molecule with an Indole Ring.

We investigated d-amino acids as potential inhibitors targeting l-peptide toxins. Among the l- and d-amino acids tested, we found that d-tryptophan (d-Trp) acted as an inhibitor of melittin-induced hemolysis. We then evaluated various Trp derivatives and found that 5-chlorotryptamine (5CT) had the largest inhibitory effect on melittin. The indole ring, amino group, and steric hindrance of an inhibitor played important roles in the inhibition of melittin activity. Despite the small size and simple molecular structure of 5CT, its IC50 was approximately 13 μg/mL. Fluorescence quenching, circular dichroism measurements, and size-exclusion chromatography revealed that 5CT interacted with Trp19 in melittin and affected the formation of the melittin tetramer involved in hemolysis. Molecular dynamics simulation of melittin also indicated that the interaction of 5CT with Trp19 in melittin affected the formation of the tetramer.

Millisecond Time-Resolved Solid-State NMR Initiated by Rapid Inverse Temperature Jumps

Elucidation of the detailed mechanisms by which biological macromolecules undergo major structural conversions, such as folding, complex formation, and self-assembly, is a central concern of biophysical chemistry that will benefit from new experimental methods. We describe a simple technique for initiating a structural conversion process by a rapid decrease in the temperature of a solution, i.e., a rapid inverse temperature jump. By pumping solutions through copper capillary tubes that are thermally anchored to heated and cooled blocks, solution temperatures can be switched from 95 to 30 °C (or lower) in about 0.8 ms. For time-resolved solid-state nuclear magnetic resonance (ssNMR), solutions can then be frozen rapidly by spraying into cold isopentane after a variable structural evolution time τe. As an initial demonstration, we use this "inverse T-jump" technique to characterize the kinetics and mechanism by which the 26-residue peptide melittin converts from its primarily disordered, monomeric state at 95 °C to its α-helical, tetrameric state at 30 °C. One- and two-dimensional ssNMR spectra of frozen solutions with various values of τe, recorded at 25 K with signal enhancements from dynamic nuclear polarization, show that both helical secondary structure and intermolecular contacts develop on the same time scale of about 6 ms. The dependences on τe of both intraresidue crosspeak patterns and inter-residue crosspeak volumes in two-dimensional spectra can be fit with a unidirectional dimerization model, consistent with dimerization being the rate-limiting step for melittin tetramer formation.

Effects of Peptide Charge, Orientation, and Concentration on Melittin Transmembrane Pores

Melittin is a short cationic peptide that exerts cytolytic effects on bacterial and eukaryotic cells. Experiments suggest that in zwitterionic membranes, melittin forms transmembrane toroidal pores supported by four to eight peptides. A recently constructed melittin variant with a reduced cationic charge, MelP5, is active at 10-fold lower concentrations. In previous work, we performed molecular dynamics simulations on the microsecond timescale to examine the supramolecular pore structure of a melittin tetramer in zwitterionic and partially anionic membranes. We now extend that study to include the effects of peptide charge, initial orientation, and number of monomers on the pore formation and stabilization processes. Our results show that parallel transmembrane orientations of melittin and MelP5 are more consistent with experimental data. Whereas a MelP5 parallel hexamer forms a large stable pore during the 5-μs simulation time, a melittin hexamer and an octamer are not fully stable, with several monomers dissociating during the simulation time. Interaction-energy analysis shows that this difference in behavior between melittin and MelP5 is not due to stronger electrostatic repulsion between neighboring melittin peptides but to peptide-lipid interactions that disfavor the isolated MelP5 transmembrane monomer. The ability of melittin monomers to diffuse freely in the 1,2-dimyristoyl-SN-glycero-3-phosphocholine membrane leads to dynamic pores with varying molecularity.

Melittin Aggregation in Aqueous Solutions: Insight from Molecular Dynamics Simulations.

Melittin is a natural peptide that aggregates in aqueous solutions with paradigmatic monomer-to-tetramer and coil-to-helix transitions. Since little is known about the molecular mechanisms of melittin aggregation in solution, we simulated its self-aggregation process under various conditions. After confirming the stability of a melittin tetramer in solution, we observed—for the first time in atomistic detail—that four separated melittin monomers aggregate into a tetramer. Our simulated dependence of melittin aggregation on peptide concentration, temperature, and ionic strength is in good agreement with prior experiments. We propose that melittin mainly self-aggregates via a mechanism involving the sequential addition of monomers, which is supported by both qualitative and quantitative evidence obtained from unbiased and metadynamics simulations. Moreover, by combining computer simulations and a theory of the electrical double layer, we provide evidence to suggest why melittin aggregation in solution likely stops at the tetramer, rather than forming higher-order oligomers. Overall, our study not only explains prior experimental results at the molecular level but also provides quantitative mechanistic information that may guide the engineering of melittin for higher efficacy and safety.

The Structure of a Melittin-Stabilized Pore

Melittin has been reported to form toroidal pores under certain conditions, but the atomic-resolution structure of these pores is unknown. A 9-μs all-atom molecular-dynamics simulation starting from a closely packed transmembrane melittin tetramer in DMPC shows formation of a toroidal pore after 1 μs. The pore remains stable with a roughly constant radius for the rest of the simulation. Surprisingly, one or two melittin monomers frequently transition between transmembrane and surface states. All four peptides are largely helical. A simulation in a DMPC/DMPG membrane did not lead to a stable pore, consistent with the experimentally observed lower activity of melittin on anionic membranes. The picture that emerges from this work is rather close to the classical toroidal pore, but more dynamic with respect to the configuration of the peptides.

Computational probe of cavitation events in protein systems

Previous all-atom simulations have identified several classes of proteins where hydrophobic de-wetting (cavitation) is at play. Here we develop and validate a computationally fast method that predicts in which protein systems water spontaneously cavitates. We implement a cubic lattice model, which incorporates the protein shape from crystallographic data and the protein-water interactions from thermodynamic data. Combining it with the previously developed coarse-grained model for water, we determine the extent of occupancy of water at protein-protein interfaces and in protein-ligand cavities. The model captures essential findings from all-atom molecular dynamics studies on the same systems by distinguishing protein cavities that dry from those that remain wet. We also interpret the origin of the cavitation inside the melittin tetramer on simple thermodynamic grounds, and show that part of the mellitin surface is sufficiently hydrophobic to trigger cavitation. Using Glauber/Kawasaki dynamics we obtain the time-scales for de-wetting events that are in agreement with those from all-atom simulations. The method can serve as an intermediate step between the necessary initial screening that identifies proteins with abundance of hydrophobic patches using bioinformatics tools (L. Hua, X. H. Huang, P. Liu, R. H. Zhou and B. J. Berne, J. Phys. Chem. B, 2007, 111, 9069), and computationally extensive studies that need to incorporate molecular details (e.g. single mutation studies of amino acid residues).

Antimicrobial peptides in toroidal and cylindrical pores

Antimicrobial peptides (AMPs) are small, usually cationic peptides, which permeabilize biological membranes. Their mechanism of action is still not well understood. Here we investigate the preference of alamethicin and melittin for pores of different shapes, using molecular dynamics (MD) simulations of the peptides in pre-formed toroidal and cylindrical pores. When an alamethicin hexamer is initially embedded in a cylindrical pore, at the end of the simulation the pore remains cylindrical or closes if glutamines in the N-termini are not located within the pore. On the other hand, when a melittin tetramer is embedded in toroidal pore or in a cylindrical pore, at the end of the simulation the pore is lined both with peptides and lipid headgroups, and, thus, can be classified as a toroidal pore. These observations agree with the prevailing views that alamethicin forms barrel-stave pores whereas melittin forms toroidal pores. Both alamethicin and melittin form amphiphilic helices in the presence of membranes, but their net charge differs; at pH ∼ 7, the net charge of alamethicin is − 1 whereas that of melittin is + 5. This gives rise to stronger electrostatic interactions of melittin with membranes than those of alamethicin. The melittin tetramer interacts more strongly with lipids in the toroidal pore than in the cylindrical one, due to more favorable electrostatic interactions.

Antimicrobial Peptides in Toroidal and Cylindrical Pores

Antimicrobial peptides (AMPs) are small, usually cationic peptides, which permeabilize biological membranes. Understanding their mechanism of action might help design better antibiotics. Using molecular dynamics (MD) simulations, we investigate the preference of alamethicin and melittin for pores of different shapes. In the simulations, an alamethicin hexamer initially embedded in a pre-formed cylindrical pore preserves the pore shape or closes the pore if glutamines in the N-terminus are not located within the pore. On the other hand, when a melittin tetramer is embedded in a toroidal pore or in a cylindrical pore, at the end of the simulations the pore is lined both with peptides and lipid headgroups, and, thus, can be classified as a toroidal pore. These observations agree with the prevailing views that alamethicin forms barrel-stave pores whereas melittin forms toroidal pores. The melittin tetramer interacts more strongly with lipids in the toroidal pore than in the cylindrical one, due to more favorable electrostatic interactions. Using an implicit membrane model, modified to include pores of different shapes, we show that melittin is better solvated in toroidal pores than in cylindrical ones.

Solvation in protein (un)folding of melittin tetramer–monomer transition

Protein structural integrity and flexibility are intimately tied to solvation. Here, we examine the effect that changes in bulk and local solvent properties have on protein structure and stability. We observe the change in solvation of an unfolding of the protein model, melittin, in the presence of a denaturant, trifluoroethanol. The peptide system displays a well defined transition in that the tetramer unfolds without disrupting the secondary or tertiary structure. In the absence of local structural perturbation, we are able to reveal exclusively the role of solvation dynamics in protein structure stabilization and the (un)folding pathway. A sudden retardation in solvent dynamics, which is coupled to the change in protein structure, is observed at a critical trifluoroethanol concentration. The large amplitude conformational changes are regulated by the local solvent hydrophobicity and bulk solvent viscosity.

Nanoscale dewetting transition in protein complex folding.

In a previous study, a surprising drying transition was observed to take place inside the nanoscale hydrophobic channel in the tetramer of the protein melittin. The goal of this paper is to determine if there are other protein complexes capable of displaying a dewetting transition during their final stage of folding. We searched the entire protein data bank (PDB) for all possible candidates, including protein tetramers, dimers, and two-domain proteins, and then performed the molecular dynamics (MD) simulations on the top candidates identified by a simple hydrophobic scoring function based on aligned hydrophobic surface areas. Our large scale MD simulations found several more proteins, including three tetramers, six dimers, and two two-domain proteins, which display a nanoscale dewetting transition in their final stage of folding. Even though the scoring function alone is not sufficient (i.e., a high score is necessary but not sufficient) in identifying the dewetting candidates, it does provide useful insights into the features of complex interfaces needed for dewetting. All top candidates have two features in common: (1) large aligned (matched) hydrophobic areas between two corresponding surfaces, and (2) large connected hydrophobic areas on the same surface. We have also studied the effect on dewetting of different water models and different treatments of the long-range electrostatic interactions (cutoff vs PME), and found the dewetting phenomena is fairly robust. This work presents a few proteins other than melittin tetramer for further experimental studies of the role of dewetting in the end stages of protein folding.

Monte Carlo simulation study of melittin: Protein folding and temperature dependence

The tetramerization of melittin, a 26-amino-acid peptide, is considered as a model for protein folding. The Monte Carlo simulation was used to study the folding arrangement of melittin, and the results are compared with the experiment. An acceptance rate of 50% for new configurations is achieved by using ranges of ±0.001 A for the translations and ±15°C for the rotations. Around 311 K, the folded structure of the protein has the greatest stability; the range from −40 to −80 shows the best ϕ angles for melittin. The final optimized structure of melittin strongly depends on the temperature. The melittin tetramer is found to have a temperature of maximum stability ranging from 35.5 to 43°C.

Structural and functional behavior of biologically active monomeric melittin

Melittin is a well-known water-soluble toxic peptide present in bee venom of Apis mellifera, capable of interacting with and disrupting cell membranes thus producing many effects on living cells. Additionally, melittin induces activation of phospholipases and calmodulin upon interaction with cellular membranes. The conformation and aggregation state adopted by melittin in solution depends on several factors including the peptide concentration, ionic strength, pH and the nature of the ions in the aqueous medium. Such conformational dependence on the peptide environment gives new insights over the currently available 3D structures of melittin and, ultimately, over its biologically functional unit. Based on crystallographic data, the melittin tetramer has been proposed as its bioactive form. Contrarily to such data, we show in this work the results obtained from molecular dynamics simulations, which clearly indicate that the tetrameric organization of melittin is not stable under biological conditions dissociating after 2.5 ns through a 10 ns trajectory. We found that the tetrameric form of melittin is stable only in conditions of high pH and high peptide concentration in the molecular dynamics simulations. Moreover, when in plasma melittin appears to be a random coil monomer, folding only upon interaction with biological membranes. In summary, these findings elucidate several properties of melittin structure and dynamics, projecting significant implications in the study of its biological function.

Observation of a dewetting transition in the collapse of the melittin tetramer

Marked hydration changes occur during the self-assembly of the melittin protein tetramer in water. Hydrophobicity induces a drying transition in the gap between simple sufficiently large (more than 1 nm(2)) strongly hydrophobic surfaces as they approach each other, resulting in the subsequent collapse of the system, as well as a depletion of water next to single surfaces. Here we investigate whether the hydrophobic induced collapse of multidomain proteins or the formation of protein oligimers exhibits a similar drying transition. We performed computer simulations to study the collapse of the tetramer of melittin in water, and observed a marked water drying transition inside a nanoscale channel of the tetramer (with a channel size of up to two or three water-molecule diameters). This transition, although occurring on a microscopic length scale, is analogous to a first-order phase transition from liquid to vapour. We find that this drying is very sensitive to single mutations of the three isoleucines to less hydrophobic residues and that such mutations in the right locations can switch the channel from being dry to being wet. Thus, quite subtle changes in hydrophobic surface topology can profoundly influence the drying transition. We show that, even in the presence of the polar protein backbone, sufficiently hydrophobic protein surfaces can induce a liquid-vapour transition providing an enormous driving force towards further collapse. This behaviour was unexpected because of the absence of drying in the collapse of the multidomain protein 2,3-dihydroxybiphenyl dioxygenase (BphC).

The structure of the melittin tetramer at different temperatures--an NOE-based calculation with chemical shift refinement.

The structure of the bee venom peptide melittin has been determined in water at pH 6.0, 50 mM sodium phosphate, at various temperatures. At all temperatures studied, the peptide is tetrameric, and consists of two helical regions (residues 2-11 and 13-23) with an unstructured C-terminal region. The connection between the helices (residues 11-13) is well defined. At 30 degrees C, the structure of the monomeric unit has been characterised using NOEs, and a family of structures is presented (root-mean-square deviation to the mean structure 1.4 A over the structured residues), with low NOE violations and good stereochemistry. The angle between the helices is 46+/-13 degrees, and the structure is very similar to the previously determined crystal structure of the aqueous tetramer. The peptide forms a tetramer that is made up from a dimer of dimers. The structure of the dimeric unit has been determined, using a novel joint refinement of intermonomer NOEs and chemical shifts. The relative position of the monomeric units in the dimer is different from that in the crystal, with less direct contact between monomers. As the temperature is raised to 70 degrees C, the peptide remains tetrameric, but the monomer units start to separate, as shown by a reduction in intermonomer NOE intensities and chemical shifts. The structural changes have been characterised: over the temperature range studied, the monomers separate by approximately 2.0 A. This movement may have implications for the mechanism by which melittin inserts into membranes.

Self-association of disulfide-dimerized melittin analogues.

Two cysteine substitutions of bee venom melittin have been synthesized to investigate the effects of disulfide cross-linking on the self-association properties of the peptide in solution. K23C melittin (mltK23C) was designed to link nonpolar surfaces of the amphipathic melittin helix on the basis of the close juxtaposition of pairs of K23 side chains in the crystal of the native melittin tetramer. K23Q/Q25C melittin (mltQ25C) was designed to link the polar surfaces of the peptide such that self-association in membrane bound states might be stabilized. The mltK23C disulfide dimer, (mltK23C)2, is highly structured at low pH under conditions where native melittin, and the mltK23C monomer, are unstructured. High-resolution NMR, circular dichroism, and fluorescence spectroscopy established that (mltK23C)2 is a helical monomer (pseudodimer) with stable helical segments between residues 2-13 and 15-25. Although the symmetrical nature of the pseudodimer prevented high-resolution structure determination, analysis of calculated hydrogen bond lengths, chemical shifts, near-UV circular dichroism, and urea denaturation demonstrated similarities with alpha-helical coiled coils and with the structure of native melittin in methanol. Stopped flow fluorescence showed that (mltK23C)2 underwent pH- and divalent anion-linked dimerization to a melittin-like pseudotetramer, indicating that a pair of disulfide bonds could be accommodated in a structure similar to the native melittin crystal structure. Despite incorporation of two disulfide bonds into the melittin tetramer, the folding free energy (DeltaGw) of [(mltK23C)2]2 was similar to that for the native melittin tetramer under the condition used. Incorporation of a disulfide bond on the polar helix face in melittin did not stabilize helical structure in the absence of self-association. Instead, this molecule underwent pH- and divalent anion-linked self-association to an ill-defined aggregate which precipitated.

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding

The tetramerization of melittin, a 26-amino acid peptide from Apis mellifera bee venom, has been studied as a model for protein folding. Melittin converts from a monomeric random coil to an alpha-helical tetramer as the pH is raised from 4.0 to 9.5, as ionic strength is increased, as temperature is raised or lowered from about 37 degrees C, or as phosphate is added. The thermodynamics of this tetramerization (termed "folding") are explored using circular dichroism. The melittin tetramer has two pKa values of 7.5 and 8.5 corresponding to protonation of the N-terminus and Lys 23, respectively. pKa values calculated with the program DelPhi (Gilson, M.K., Sharp, K.A., & Honig, B.H., 1987, J. Comp. Chem. 9, 327-335; Gilson, M.K. & Honig, B.H., 1988a, Proteins 3, 32-52; Gilson, M.K. & Honig, B.H., 1988b, Proteins 4, 7-18) agree with experimental titration data. Greater electrostatic repulsion of these protonated groups destabilizes the tetramer by 3.6 kcal/mol at pH 4.0 compared to pH 9.5. Increasing the concentration of NaCl in the solution from 0 to 0.5 M stabilizes the tetramer by 5-6 kcal/mol at pH 4.0. The effect of NaCl is modeled with a ligand-binding approach. The melittin tetramer is found to have a temperature of maximum stability ranging from 35.5 to 43 degrees C depending on the pH, unfolding above and below that temperature. delta Cp0 for folding ranges from -0.085 to -0.102 cal g-1 K-1, comparable to that of other small globular proteins (Privalov, P.L., 1979, Adv. Protein Chem. 33, 167-241). delta H0 and delta S0 are found to decrease with temperature, presumably due to the hydrophobic effect (Kauzmann, W., 1959, Adv. Protein Chem. 14, 1-63). Phosphate is found to perturb the equilibrium substantially with a maximal effect at 150 mM, stabilizing the tetramer at pH 7.4 and 25 degrees C by 4.6 kcal/mol. The enthalpy change due to addition of phosphate (-7.5 kcal/mol at 25 degrees C) can be accounted for by simple dielectric screening. Both circular dichroism and crystallographic results suggest that phosphate may bind Lys 23 at the ends of the elongated tetramer. These detailed measurements give insight into the relative importance of various forces for the stability of melittin in the folded form and may provide an experimental standard for future tests of computational energetics on this simple protein system.

Anisotropy decays of indole, melittin monomer and melittin tetramer by frequency-domain fluorometry and multi-wavelength global analysis

We used frequency-domain fluorescence spectroscopy to measure the fluorescence lifetime and anisotropy decays of indole in propylene glycol, and of the tryptophan emission of melittin monomer and tetramer in water solutions at 5 degrees C. We obtained an increase in resolution of the anisotropy decays by using multiple excitation wavelengths, chosen to provide a range of fundamental anisotropy values. The multi-excitation wavelength anisotropy decays were analyzed globally to recover a single set of correlation times with wavelength-dependent anisotropy amplitudes. Simulated data and kappaR2 surfaces are shown to reveal the effect of multi-wavelength data on the resolution of complex anisotropy decays. For both indole and melittin, the anisotropy decays are heterogeneous and require two correlation times to fit the frequency-domain data. For indole in propylene glycol at 5 degrees C we recovered correlation times of 0.59 and 4.10 ns, which appear to be characteristic of the rigid and asymmetric indole molecule. For melittin monomer the correlation times were 0.13 and 1.75 ns, and for melittin tetramer 0.12 and 3.96 ns. The shorter and longer correlation times of melittin are due to segmental motions and overall rotational diffusion of the polypeptide.

Fluorescence and 13C NMR determination of side-chain and backbone dynamics of synthetic melittin and melittin analogues in isotropic solvents.

The dynamics in isotopic solvents of selectively 13C labeled synthetic melittin and three analogues have been investigated by using NMR and fluorescence techniques both separately and in combination. In conjunction with the "model-free" approach to interpretation of NMR relaxation data [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4570], the availability of steady-state fluorescence anisotropy and lifetime data augment T1, T2, and NOE data to provide quantitative information about fluorophore dynamics in these peptides. A method is presented for using combined fluorescence and NMR data to obtain technique- and model-independent values for parameters describing local motion of 13C-labeled fluorophores in peptides and proteins. The dynamics of melittin and melittin analogues are found to be consistent with structural characteristics inferred from CD, fluorescence, and NMR spectral information presented in the preceding paper (Weaver et al., 1989). In particular, the mobility of the random coil peptide monomers is shown to be quite similar, while side-chain as well as peptide backbone motion in the aggregated or oligomeric species differs markedly among the analogues. For melittin itself, experimentally determined overall rotational correlation times for the monomer and tetramer agree very well with values predicted on the basis of solvent-accessible protein surface area. The local dynamics of selectively 13C-labeled Trp-19 and Gly-12 residues of melittin are also found to be consistent with peptide structure. In random coil melittin monomer, a specific model for the motion indicates that the Trp side chain moves through an approximate angle of +/- 71 degrees about the beta-gamma bond with a correlation time of 159 +/- 24 ps. In melittin tetramer, the indole moiety is spatially more confined with a flip angle of +/- 37 degrees, yet demonstrates an increased rate of motion with a correlation time of 56 +/- 8 ps. The constrained mobility of the Trp-19 side chain is consistent with motional constraints inferred from the X-ray structure of melittin tetramer. These results show that protein side-chain motion, even of moieties as large as indole, can occur on the picosecond time scale and that these motions are reasonably similar to those inferred from molecular dynamics simulations.

Morphological changes of phosphatidylcholine bilayers induced by melittin: vesicularization, fusion, discoidal particles

Morphological changes induced by the melittin tetramer on bilayers of egg phosphatidylcholine and dipalmitoylphosphatidylcholine have been studied by quasi-elastic light scattering, gel filtration and freeze-fracture electron microscopy. It is concluded that melittin similarly binds and changes the morphology of both single and multilamellar vesicles, provided that their hydrocarbon chains have a disordered conformation, i.e., at temperatures higher than that of the transition, T m. When the hydrocarbon chains are ordered (gel phase), only small unilamellar vesicles are morphologically affected by melittin. However after incubation at T > T m, major structural changes are detected in the gel phase, regardless of the initial morphology of the lipids. Results from all techniques agree on the following points. (i) At low melittin content, phospholipid-to-peptide molar ratios, R i > 30, heterogeneous systems are observed, the new structures coexisting with the original ones. (ii) For lipids in the fluid phase and R i > 12, the complexes formed are large unilamellar vesicles of about 1300 ± 300 A ̊ diameter and showing on freeze-fracture images rough fracture surfaces. (iii) For lipids in the gel phase, T < T m after passage above T m, and for 5 < R i < 50, disc-like complexes are observed and isolated. They have a diameter of 235 ± 23 A ̊ and are about one bilayer thick; their composition corresponds to one melittin for about 20 ± 2 lipid molecules. It is proposed that the discs are constituted by about 1500 lipid molecules arranged in a bilayer and surrounded by a belt of melittin in which the mellitin rods are perpendicular to the bilayer. (iv) For high amounts of melittin, R i < 2, much smaller and more spherical objects are observed. They are interpreted as corresponding to lipid-peptide comicelles in which probably no more bilayer structure is left. It is concluded that melittin induces a reorganization of lipid assemblies which can involve different processes, depending on experimental conditions: (i) vesicularization of multibilayers; (ii) fusion of small lipid vesicles; (iii) fragmentation into discs and micelles. Such processes are discussed in connexion with the mechanism of action of melittin: the lysis of biological membranes and the synergism between melittin and phospholipases.

Studies of the fluorescence from tryptophan in melittin.

The fluorescence lifetime and rotational correlation time of the tryptophan residue in melittin, as both a monomer and tetramer, have been measured between pH 6 and 11. The fluorescence decays are non-exponential and give lifetimes of 0.7 +/- 0.1 ns and 3.1 +/- 0.1 ns. This emission is consistent with a model in which the tryptophan residue is in slightly different environments in the protein. In a dilute solution of monomer the mean fluorescence lifetime is 2.3 +/- 0.1 ns, below pH 10, but falls to 1.7 ns at higher pH. In contrast, the melittin tetramer has a mean fluorescence lifetime of only 2.2 ns at pH 6, which falls to 1.9 ns by pH 8, and falls again above pH 10 to the same value as in monomeric melittin. The behaviour between pH 6 and 8 is explained as the quenching of the Trp residue by lysine groups, which are near to the Trp in the tetramer but in the monomer, are too distant to quench. Fluorescence anisotropy decays show that the Trp residue has considerable freedom of motion and the range of "wobbling" motion is 35 +/- 10 degrees in the tetramer.