Assembly of the alpha-toxin-hexamer of Staphylococcus aureus in the liposome membrane

It has been shown that the access of the alpha-toxin of Staphylococcus aureus to the target membrane and assembly of the hexamer can be monitored independently by respectively measuring the fluorescence energy transfer from the tryptophan residue(s) of the toxin to the dansylated phosphatidylethanolamine in the liposome membrane and the fluorescence increment of the toxin at 336 nm (Ikigai, H., and Nakae, T., (1987) J. Biol. Chem. 262, 2150-2155). Measurement of these parameters under various conditions showed the following results: when phosphatidylcholine (PC) liposomes composed of saturated fatty acids were mixed with the toxin, the fluorescence energy transfer occurred below, at, and above the transition temperature of the lipid, but the change of fluorescence at 336 nm was never detectable; when PC-liposomes containing unsaturated fatty acids were used, both the fluorescence energy transfer and the fluorescence increment of 336 nm were observed. These results suggested that the toxin-membrane interaction occurs in PC-membranes containing saturated and/or unsaturated fatty acids and that the oligomerization occurs only in the presence of PC containing unsaturated fatty acid(s). This conclusion was supported by the results of quantitative determination of the toxin-hexamer assembly and leakage of carboxyfluorescein from PC-liposomes under conditions similar to the above.     

Staphylococcal alpha-toxin: a study of membrane penetration and pore formation

Cell lysis by staphylococcal alpha-toxin, a potent virulence factor of most pathogenic strains of Staphylococcus aureus, follows a three-step sequence: binding of toxin to the membrane, leaking of ions caused by membrane injury, and rupturing of the membrane caused by osmotic swelling. The membrane injury step is composed of two separate events, membrane penetration and membrane perturbation. The membrane penetration event involves conversion of the soluble toxin monomer into an amphipathic molecule, which inserts into the lipid bilayer of the membrane. The membrane perturbation event involves association of the toxin monomers, in the plane of the membrane, to form hexameric transmembrane pores. In this study, we demonstrate that, in an asolectin liposome system, controlling the pH of the external buffer permits these two events to be temporally resolved. Using Controlled-Pore Glass bead-purified alpha-toxin, four events are measured as a function of pH: (a) release of potassium from prelabeled asolectin vesicles, (b) conversion of the toxin to a globally hydrophobic molecule, (c) binding of detergent by the toxin, and (d) labeling of the toxin with photoactivable, radiolabeled, hydrophobic probes. Two of these events, potassium release and conversion to a net hydrophobic state, are paired in that, for the event to occur, each requires a pH of 4.6 or less. In contrast, photolabeling with the membrane probes PC I and PC II (where PC represents phosphatidylcholine) is easily detectable at pH values as high as 5.0 and 6.0. These results demonstrate that, as the pH is lowered, two distinct changes in the physical properties of alpha-toxin occur. The first, which occurs under mild acidic conditions, converts the toxin from a water-soluble molecule into an amphipathic molecule. The second, requiring relatively more acidic conditions, converts the amphipathic toxin molecule into a globally hydrophobic molecule. Correlated with these physical changes in the alpha-toxin molecule is the acquisition of two new biological properties. The conversion of alpha-toxin into an amphipathic conformation correlates with the acquisition of the biological property of the reversible penetration into the bilayer of the asolectin liposome membrane, as evidenced by labeling with the photoactivable probes. At lower pH, the conversion of the toxin into a globally hydrophobic molecule correlates with the biological property of causing damage to the cell membrane, as measured by the release of internal potassium ions, presumably by the formation of transmembrane hexamer pores.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization and environment of tryptophans in soluble and membrane-bound states of a pore-forming toxin from Staphylococcus aureus

The location and environment of tryptophans in the soluble and membrane-bound forms of Staphylococcus aureus alpha-toxin were monitored using intrinsic tryptophan fluorescence. Fluorescence quenching of the toxin monomer in solution indicated varying degrees of tryptophan burial within the protein interior. N-Bromosuccinimide readily abolished 80% of the fluorescence in solution. The residual fluorescence of the modified toxin showed a blue-shifted emission maximum, a longer fluorescence lifetime as compared to the unmodified and membrane-bound alpha-toxin, and a 5- to 6-nm red edge excitation shift, all indicating a restricted tryptophan environment and deeply buried tryptophans. In the membrane-bound form, the fluorescence of alpha-toxin was quenched by iodide, indicating a conformational change leading to exposure of some tryptophans. A shorter average lifetime of tryptophans in the membrane-bound alpha-toxin as compared to the native toxin supported the conclusions based on iodide quenching of the membrane-bound toxin. Fluorescence quenching of membrane-bound alpha-toxin using brominated and spin-labeled fatty acids showed no quenching of fluorescence using brominated lipids. However, significant quenching was observed using 5- and 12-doxyl stearic acids. An average depth calculation using the parallax method indicated that the doxyl-quenchable tryptophans are located at an average depth of 10 A from the center of the bilayer close to the membrane interface. This was found to be in striking agreement with the recently described structure of the membrane-bound form of alpha-toxin.     

Characterisation of the calcium-binding C-terminal domain of Clostridium perfringens alpha-toxin

Alpha-toxin is the key determinant in gas-gangrene. The toxin, a phospholipase C, cleaves phosphatidylcholine in eukaryotic cell membranes. Calcium ions have been shown to be required for the specific binding of toxin to membranes prior to phospholipid cleavage. Reported X-ray crystallographic structures of the toxin show that the C-terminal domain has a fold that is analogous to the eukaryotic calcium and membrane-binding C2 domains. We report the binding sites for three calcium ions that have been identified, by crystallographic methods, in the C-terminal domain of the protein close to the postulated membrane-binding surface. The position of these ions at the tip of the domain, and their function (to facilitate membrane binding) is similar to that of calcium ions observed bound to C2 domains. Using the optical spectroscopic techniques of circular dichroism (CD) and fluorescence spectroscopy, pronounced changes to both near and far-UV CD and tryptophan emission fluorescence upon addition of calcium to the C-terminal domain of alpha-toxin have been observed. The changes in near-UV CD, fluorescence enhancement and a 2 nm blue-shift in the fluorescence emission spectrum are consistent with tryptophan residue(s) becoming more immobilised in a hydrophobic environment. Calcium binding appears to be low-affinity: Kd approximately 175-250 microM at pH 8 assuming a 1:1 stoichiometry. as measured by spectroscopic methods.     

Effect of fatty acyl domain of phospholipids on the membrane-channel formation of Staphylococcus aureus alpha-toxin in liposome membrane.

By use of carboxyfluorescein-loaded multilamellar liposomes prepared from synthetic phosphatidylcholine (PC) or sphingomyelin and cholesterol in a molar ratio of 1:1, we studied whether or not fatty acyl domain of the phospholipids affects the membrane-damaging action (or channel formation) of Staphylococcus aureus alpha-toxin on the phospholipid-cholesterol membranes. Our data indicated: (1) that toxin-induced carboxyfluorescein-leakage from the liposomes composed of saturated fatty acyl residue-carrying PC and cholesterol was decreased with increasing chain length of the acyl residues between 12 and 18 carbon atoms, although toxin-binding to the liposomes was not significantly affected by the length of fatty acyl residue; (2) that unsaturated fatty acyl residue in PC or sphingomyelin molecule conferred higher sensitivity to alpha-toxin on the phospholipid-cholesterol liposomes, compared with saturated fatty acyl residues; and (3) that hexamerization of alpha-toxin, estimated by SDS-polyacrylamide gel electrophoresis, occurred more efficiently on the liposomes composed of PC with shorter fatty acyl chain or unsaturated fatty acyl chain. Thus, hydrophobic domain of the phospholipids influences membrane-channel formation of alpha-toxin in the phospholipid-cholesterol membrane, perhaps by modulating packing of phospholipid, cholesterol and the toxin in membrane.     

Interaction of Staphylococcal α-Toxin with Artificial and Natural Membranes

Comparison of hemolytic activity and chromate-releasing activity of partially purified preparations of staphylococcal alpha-toxin indicated the presence of a lytic factor other than alpha-toxin. This lytic release factor (RF) was isolated from the preparations and was shown to be active against both lipid spherules and erythrocytes. Heat-purified alpha-toxin (HP alpha-toxin) disrupted spherules, with the formation of fragments which always showed the presence of ring structures similar in dimensions (ca. 90 A) to pure alpha 12S-toxin. The interaction of HP alpha-toxin with spherules was accompanied by loss of hemolytic activity and adsorption of toxic protein. The alpha 12S-toxin, although only weakly hemolytic, was shown to be lytic for spherules. An alpha 12S-free toxin rapidly disrupted spherules, with formation of fragments with attached rings similar in dimensions to the alpha 12S molecule. Lipid monolayer experiments showed that HP alpha-toxin could penetrate lipid monolayers by virtue of a hydrophobic interaction. Effects of HP alpha-toxin on rabbit and human erythrocyte ghosts were similar to its effects on spherules, in that rings appeared on membrane fragments. Toxin-lysed rabbit erythrocytes showed similar rings on the resulting membrane fragments. However, rings were not seen on toxin-treated rabbit erythrocytes in the prelytic lag phase; this result and the fact that human erythrocytes are largely insensitive to alpha-toxin were interpreted as evidence against a lytic mechanism involving ring formation as the primary event. Rings were interpreted as toxin polymers similar to alpha 12S molecules, formed from specifically orientated active toxin molecules at the surface of lipid structures. Possible mechanisms for toxin lysis of spherules and erythrocytes are discussed.     

Membrane-active properties of alpha-MSH analogs: aggregation and fusion of liposomes triggered by surface-conjugated peptides.

Reaction of the melanotropin hormone analogs [Nle(4),D-Phe(7)]-alpha-MSH and [Nle(4),D-Phe(7)]-alpha-MSH(4-10), which were extended at their N-terminus by a thiol-functionalized spacer arm, with preformed liposomes containing thiol-reactive (phospho)lipid derivatives resulted in the aggregation of the vesicles and in a partial leakage of their inner contents. This aggregation/leakage effect, which was only observed when the peptides were covalently conjugated to the surface of the liposomes, was correlated with the fusion of the vesicles as demonstrated by the observed decrease in resonance energy transfer between probes in a membrane lipid mixing assay. A limited fusion was confirmed by monitoring the mixing of the liposome inner contents (formation of 1-aminonaphthalene-3,6,8-trisulfonic acid/p-xylene bis(pyridinium bromide) complex). The membrane-active properties of the peptides could be correlated with changes in the fluorescence emission spectra of their tryptophan residue, which suggested that after their covalent binding to the outer surface of the liposomes they can partition within the core of the bilayers. A blue shift of 10 nm was observed for [Nle(4),D-Phe(7)]-alpha-MSH which was correlated with an increase in fluorescence anisotropy and with changes in the accessibility of the coupled peptide as assessed by the quenching of fluorescence of its tryptophan residue by iodide (Stern-Volmer plots). These results should be related to the previously described capacity of alpha-MSH, and analogs, to interact with membranes and with the favored conformation of these peptides which, via a beta-turn, segregate their central hydrophobic residues into a domain that could insert into membranes and, as shown here, trigger their destabilization.     

Interaction of melittin with membrane cholesterol: a fluorescence approach

We have monitored the organization and dynamics of the hemolytic peptide melittin in membranes containing cholesterol by utilizing the intrinsic fluorescence properties of its functionally important sole tryptophan residue and circular dichroism spectroscopy. The significance of this study is based on the fact that the natural target for melittin is the erythrocyte membrane, which contains high amounts of cholesterol. Our results show that the presence of cholesterol inhibits melittin-induced leakage of lipid vesicles and the extent of inhibition appears to be dependent on the concentration of membrane cholesterol. The presence of cholesterol is also shown to reduce binding of melittin to membranes. Our results show that fluorescence parameters such as intensity, emission maximum, and lifetime of membrane-bound melittin indicate a change in polarity in the immediate vicinity of the tryptophan residue probably due to increased water penetration in presence of cholesterol. This is supported by results from fluorescence quenching experiments using acrylamide as the quencher. Membrane penetration depth analysis by the parallax method shows that the melittin tryptophan is localized at a relatively shallow depth in membranes containing cholesterol. Analysis of energy transfer results using melittin tryptophan (donor) and dehydroergosterol (acceptor) indicates that dehydroergosterol is not randomly distributed and is preferentially localized around the tryptophan residue of membrane-bound melittin, even at the low concentrations used. Taken together, our results are relevant in understanding the interaction of melittin with membranes in general, and with cholesterol-containing membranes in particular, with possible relevance to its interaction with the erythrocyte membrane.     

Effect of cholesterol on the interaction of the amphibian antimicrobial peptide DD K with liposomes

DD K is an antimicrobial peptide previously isolated from the skin of the amphibian Phyllomedusa distincta. The effect of cholesterol on synthetic DD K binding to egg lecithin liposomes was investigated by intrinsic fluorescence of tryptophan residue, measurements of kinetics of 5(6)-carboxyfluorescein (CF) leakage, dynamic light scattering and isothermal titration microcalorimetry. An 8 nm blue shift of tryptophan maximum emission fluorescence was observed when DD K was in the presence of lecithin liposomes compared to the value observed for liposomes containing 43 mol% cholesterol. The rate and the extent of CF release were also significantly reduced by the presence of cholesterol. Dynamic light scattering showed that lecithin liposome size increase from 115 to 140 nm when titrated with DD K but addition of cholesterol reduces the liposome size increments. Isothermal titration microcalorimetry studies showed that DD K binding both to liposomes containing cholesterol as to liposomes devoid of it is more entropically than enthalpically favored. Nevertheless, the peptide concentration necessary to furnish an adjustable titration curve is much higher for liposomes containing cholesterol at 43 mol% (2 mmol L(-1)) than in its absence (93 micromol L(-1)). Apparent binding constant values were 2160 and 10,000 L mol(-1), respectively. The whole data indicate that DD K binding to phosphatidylcholine liposomes is significantly affected by cholesterol, which contributes to explain the low hemolytic activity of the peptide.     

Interaction of terbium and calcium with chicken cystatin

The emission intensity of the fluorescent lanthanide, terbium, is shown to be enhanced upon binding to chicken cystatin. Fluorescence titrations indicate the presence of a single high affinity binding site per molecule. Binding of the terbium results in a 29% quenching of the fluorescence of the single tryptophan residue in the molecule. Calcium displaces the terbium from cystatin as judged by the decrease of terbium fluorescence in competition titrations. Similar titrations with magnesium or strontium demonstrate that the metal binding site of cystatin exhibits specificity for calcium or terbium. Analysis of the N-terminal sequence of chicken cystatin suggests the presence of a putative consensus sequence for a metal binding site between residues 13 and 24. Calcium causes a 17% decrease in the tryptophan fluorescence of cystatin, indicating that an induced conformational change accompanies metal binding. The increased quenching observed with terbium appears to be the result of resonance energy transfer from tryptophan to terbium. From the critical distance for energy transfer from tryptophan to terbium, it is estimated that the terbium binding site lies approximately 12 A from the single tryptophan residue in the molecule.     

Membrane electropermeabilization effects of frequency and membrane surface order on liposome leakage

Liposomes containing fluorescence marker were exposed to an alternating electric field of 80 V peak to peak square electric waves at different frequencies 0.01, 1, and 100 kHz to perturb the liposome permeation. The efflux of fluorescence dye after application of the electric field was measured by recording the fluorescence emission due to the complex formation reaction between the fluorescence dye and calcium ions in the bulk medium solution. Two independent sets of experiments were conducted: 1) calcium ions were present during electropulsation; and 2) they were added after electric field application. Two parameters, fluorescence emission intensity and increment of temperature of the solution in the chamber, were studied. The effect of membrane surface order on the fluorescence dye leakage from the liposomes was studied by addition of urea at threshold concentration before the liposomes sealed. The data demonstrate the existence of frequency dependency window at 1 kHz. Furthermore, the data were interpreted according to the theory of interactions of electromagnetic fields with highly polarized and deformed materials such as liposome particles. The urea caused an enhancement of the fluorescence dye leakage at frequency of 100 kHz. This effect could be explained as a decrease of the membrane binding rigidity due to the disordering effect of urea on the membrane lipid surface. Our conclusion is that the frequency and the membrane surface order are additional parameters that influence the processes of membrane electropermeabilization.     

Slow bleach-induced cooperative fluorescence changes in bovine photoreceptor disk membranes

The increase of protein fluorescence in suspensions of bovine photoreceptor disk membrane fragments was investigated under various conditions. The increment of fluorescence on bleaching was dependent on temperature, being about 10% at 10°C and 50% at 40°C. The time course of fluorescence increase also depended on temperature, and the activation energy was estimated to be about 14 kcal/mole. The relationship between the extent of fluorescence increase and the degree of bleaching was not stoichiometric. It was concluded that the environment of tryptophan residues of unbleached rhodopsin molecule(s) located near a bleached rhodopsin molecule is cooperatively modified upon bleaching (to a more hydrophobic environment).     

Fluorescence of tryptophan residues in firefly luciferases and enzyme--substrate complexes

Quenching of tryptophan fluorescence of Luciola mingrelica (single tryptophan residue, Trp-419) and Photinus pyralis (two tryptophan residues, Trp-417 and Trp-426) luciferases with different quenchers (I-, Cs+, acrylamide) was studied. The conserved Trp-417(419) residue was shown to be not accessible to charged particles, and positively and negatively charged amino acid residues are located in close vicinity to it. We found previously unreported effective energy transfer from this tryptophan to luciferin during the quenching of the tryptophan fluorescence. The distance between the luciferin molecule and Trp-417(419) was calculated: 11-15 and 12-17 A for P. pyralis and L. mingrelica luciferases, respectively. The role of the conserved Trp residue in the catalysis is discussed. ATP and AMP are also quenchers of the tryptophan fluorescence of the luciferases. In this case, an allosteric mechanism of the interaction of Trp-417(419) with an excess of ATP (AMP) is proposed.     

Unfolding events in the water-soluble monomeric Cry1Ab toxin during transition to oligomeric pre-pore and membrane-inserted pore channel

The insecticidal crystal (Cry) proteins produced by Bacillus thuringiensis undergo several conformational changes from crystal inclusion protoxins to membrane-inserted channels in the midgut epithelial cells of the target insect. Here we analyzed the stability of the different forms of Cry1Ab toxin, monomeric toxin, pre-pore complex, and membrane-inserted channel, after urea and thermal denaturation by monitoring intrinsic tryptophan fluorescence of the protein and 1-anilinonaphthalene-8-sulfonic acid binding to partially unfolded proteins. Our results showed that flexibility of the monomeric toxin was dramatically enhanced upon oligomerization and was even further increased by insertion of the pre-pore into the membrane as shown by the lower concentration of chaotropic agents needed to achieve unfolding of the oligomeric species. The flexibility of the toxin structures is further increased by alkaline pH. We found that the monomer-monomer interaction in the pre-pore is highly stable because urea promotes oligomer denaturation without disassembly. Partial unfolding and limited proteolysis studies demonstrated that domains II and III were less stable and unfold first, followed by unfolding of the most stable domain I, and also that domain I is involved in monomer-monomer interaction. The thermal-induced unfolding and analysis of energy transfer from Trp residues to bound 1-anilinonaphthalene-8-sulfonic acid dye showed that in the membrane-inserted pore domains II and III are particularly sensitive to heat denaturation, in contrast to domain I, suggesting that only domain I may be inserted into the membrane. Finally, the insertion into the membrane of the oligomeric pre-pore structure was not affected by pH. However, a looser conformation of the membrane-inserted domain I induced by neutral or alkaline pH correlates with active channel formation. Our studies suggest for the first time that a more flexible conformation of Cry toxin could be necessary for membrane insertion, and this flexible structure is induced by toxin oligomerization. Finally the alkaline pH found in the midgut lumen of lepidopteran insects could increase the flexibility of membrane-inserted domain I necessary for pore formation.     

Iodination of a tyrosyl residue in staphylococcal alpha-toxin.

Iodination of staphylococcal alpha-toxin by the lactoperoxidase method resulted in the maximal incorporation of about 2.5 atoms of iodine per molecule of alpha-toxin. The iodination primarily involved a single tyrosine residue as shown by analysis of both cyanogen bromide and tryptic peptides. Iodination at a level of 1.2 iodine atoms per alpha-toxin molecule led to a dramatic decrease in the hemolytic and lethal activities, although no decrease in the binding of iodinated toxin to rabbit erythrocytes was observed (Cassidy and Harshman (1976), Biochemistry, the following paper in this issue). Monoiodinated alpha-toxin was found to have 15% of the specific hemolytic activity of native alpha-toxin. Incubation of rabbit erythrocytes with iodinated alpha-toxin led to a significant protection from the hemolytic activity of native alpha-toxin added later. The results show the modification of a single unique tyrosyl residue in alpha-toxin permits the resolution of alpha-toxin's biological activities from its cell binding activity.     

Fluorescence energy transfer between cobra alpha-toxin molecules bound to the acetylcholine receptor

An approach was developed with steady state fluorescence energy transfer measurements to examine the spatial relationship between the two alpha-toxins bound to the acetylcholine receptor. By taking advantage of the slow dissociation rates of alpha-toxins (Naja naja siamensis 3) from the receptor and of the equal probability with which alpha-toxins bind to the two alpha-toxin-binding sites, we derived an equation which allows prediction of a "true" efficiency of transfer based on the relationship between fractional site occupancy and the observed transfer efficiency ascertained from donor quenching. Using this approach, we examined the efficiency of energy transfer between two fluorescently labeled alpha-toxins, N epsilon-fluorescein isothiocyanate lysine 23 alpha-toxin and monolabeled tetramethylrhodamine isothiocyanate alpha-toxin bound to the receptor from the Torpedo californica electric organ. Significantly greater (32 versus 14%) energy transfer was observed with the membrane-associated than with the solubilized receptor, suggesting that transfer between fluorophores on separate receptor molecules is greater than that occurring intramolecularly between the two sites on the receptor. The magnitude of the distances calculated from the intrareceptor energy transfer efficiency combined with the considerable inter-receptor energy transfer indicate that the fluorophores would reside on the outer perimeter of the receptor molecule rather than near the central axis perpendicular to the plane of the membrane.     

Spatial proximity of ATP-sensitive tryptophanyl residue(s) and Cys-697 in myosin ATPase.

The reactive thiol Cys-697 (SH2) in myosin ATPase was labeled with a fluorescent analog of maleimide, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) (Hiratsuka, T. (1992) J. Biol. Chem. 267, 14941-14948). Although the tryptophan fluorescence of myosin subfragment-1 (S-1) was slightly affected by incorporation of the MIANS fluorophore, the tryptophan fluorescence of the resultant S-1 derivative (MIANS-S-1) was enhanced by ATP in a manner similar to that of unlabeled S-1. The quenching of tryptophan fluorescence of MIANS-S-1 was shown to result from a transfer of the excitation energy from tryptophanyl residue(s) to the MIANS fluorophore attached to SH2, which absorbed and fluoresced maximally at 325 and 418 nm, respectively. The energy transfer measurements were performed in the presence of acrylamide and compared to those performed in the absence of the quencher. The energy transfer efficiencies were found to be unaltered by acrylamide, indicating that the observed fluorescence energy transfer is originated exclusively from the tryptophanyl residue(s) that are not affected by acrylamide, i.e. the ATP-sensitive tryptophanyl residue(s) of S-1 (Torgerson, P. M. (1984) Biochemistry 23, 3002-3007). The distance between the tryptophanyl residue(s) and Cys-697 was calculated to be 27 A assuming a single donor-acceptor pair. Trp-510 is proposed to be one of the ATP-sensitive tryptophanyl residues.     

Study of reactivity of tryptophan residues in serum albumins and lysozyme by N-bromosuccinamide fluorescence quenching

The kinetics of quenching by N-bromosuccinamide of the fluorescence of tryptophan residues in various proteins and peptides were studied using the stopped-flow technique. Human serum albumin, which has a single tryptophan residue located in a hydrophobic fold, showed biphasic kinetics; one component was second order and the other first order, the rate for the latter component being independent of the concentration of quencher. Bovine serum albumin, which has in addition a tryptophan residue at the surface of the molecule, showed triphasic kinetics; two components corresponded to those for human serum albumin, and there was a faster second-order component resulting from the surface tryptophan. It is concluded that reaction with the buried tryptophan involves the initial second-order formation of a complex in which the quencher is located at the mouth of a hydrophobic fold, and that this is followed by a first-order conformational change which allows interaction to occur between the quencher and the tryptophan. The kinetics of lysozyme fluorescence quenching at pH 5.4 showed two relaxations whose rates were proportional to the N-bromosuccinamide concentration. The results of kinetic and titration experiments suggest that a molecule of lysozyme contains at least two groups of tryptophan residues of significantly different reactivities. The faster component probably reflects the bromination of the tryptophan residues at the active site. An octapeptide consisting of residues 22 to 29 of glucagon showed essentially the same quenching kinetics as glucagon itself, and Leu-Trp-Met showed the same behavior as Gly-Trp-Gly. The results indicate that quenching by N-bromosuccinamide provides a useful indication of the accessibility of tryptophan residues, and that side reactions do not significantly affect the kinetics.     

The role of tryptophan in structural and functional properties of equinatoxin II.

A pore-forming, cytolytic and lethal polypeptide, equinatoxin II, from the sea anemone Actinia equina, was subjected to oxidation with N-bromosuccinimide to study the role of five present tryptophan residues in structure-function relationships. In the folded toxin molecule, 1-2 tryptophan residues were readily susceptible to oxidation with N-bromosuccinimide, whereas modification of a single residue resulted in complete impairment of the toxin lethal and hemolytic activities as well as the ability of an oxidized toxin to precipitate with serum lipoproteins. CD and fluorescence spectra indicated a slight alteration of a toxin secondary structure following N-bromosuccinimide treatment. Incubation with sphingomyelin of the toxin prior to oxidation did not prevent subsequent modification with N-bromosuccinimide and loss of its activities, indicating that the modified tryptophan residue is not directly involved in toxin binding and insertion into lipid membranes. It was concluded that the modified tryptophan residue is essential for the structure of equinatoxin II.     

A fluorescence approach of the gamma radiation effects on gramicidin A inserted in liposomes.

The fluorescence of tryptophan residues of gramicidin A (gA), bound to phosphatidylcholine liposomes contains valuable information about local changes in the environment of the molecule induced by gamma radiation. With this work, we aim to demonstrate that the gamma radiation effect on the peptide involves the action of free radicals, derived from water radiolysis and the process of lipid peroxidation. Basically, the methodology consists of the analysis of UV and fluorescence emission spectra of the peptide liposome complexes under control conditions and upon gamma irradiation. Free radical production was impaired by the removal of molecular oxygen or the presence of ethanol in the liposome suspension. The intensity of the tryptophan fluorescence was recorded as a function of the gamma radiation dose in the range of 0-250 Gy and the data were fitted with a single decay exponential function containing an additional constant term (named residual fluorescence). The correlation between the decrease in tryptophan fluorescence emission (D(c) = 80 +/- 10 Gy) and increase in gamma radiation dose indicates the partial damage of the tryptophan side chains of gA. O(2) removal or ethanol addition partially reduced the decay of the tryptophan fluorescence emission involving an indirect action of gamma radiation via a water radiolysis mechanism. The residual fluorescence emission (A(0)) increases in O(2)-free buffer (98 +/- 13) and in 10% ethanol-containing buffer (74 +/- 34) compared to control conditions (23 +/- 5). Varying the dose rate between 1-10 Gy/min at a constant dose of 50 Gy, an inverse dose-rate effect was observed. Thus, our study provides evidence for the lipid peroxidation effect on the tryptophan fluorescence. In conclusion, this article sustains the hypothesis of the connection between the lipid peroxidation and structural changes of membrane proteins induced by gamma radiation. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.