Crystallization and preliminary crystallographic analysis of fragaceatoxin C, a pore-forming toxin from the sea anemone Actinia fragacea.

Sea anemones produce water-soluble toxins that have the ability to interact with cell membranes and form pores within them. The mechanism of pore formation is based on an initial binding step followed by oligomerization and membrane insertion. Although the final structure of the pore remains unclear, biochemical studies indicate that it consists of a tetramer with a functional radius of approximately 1.1 nm. Since four monomers seem to be insufficient to build a pore of this size, the currently accepted model suggests that lipids might also participate in its structure. In this work, the crystallization and preliminary crystallographic analysis of two crystal forms of fragaceatoxin C (FraC), a newly characterized actinoporin from Actinia fragacea, are described. The crystals diffracted up to 1.8 A resolution and the preliminary molecular-replacement solution supports an oligomeric structure of about 120 A in diameter.

Differential interaction of equinatoxin II with model membranes in response to lipid composition.

Equinatoxin II is a 179-amino-acid pore-forming protein isolated from the venom of the sea anemone Actinia equina. Large unilamellar vesicles and lipid monolayers of different lipid compositions have been used to study its interaction with membranes. The critical pressure for insertion is the same in monolayers made of phosphatidylcholine or sphingomyelin (approximately 26 mN m(-1)) and explains why the permeabilization of large unilamellar vesicles by equinatoxin II with these lipid compositions is null or moderate. In phosphatidylcholine-sphingomyelin (1:1) monolayers, the critical pressure is higher (approximately 33 mN m(-1)), thus permitting the insertion of equinatoxin II in large unilamellar vesicles, a process that is accompanied by major conformational changes. In the presence of vesicles made of phosphatidylcholine, a fraction of the protein molecules remains associated with the membranes. This interaction is fully reversible, does not involve major conformational changes, and is governed by the high affinity for membrane interfaces of the protein region comprising amino acids 101-120. We conclude that although the presence of sphingomyelin within the membrane creates conditions for irreversible insertion and pore formation, this lipid is not essential for the initial partitioning event, and its role as a specific receptor for the toxin is not so clear-cut.

Interaction of wheat alpha-thionin with large unilamellar vesicles.

The interaction of the wheat antibacterial peptide alpha-thionin with large unilamellar vesicles has been investigated by means of fluorescence spectroscopy. Binding of the peptide to the vesicles is followed by the release of vesicle contents, vesicle aggregation, and lipid mixing. Vesicle fusion, i.e., mixing of the aqueous contents, was not observed. Peptide binding is governed by electrostatic interactions and shows no cooperativity. The amphipatic nature of wheat alpha-thionin seems to destabilize the membrane bilayer and trigger the aggregation of the vesicles and lipid mixing. The presence of distearoylphosphatidylethanolamine-poly(ethylene glycol 2000) (PEG-PE) within the membrane provides a steric barrier that inhibits vesicle aggregation and lipid mixing but does not prevent leakage. Vesicle leakage through discrete membrane channels is unlikely, because the release of encapsulated large fluorescent dextrans is very similar to that of 8-aminonaphthalene-1,3,6,trisulfonic acid (ANTS). A minimum number of 700 peptide molecules must bind to each vesicle to produce complete leakage, which suggests a mechanism in which the overall destabilization of the membrane is due to the formation of transient pores rather than discrete channels.

Differential effects of five types of antipathogenic plant peptides on model membranes.

The effects of five antipathogenic plant peptides, wheat alpha-thionin, potato PTH1 defensin, barley LTP2 lipid transfer protein, and potato tuber DL1 and DL2 defensins, have been tested against phospholipid vesicles (liposomes). Wheat thionin very actively induces aggregation and leakage of negatively charged vesicles. LTP2 displays the same activities, although to a limited extent. Under certain conditions PTH1 and DL2 induce vesicle aggregation, but not leakage. Potato defensin DL1 failed to show any effect on liposomes. The same peptides have been assayed against a plant pathogenic bacterium, both the membrane-active and -inactive compounds having efficient antibacterial action.

Membrane topology of the colicin A pore-forming domain analyzed by disulfide bond engineering.

Four colicin A double-cysteine mutants possessing a disulfide bond in their pore-forming domain were constructed to study the translocation and the pore formation of colicin A. The disulfide bonds connected alpha-helices 1 and 2, 2 and 10, 3 and 9, or 3 and 10 of the pore-forming domain. The disulfide bonds did not prevent the colicin A translocation through the Escherichia coli envelope. However, the mutated colicins were able to exert their in vivo channel activity only after reduction of their disulfide bonds. In vitro studies with brominated phospholipid vesicles and planar lipid bilayers revealed that the disulfide bond that connects the alpha-helices 2 and 10 prevented the colicin A membrane insertion, whereas the other double-cysteine mutants inserted into lipid vesicles. The disulfide bonds that connect either the alpha-helices 1 and 2 or 3 and 10 were unable to prevent the formation of a conducting channel in presence of membrane potential. These results indicate that alpha-helices 1, 2, 3, and 10 remain at the membrane surface after application of a membrane potential.

Early and delayed stages in the solubilization of purple membrane by a polyoxyethylenic surfactant.

The purpose of this paper is to explore the reasons by which purple membrane solubilization by detergents takes hours, or even days, to reach equilibrium, while most biomembranes are solubilized in a matter of seconds, or minutes. With that aim, changes in the purple membrane absorption spectrum produced by hydrogenated Triton X-100 under equilibrium conditions (24 h) have been compared to those caused by the same surfactant in the minute, second and sub-second time scale. It is found that the various processes that accompany, or lead to, solubilization are already detected, and even reach an apparent equilibrium, in the 10 s that follow detergent addition. No new phenomena are detected in the following minutes, or hours, that are relevant to the process under study. This leads to the conclusion that the long solubilization process consists of the repeated operation of simple phenomena that are relatively fast in themselves. A hypothesis is proposed according to which the tight crystalline organization of the purple membrane prevents the insertion of detergent monomers in the lipid bilayer; instead, the surfactant would bind the periphery of the patches, i.e., the hydrocarbon-water contact region, and solubilization would take place gradually, from the periphery towards the core of the membrane patches, at a progressively lower rate as the amounts of free detergent and detergent-binding sites are decreased by the previous solubilization steps.

Uncoupled steps of the colicin A pore formation demonstrated by disulfide bond engineering.

Four disulfide bonds were engineered into the pore-forming domain of colicin A to probe the conformational changes associated with its membrane insertion and channel formation. The soluble pore-forming domain consists of 10 alpha-helices with two outer layers (helices 1, 2, and 3-7, respectively) sandwiching a middle layer of three helices (8-10). Helices 8 and 9 form a hairpin which is completely buried and consists of hydrophobic and neutral residues only. This helical hairpin has been hypothesized to be the membrane anchor. Each double-cysteine mutant possessing an individual disulfide bond, cross-linking either helices 1 to 9 (H1/H9), 5 to 6 (H5/H6), 7 to 8 (H7/H8), or 9 to 10 (H9/H10), respectively, is unable to promote K+ efflux from sensitive Escherichia coli cells. Activity can be restored by addition of a reducing agent. In vitro studies with brominated lipid vesicles and planar lipid bilayers show that the disulfide bond which connects the helices 1 to 9 prevents colicin A membrane insertion, whereas the other disulfide bond mutants insert readily into lipid vesicles. All of the engineered bridges prevented the formation of a conducting channel in the presence of a membrane potential. This novel approach indicates that membrane insertion and channel formation are two separate steps. Moreover, the effects of the distance constraints introduced by the different disulfide bonds on colicin A activity indicate that the helical pair 1 and 2 moves away from the other helices upon membrane insertion. Helices 3-10 remain associated together. As a consequence, the results imply that the helical hairpin lies parallel to the membrane surface. In contrast, induction of the colicin channel by the membrane potential requires a profound reorganization of the helices association. These results are discussed in light of several proposed models of the membrane-bound colicin and channel structures.

The role of electrostatic charge in the membrane insertion of colicin A. Calculation and mutation.

The bacterial toxin colicin A binds spontaneously to the surfaces of negatively charged membranes. The surface-bound toxin must subsequently, however, become an acidic 'molten globule' before it can fully insert into the lipid bilayer. Clearly, electrostatic interactions must play a significant role in both events. The electrostatic field around the toxin in solution was calculated using the finite-difference Poisson-Boltzmann method of the Delphi programme and the known X-ray structure. A large positively charged surface was identified which could be involved in the binding of colicin to negatively charged membranes. The applicability of the result was tested by also calculating the fields around modelled structures of the closely related colicins B and N. Surprisingly, colicin N showed a similar charge distribution in spite of its isoelectric point of pI 10.20 (colicin A has pI 5.44). One reason for this is the strong conservation of certain negative charges in all colicins. There is a single highly conserved aspartate residue (Asp78) on the positively charged face which provides a small but discrete region of negative charge. This residue, Asp78, was replaced by asparagine in the mutant D78N. D78N binds faster to negatively charged vesicles but inserts only half as fast as the wild-type protein into the membrane core. This indicates that, first, the initial membrane binding has a significant electrostatic component and, second, that the isolated charge on Asp78 plays a role in the formation of the insertion intermediate.

Fluorescence energy transfer distance measurements. The hydrophobic helical hairpin of colicin A in the membrane bound state.

The ion-channel-forming C-terminal fragment of colicin A binds to negatively charged lipid vesicles and provides an example of the insertion of a soluble protein into a lipid bilayer. The soluble structure is known and consists of a ten-helix bundle containing a hydrophobic helical hairpin. In this study fluorescence resonance energy transfer spectroscopy was used to determine the position of this helical hairpin in the membrane bound state. An extrinsic probe, N'-(iodoacetyl)-N'-(5-sulpho-1-naphthyl)ethylenediamine (I-AEDANS) was attached to mutant proteins each of which bears a unique cysteine residue. Five mutants I26C (helix 1), F105C (between helices 4 and 5), G166CJ (helix 8), A169C (helix 8-9), G176C (helix 9) were used. All mutants show wild-type binding activity to phosphatidylglycerol vesicles as judged by fluorescence polarization anisotropy, emission wavelength changes and brominated lipid quenching. The three tryptophan residues were used as a compound donor to AEDANS in resonance energy transfer distance determinations. The distances obtained for the soluble form were equal to those found in the crystal structure. On adding vesicles under conditions where intermolecular transfer was avoided the indicated distances increased; I26(10.9 A) F105(3.4 A), G166(3.3 A), A169(1.9 A) and G176(2.9 A). This confirms that, in the absence of a membrane potential, helices 1 and 2 open out onto the membrane surface whilst the helical hairpin remains closely packed against the rest of the structure. The insertion of this hairpin is thus not the driving force behind colicin membrane binding.

Interaction of the colicin-A pore-forming domain with negatively charged phospholipids.

The interaction of colicin-A thermolytic fragment with negatively charged liposomes was studied by fluorescence spectroscopy. 1,2-Dioleoyl-sn-glycero-3-phospho-1-sn-glycerol (Ole2GroPGro) containing liposomes do not significantly alter the fluorescence properties of the protein, and thus cannot give much information about this interaction. 1,2-Bis(9,10-dibromooleoyl-sn-glycero-3-phospho-1-sn-glycerol (Br4Ole2GroPGro) is easily synthesized by addition of bromine atoms to the double bond located at the mid-point of the fatty-acid acyl chain of Ole2GroPGro. The brominated phospholipid forms vesicles that strongly quench the protein fluorescence emission. The results presented here show that conversion of Ole2GroPGro to Br4Ole2GroPGro does not change either the affinity for the protein or the extent of lipid binding. This observation allows for the estimation of the distribution of the quenching phospholipid molecules around the fluorophores [Yeager, M. D. & Feigenson G. W. (1990) Biochemistry 29, 4380-4392]. Binding of the protein to the vesicles is an irreversible process, since inserted molecules do not dissociate from the vesicle. From steady-state measurements, it can be concluded that in the membrane-bound form, the tryptophans are located within quenching distance of the bromine atoms, i.e. close to the lipid head-group/hydrocarbon boundary, completely accessible to the quencher, protected from the polar phase and that the maximum number of phospholipid molecules in contact with the fluorescent domain of the protein is nine.

pH-dependent stability and membrane interaction of the pore-forming domain of colicin A.

Thermal stability of the pore-forming domain of colicin A was studied by high sensitivity differential scanning calorimetry and circular dichroism spectroscopy. In the pH range between 8 and 5, the thermal denaturation of the protein in solution occurs at 66-69 degrees C and is characterized by the calorimetric enthalpy of approximately 90 kcal/M. At pH below 5, there is a rapid pH-dependent destabilization of the pore-forming domain resulting in the lowering of the midpoint denaturation temperature and a decrease in the calorimetric enthalpy of denaturation. Circular dichroism spectra in the near and far ultraviolet show that the thermotropic transition is associated with collapse of the native tertiary structure of the pore-forming domain, although a large proportion of the helical secondary structure remains preserved. The present data indicate some similarity also between acid-induced and temperature-induced denaturation of the pore-forming domain of colicin A. Association of the pore-forming domain with phospholipid vesicles of dioleoylphosphatidylglycerol results in total disappearance of the calorimetric transition, even at pH values as high as 7. Since lipid binding also induces collapse of the near ultraviolet circular dichroism spectrum, these data indicate that interaction with the membrane facilitates a conformational change within the pore-forming domain to a looser (denaturated-like) state. These findings are discussed in relation to the recent model (van der Goot, F. G., Gonzalez-Manas, J. M., Lakey, J. H., Pattus, F. (1991) Nature 354, 408-410) which postulates that a flexible "molten globule" state is an intermediate on the pathway to membrane insertion of colicin A.

Brominated phospholipids as a tool for monitoring the membrane insertion of colicin A.

The intrinsic fluorescence of the colicin A thermolytic fragment does not change after insertion into normal phospholipid vesicles and is thus an unsuitable probe for monitoring the membrane insertion process. In this paper, we report the results of studies on the quenching of this fluorescence by brominated dioleoylphosphatidylglycerol (Br-DOPG) vesicles. Bromine atoms located at the midpoint of the phospholipid acyl chain quench the tryptophan fluorescence, indicating contact between fluorophores of the protein and the bilayer's hydrophobic core. Addition of Br-DOPG vesicles to a protein solution quenches the tryptophan fluorescence in a time-dependent manner. This quenching can be fitted to a single-exponential function, and thus interpreted as a one-step process. This allows calculation of an apparent rate constant of protein insertion into the membrane. Parameters known to affect the insertion of the thermolytic fragment into phospholipid monolayers or vesicles (pH and negative charge density) also affect the rate constant in comparable ways. In addition to the information gained concerning membrane exposure in the steady state, this approach provides the first real-time method for measuring the insertion of colicin into membranes. It is highly quantitative and can be used on all versions of the protein, e.g., full size, proteolytic fragments, and mutants. Brominated lipids provide experimental conditions identical to normal lipids and allow for great flexibility in protein/lipid ratios and concentrations. The kinetic analysis shows clearly the existence of a two-step process involving a rapid adsorption of the protein to the lipid surface followed by a slow insertion.

The membrane insertion of colicins.

Pore-forming toxins, such as colicin A, are water-soluble proteins that insert into lipid bilayers. The water-soluble structure of Colicin A is known at a high resolution and this review describes the kinetic and structural steps involved in its soluble-to-membrane bound transformation.

A 'molten-globule' membrane-insertion intermediate of the pore-forming domain of colicin A.

The 'molten' globular conformation of a protein is compact with a native secondary structure but a poorly defined tertiary structure. Molten globular states are intermediates in protein folding and unfolding and they may be involved in the translocation or insertion of proteins into membranes. Here we investigate the membrane insertion of the pore-forming domain of colicin A, a bacteriocin that depolarizes the cytoplasmic membrane of sensitive cells. We find that this pore-forming domain, the insertion of which depends on pH, undergoes a native to molten globule transition at acidic pH. The variation of the kinetic constant of membrane insertion of the protein into negatively charged lipid vesicles as a function of the interfacial pH correlates with the appearance of the acidic molten globular state, indicating that this state could be an intermediate formed during the insertion of colicin A into membranes.

On the mechanism of bacteriorhodopsin solubilization by surfactants.

Purple membrane bacteriorhodopsin can be easily solubilized by Triton X-100 and other detergents, but not by deoxycholate. In order to understand this behavior, we have examined the effects of a variety of surfactants. We show that detergents containing the cholane ring (cholate, taurocholate, 3[(3-cholamidopropyl)diethyl-ammonio]propanesulfonic acid...) are virtually unable to solubilize native bacteriorhodopsin. However, when the protein is reconstituted in dimyristoyl phosphatidylcholine and solubilization is assayed at a temperature such that bacteriorhodopsin is in the form of monomers, solubilization by cholane detergents does occur. We propose that steric factors prevent access of the rigid planar surfactant molecules to the hydrophobic protein regions. These are perhaps located in the monomer-monomer interface, whose solvation by surfactants is essential for solubilization to occur. We note that the capacity of some detergents to solubilize bacteriorhodopsin is always associated within the same range of surfactant concentrations with bleaching (partial or total) of the protein chromophore. The detergent-induced bleaching is at least partially reversible, suggesting that free retinal remains associated to some membrane components. While some surfactant molecules remain tightly bound to the membrane protein, cholane detergents can be completely removed from bacteriorhodopsin. Our results indicate that a structure-function relationship exists for detergents applied to the solubilization of bacteriorhodopsin.

Binding of Triton X-100 to bovine serum albumin as studied by surface tension measurements.

A previously published computerized drop-weight technique for surface tension measurements, not involving the use of radioactively labelled compounds, has been applied to the study of detergent binding to proteins. The procedure is based on the observation that the protein-surfactant complex is no longer surface-active. As an example, the binding of Triton X-100 to bovine serum albumin has been studied, and the results were found to be in good agreement with those obtained through established but less convenient methods. Our procedure should be useful for measurements of detergent binding to biomembranes.

Kinetics of purple membrane dark-adaptation in the presence of Triton X-100.

The kinetics of purple membrane dark adaptation were studied at pH 5 and 7, in the presence and absence of the nonionic detergent Triton X-100. The effect of both sublytic and lytic surfactant concentrations has been considered. Our results show that: (a) dark adaptation is faster at pH 5 than at pH 7, (b) dark adaptation is slower, and of smaller amplitude, in the presence than in the absence of Triton X-100. The data may be interpreted in terms of a simple first-order kinetic model, according to which light-dark adaptation would depend basically on the equilibrium between the 13-cis- and the all-trans-isomers. The experiments also suggest that at pH 5, but not at pH 7, solubilizing surfactant concentrations produce a considerable increase in the velocity of the dark adaptation reaction, perhaps through changes in the microenvironment of a protonable group.

Detergent solubilization of phospholipid vesicle. Effect of electric charge.

In order to explore the effect of electric charge on detergent solubilization of phospholipid bilayers, the interaction of nine electrically charged surfactants with neutral or electrically charged liposomes has been examined. The detergents belonged to the alkyl pyridinium, alkyl trimethylammonium or alkyl sulphate families. Large unilamellar liposomes formed by egg phosphatidylcholine plus or minus stearylamine or dicetyl phosphate were used. Solubilization was assessed as a decrease in light-scattering of the liposome suspensions. The results suggest that electrostatic forces do not play a significant role in the formation of mixed micelles and that hydrophobic interactions are by far the main forces involved in solubilization. In addition, from the study of thirty different liposome-surfactant systems, we have derived a series of empirical rules that may be useful in predicting the behaviour of untested surfactants: (i) the detergent concentration producing the onset of solubilization (Don) decreases as the alkyl chain length increases; the decrease follows a semi-logarithmic pattern in the case of alkyl pyridinium compounds; (ii) for surfactants with critical micellar concentrations (cmc) less than 6 x 10(-3) M, Don. is independent of the nature of the detergent and the bilayer composition; for detergents having cmc greater than 6 x 10(-3) M, Don. increases linearly with the cmc; and (iii) Don. varies linearly with the surfactant concentration that produces maximum solubilization.

The interaction of Triton X-100 with purple membranes. Detergent binding, spectral changes and membrane solubilization.

The interaction of the non-ionic surfactant Triton X-100 with Halobacterium purple membranes has been examined at sublytic and lytic surfactant concentrations. These membranes present a number of important peculiarities in their behaviour towards the surfactant. Although solubilization is a very slow process, with a half-time of the order of hours, detergent binding appears to occur at the same fast rate as that found in other membranes. Lipids are solubilized more easily than proteins, so that hardly any protein is solubilized at surfactant concentrations at which about 75% of the lipid is in the form of detergent-mixed micelles; once started, protein solubilization takes place within a narrow range of surfactant concentrations. Retinal provides a built-in probe to monitor detergent-induced conformational changes by spectroscopy in the visible range. No spectral variation is detected at the prelytic stage, i.e. when detergent is incorporated into the membrane in monomeric form. Membrane disruption is accompanied by a blue shift in the absorption maximum, retinal isomerization (from all-trans to 13-cis), and a decrease in specific absorbance (bleaching). Increasing detergent concentrations after solubilization is completed do not produce further shifts in the spectral maximum, but the specific absorbance is progressively decreased. It is shown that Triton X-100 has a complex effect on the retinal chromophore, modifying its configuration and microenvironment (changes in maximum wavelength) and promoting hydrolysis of the retinal-bacteriorhopsin Schiff's base (bleaching).

Membrane solubilization by the non-ionic detergent triton X-100. A comparative study including model and cell membranes.

The solubilizing effects of the non-ionic detergent Triton X-100 have been examined on three membranous systems, namely rabbit sarcoplasmic reticulum, Halobacterium purple membrane and gramicidin A-phosphatidylcholine liposomes. The loss of membrane structure has been assessed through changes in suspension turbidity, while chemical analysis has revealed the differential solubilization of proteins and lipids. Solubilization data obtained on the above three systems are compared with previously published values concerning other membrane preparations. Also, solubilization of sarcoplasmic reticulum by Triton X-100 is monitored by Fourier-transform infrared spectroscopy and, similarly, purple membrane-surfactant interaction is studied using visible spectroscopy. The biochemical and spectroscopic data may be rationalized assuming a three-stage model of membrane-detergent interaction, incorporation of surfactant monomers into the membrane; disruption of the bilayer into mixed micelles, and separation of lipid and protein.