Báo cáo khoa học: Interaction of the P-type cardiotoxin with phospholipid membranes pptx

To assess possible modes of CTII/membrane interaction 31P- and 1H-NMR spectroscopy was used to study binding of the toxin and its effect onto multilamellar vesicles MLV composed of either

Interaction of the P-type cardiotoxin with phospholipid membranes

Peter V Dubovskii, Dmitry M Lesovoy, Maxim A Dubinnyi, Yuri N Utkin and Alexander S Arseniev Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation

The cardiotoxin (cytotoxin II, or CTII) isolated from cobra

snake (Naja oxiana) venom is a 60-residue basic

membrane-active protein featuring three-finger beta sheet fold To assess

possible modes of CTII/membrane interaction 31P- and

1H-NMR spectroscopy was used to study binding of the

toxin and its effect onto multilamellar vesicles (MLV)

composed of either zwitterionic or anionic phospholipid,

dipalmitoylglycerophosphocholine (Pam2Gro-PCho) or

dipalmitoylglycerophosphoglycerol (Pam2Gro-PGro),

res-pectively The analysis of1H-NMR linewidths of the toxin

and31P-NMR spectral lineshapes of the phospholipid as a

function of temperature, lipid-to-protein ratios, and pH

values showed that at least three distinct modes of CTII

interaction with membranes exist: (a) nonpenetrating mode;

in the gel state of the negatively charged MLV the toxin

is bound to the surface electrostatically; the binding

to Pam2Gro-PCho membranes was not observed; (b)

penetrating mode; hydrophobic interactions develop due to penetration of the toxin into Pam2Gro-PGro membranes in the liquid-crystalline state; it is presumed that in this mode CTII is located at the membrane/water interface deepening the side-chains of hydrophobic residues at the tips of the loops 1–3 down to the boundary between the glycerol and acyl regions of the bilayer; (c) the penetrating mode gives way to isotropic phase, stoichiometrically well-defined CTII/ phospholipid complexes at CTII/lipid ratio exceeding a threshold value which was found to depend at physiological

pH values upon ionization of the imidazole ring of His31 Biological implications of the observed modes of the toxin– membrane interactions are discussed

Keywords: cytotoxin II (cardiotoxin); membrane binding mode; multilamellar phospholipid vesicles; 31P-NMR; isotropic phase

Understanding the physical principles underlying

mem-brane protein structure and dynamics advanced rapidly

during the past decade A number of high resolution

membrane protein structures available is growing

How-ever, all of them are either helical bundles or b-barrels [1]

An important question is whether new motifs would

emerge The positive answer has been obtained by

consid-ering insertion of cytotoxins (CTs) into membranes

CTs (Fig 1A) are single chain all b-sheet proteins with a

common fold provided by four disulfide linkages forming a

globular head from which three major loops emerge [2,3]

Due to their cytotoxic and hemolytic activities it was

suggested [4,5] that CTs act on biological membranes This

has been demonstrated comprehensively with membrane

models such as micelles, monolayers, liposomes [6–8]

Surface [9,10] and transbilayer [11–13] modes of the

insertion of CTs into membranes were suggested CTs were

classified into P- and S-types [6] The S-type CTs contain

Ser28 and are thought to insert only loop I into membranes The Pro30 residue is typical of the P-type CTs which interact with membranes by all three loops [14] Lytic activity of CT molecules has been ascribed to a change

in their orientation at the membrane surface [10] or in the positioning from a surface location to a transbilayer one [13]

A recent theoretical study of the binding of S- and P-type CTs to membranes suggested that P-type CTs are inserted into membranes via the tips of the three loops while S-type ones are inserted through the tip of the loop I only [15] Experimental data supporting this hypothesis have been found for the interaction of the P-type CTII (Fig 1B) with micelles [16] and phospholipid vesicles [17] The effect of CTII on phospholipid membranes was not studied A wide-line31P-NMR spectroscopy was used for this purpose in this work

Different phospholipid phases characterized by specific modes of molecular motion result in specific line shapes

of chemical shift anisotropy (CSA)-dominated wideline

31P-NMR spectra [7] The induction by CTs of bilayer-to-isotropic phase transitions in membranes composed of anionic [18] or zwitterionic phospholipids [13] has been shown An analysis of redistribution of the intensities within powder type31P-NMR spectra of MLV as a result of their deformation by the magnetic field of the spectrometer was performed and modulation of this effect by peptides [19], CTs [20] in particular, was studied In the present work CTII/phospholipid interactions for MLV composed of either zwitterionic Pam2Gro-PCho or anionic Pam2 Gro-PGro were analysed with this technique These data were

Correspondence to A S Arseniev, Shemyakin & Ovchinnikov Institute

of Bioorganic Chemistry, 16/10 Miklukho-Maklaya str.,

V-437 Moscow, 117997 Russia Fax: + 7 95 335 50 33,

E-mail: aars@nmr.ru

Abbreviations: CSA, chemical shift anisitropy; CT, cytotoxin; L/P,

lipid to protein molar ratio; MLV, multilamellar vesicles; NaOAc,

sodium acetate; PtdCho, phosphatidylcholine; Pam 2 Gro-PCho,

dipalmitoylglycerophosphocholine; PtdGro, phosphatidylglycerol;

Pam 2 Gro-PGro, dipalmitoylglycerophosphoglycerol.

(Received 3 December 2002, revised 4 March 2003,

accepted 18 March 2003)

supplemented by1H-NMR data on the toxin binding to

MLV Finally, conclusions about the modes of CTII/

membrane interaction and their relation to biological

activity were drawn

Materials and methods

Purification of CTII

CTII from Naja oxiana snake venom was purified as

described previously [21] Analytical RP-HPLC showed

that purity of the CTII preparations was 98% The

phospholipase A2activity of CTII was found to be

negli-gibly small In the presence of CTII and EDTA, the effects

of phospholipid degradation was not detected at all, even

after long incubation (> a week)

Sample preparation

31P-NMR spectroscopy The phospholipids used in the

work, namely 1,2-dipalmitoyl-sn-glycero-3-phosphocholine

(Pam2Gro-PCho) and

1,2-dipalmitoyl-sn-glycero-3-[phos-pho-rac-(1-glycerol)] (Pam2Gro-PGro) were obtained

com-mercially (Avanti Polar Lipids, Alabaster, AL, USA) and

used without any further purification.2HO (99.9%) was

from IZOTOP (St Petersburg, Russian Federation), sodium acetate (NaOAc), EDTA and KCl were from REACHIM (Moscow, Russian Federation)

(0.2M) containing 10 mM EDTA, 150 mM KCl, in2H2O (direct pH meter reading of 5.5) was used for the hydration

of phospholipids and dissolution of CTII

Samples containing CTII/phospholipid mixtures were prepared in the following way NMR tubes (5 mm outer diameter

loaded with 20 mg of the phospholipid powder Fol-lowing this, buffer (the amount of buffer added provided at least 200 mol of water per mol of phos-pholipid) or CTII dissolved in the buffer [to provide a desired lipid to protein (L/P) molar ratio] was added The dispersion obtained was cycled thermally in the range 20–50C and agitated mechanically to ensure its homogeneity

For experiments on pH titration, CTII/Pam2Gro-PGro mixture (20 mg of Pam2Gro-PGro,2H2O/Pam2Gro-PGro

of 600 : 1 mol : mol) was prepared in 2H2O containing

10 mM EDTA, 150 mM KCl without buffer The pH was adjusted by adding small aliquots of concentrated NaOH or HCl solutions and pH values are given as direct

pH meter readings

1H-NMR spectroscopy One milligram of CTII was dissolved in 0.5 mL of 10 mM NaOAc (pH 5.5) buffer containing 95% H2O, 5% 2H2O, 10 mM KCl and 1 mM EDTA The 25 mM stock solution of Pam2Gro-PCho or Pam2Gro-PGro MLV were prepared in the same buffer After addition of the required amount of lipid to the toxin solution, the mixture was cycled thermally and vortexed before measurement as described above

NMR data acquisition NMR spectra were obtained on an 11.4 T Bruker

DRX-500 spectrometer (Germany) with 1H- and31P-resonance frequencies at x0/2p¼ 500.13 and 202.5 MHz, respect-ively, using a standard broad-band 5 mm probehead The magnetic field of the spectrometer was locked during acquisition through 2H2O contained in the samples The temperature of the samples was controlled by dry air and monitored by the VT-system (BVT 3000) to an accuracy of

± 0.1C Chemical shifts were referenced to external 85%

H3PO4 or internal HDO signal in the31P- and1H-NMR spectra, respectively The WATERGATE scheme was used for water signal suppression in the1H-NMR spectra [22]

31P-NMR spectra were recorded with the spin ½ Hahn echo pulse sequence with full phase cycling of both transmitter and receiver [23] using 90 pulse of 8 ls and interpulse delay of 40 ls The repetition time was 3–8 s, the longer time being used at higher temperatures A total of 1000–3000 scans were accumulated for each spectrum of

60 606 Hz spectral width During acquisition, broadband

31P-1H decoupling was applied The spectral processing was carried out with WINNMR software supplied by the spectrometer manufacturer (Bruker) The 2K time domain points were zero-filled to 4K data points multiplied

by the Lorentzian (broadening factor of 1–5 Hz; this distorted minimally the line-shape of spectra) and Fourier transformed

Fig 1 Schematic representation of three-fingered b-sheet structure of

CTII (A) and its amino-acid sequence (B) In (A) the finger numbers, the

N- and C-termini together with the hydrophilic residues situated at the

membrane/water interface (dotted line) are marked In (B) the disulfide

bridges are shown in connecting lines, residues at the tips of the three

loops interacting with dodecylPCho micelle at pH 5.5 [16] are shown

in bold type.

Line shape simulation

Theoretical31P-NMR spectra were calculated and fitted to

the experimental ones within programMATHEMATICA

(ver-sion 4.0, Wolfram Research) Theoretical spectra were

represented as the convolution of the Lorentzian, Gaussian

(or their linear combination) and function of angular

distribution for ellipsoids [19] This procedure takes into

account that MLV of phospholipids in the magnetic field of

the spectrometer adopt ellipsoidal shapes [19,24] In case

where the experimental spectra were combinations of

isotropic and broad lines, the adjustable parameters in the

simulation protocol were: components of the tensor of

chemical shift anisotropy, integral intensity, the semiaxis

ratio of ellipsoidal MLV, halfwidths of Lorentzian or

Gaussian lineshapes and their proportion At the attained

level of signal-to-noise ratio this procedure ensured the error

of the spectral decomposition into isotropic and broad

components to be within 2–4%

Molecular graphics

Fig 1 was drawn with theMOLMOLprogram [25]

Results and discussion

In the present study 31P-NMR spectroscopy was used to

evaluate the effects of CTII on MLV and thus, in

combination with the structural data on the protein

[16,21], to investigate its membrane binding modes

The extent of the toxin binding to MLV was determined

by1H-NMR spectroscopy as the first step

Binding of CTII to Pam2Gro-P Gro vs Pam2Gro-P Cho MLV

1H-NMR spectra The binding of peptides to lipid vesicles

is seen in the1H-NMR spectra of peptide/lipid mixtures as

broadening and/or chemical shift changes of the peptide

proton signals because the binding affects overall rotational

correlation time and environment of a peptide [26] The

signals in the1H-NMR spectra of CTII in aqueous solution

at 30C are sharp (Fig 2) Their spectral intensities were

attenuated by the addition of Pam2Gro-PGro MLV while

line widths and chemical shifts remained unchanged (Fig 2,

from top to bottom) Evidently, this corresponds to the case

when the exchange rate between free and lipid bound toxin

falls into the slow time scale (with an upper boundary of the

order of s)1) In this case only the ÔfreeÕ peptide is seen

spectroscopically while the1H-NMR signals of the

mem-brane-bound CTII are extremely broad due to low overall

rotational correlation time of the complex When the L/P

ratio becomes > 10 : 1, CTII signals in the spectra are

not observed (Fig 2, bottom), i.e all CTII is bound to

Pam2Gro-PGro It is of note that the31P-NMR spectra of

the samples corresponding to L/P¼ 7, 10, 12 (Fig 2) were

similar to spectrum taken at L/P¼ 14 (see below)

bilayer structure of Pam2Gro-PGro membranes in the gel

phase is intact at these conditions Raising the temperature

above the gel to liquid crystalline phase transition of

Pam2Gro-PGro ( 41 C) at L/P ¼ 14 : 1 results in the

domination of the isotropic signal in the31P-NMR spectra

However no signal of free CTII was observed in1H-NMR

spectrum under these conditions This means that the transition of Pam2Gro-PGro to an isotropic phase is not accompanied by a redistribution of CTII between aqueous and lipid phases

The titration of CTII with MLV of zwitterionic Pam2Gro-PCho at 30 and 50C, i.e at the temperatures

of the gel and liquid crystalline phase, respectively, to an L/P ratio of 50 : 1 did not influence high-resolution1H-NMR spectra of the toxin (spectra are not shown) This indicates that binding of CTII to MLV of Pam2Gro-PCho is negligibly small and, thus, electrostatic attraction of the positively charged CTII molecule to a negatively charged membrane is required for the binding of the toxin

31P-NMR spectra The partitioning of the P-type CTs into zwitterionic membranes was shown to depend upon surface pressure in the outer membrane leaflet [27] CTs partitioned into lyso-PtdCho micelles [6], sonicated vesicles of sphingo-myelin [28] or egg PtdCho [17] but not 100 nm unilamellar extruded vesicles of dimyristoyl glycero phosphocholine

5

(P V Dubovskii & M A Dubinnyi, unpublished obser-vations).1H-NMR data of the present study showed that CTII remains in aqueous phase of Pam2Gro-PCho MLV Indeed, the 31P-NMR spectra of Pam2Gro-PCho/CTII dispersions (spectra are not shown) were found to be identical to those of pure Pam2Gro-PCho dispersions in the temperature range studied (30–55C) Thus, both

1H-NMR and 31P-NMR data argue for the absence of CTII binding to Pam2Gro-PCho

The influence of CTII binding to MLV of Pam2 Gro-PGro on the lineshapes of the 31P-NMR spectra of the phospholipid was studied at different temperatures and L/Ps These observations were made in the temperature interval (30–55C) encompassing the gel-to-liquid crystal transition of Pam2Gro-PGro bilayers ( 41 C), while the L/P ratio varied from 7 to 200

In Fig 3, the 31P-NMR spectra of the MLV of pure Pam2Gro-PGro and in the presence of CTII (L/P¼ 129 : 1) are compared The spectra in the gel state (30C) exhibit no difference while in the spectra corresponding to the liquid

Fig 2.1H-NMR spectra of CTII in the presence of increasing amounts

of Pam 2 Gro-PGro MLV (from top to bottom) at 30 °C The L/P ratio is indicated on the right The impurity peaks are marked with asterisks.

crystalline state (50C) the redistribution of the intensities

of the high-field peak and the low-field shoulder are clearly

seen It is known that the negative diamagnetic susceptibility

anisotropy of the fatty acyl chains of the phospholipid

molecules causes a preferential orientation of the long

molecular axis perpendicular to the external magnetic field

[24,29,30] As a result, MLV of phospholipids adopt shape

of a prolate ellipsoid instead of a sphere Spectral

simula-tions of the lineshape of the 31P-NMR spectra allow to

extract the ratio of ellipsoid long/short axis (c/a) [31,32]

This ratio is dependent upon hydration of Pam2Gro-PGro

which changes with the amount of CTII added To elucidate

the effect of CTII on the deformation of MLV at 50C, the

c/a values calculated for MLV of Pam2Gro-PGro solvated

by either buffer alone or buffered toxin solution are plotted

in Fig 4A against L/P The addition of the buffer or CTII

aliquots resulted in the same phospholipid hydration in

both samples

The decrease of L/P below of 20 : 1 induces nearly

spherical shape of Pam2Gro-PGro MLV (c/a 1), i.e

CTII inhibits the deformation of MLV by the magnetic field

(Fig 4A)

Addition of CTII to MLV of Pam2Gro-PGro in the

liquid crystalline phase results in the appearance of the

isotropic signal in the31P-NMR spectra Dependence of

the amount of isotropic signal in the31P-NMR spectra of

Pam2Gro-PGro upon amount of CTII added was

deter-mined at 50C (Fig 4B) The main feature of this

dependence is an existence of a threshold of the L/P ratio

below which the transition of bilayer to isotropic phase is

initiated At L/P > 20 the amount of the isotropic signal is

zero within an experimental error ( 3%) At lower L/P

ratios the amount of isotropic signal increases nearly

proportionally with amount of CTII added The complete

transformation of bilayer into isotropic phase at 50C is

Fig 3.31P-NMR spectra of Pam 2 Gro-PGro

MLV (water/Pam 2 Gro-PGro ¼ 230 : 1,

mol/mol) in the presence (left) of CTII (L/P ¼

129 : 1) and its absence (right) at 30 °C

(bottom), 50 °C (top) The spectra are scaled to

the intensity of the high-field peak The

com-puter-simulated shapes of MLV at 50 C are

shown in the top.

Fig 4 Change in the ellipticity c/a of Pam 2 Gro-PGro MLV with an amount of CTII (L/P and L/2H 2 O) or buffer (L/2H 2 O) added (A) and the amount of isotropic signal in the31P-NMR spectra of Pam 2 Gro-PGro/CTII dispersions (B) The experiments were carried out at 50 C.

observed at L/P ratio of 10 : 1 (Fig 4B) It is interesting

to note that under these conditions (pH 5.5) CTII bears a

net positive charge of 10 and thus CTII/Pam2Gro-PGro

complexes forming isotropic phase are electrically neutral

The temperature variation of 31P-NMR spectra of

Pam2Gro-PGro in the presence of CTII at L/P ratio of

14 : 1 is shown in Fig 5

The decomposition of the spectra (see example in Fig 5)

gives the amount of the isotropic component Its

tempera-ture dependence is featempera-tured by a sigmoidal shape (Fig 6A)

The temperature of the half-transition of bilayer to isotropic

phase (Fig 6A) coincides that of the gel-to-liquid crystal

transition of Pam2Gro-PGro MLV The CSA values of

Pam2Gro-PGro bilayers in the presence of CTII across the

whole temperature range studied (30–55C) were similar to

their respective values in the absence of the toxin Thus the

average conformation of the head group in Pam2Gro-PGro

bilayers is insensitive to the presence of CTII under the

chosen experimental conditions This does not contradict a

suggestion of an insertion of CTII between the negatively

charged polar PtdGro groups Similar observations were

made in studies of positively charged peptides such as

polymyxin B [33], melittin [34], gramicidin S [35]

cases an increase of peptide concentration in PtdGro bilayers

did not affect

7 the31P CSA values of bilayers but lead to the

occurence of an isotropic peak in the31P NMR spectra

Under conditions chosen (L/P ¼ 14 : 1) the magnetic

field induced deformation of MLV of Pam2Gro-PGro was

determined in the temperature range of 30–55C and the

values obtained were compared to those for pure Pam2

Gro-PGro (Fig 6B) It is seen that CTII extinguishes the

temperature dependence of the deformation of Pam2

Gro-PGro MLV observed in the absence of the toxin These data

support the above conclusion on hampering of the

defor-mation of Pam2Gro-PGro MLV in the magnetic field by

CTII

pH-dependence of the CTII/Pam2Gro-PGro interaction

The only group of CTII which changes its ionogenic state in

the physiological pH range is the imidazole ring of His31

residue (Fig 1A) This group is at the membrane-binding site of CTII [16] and thus might influence deepening of CTII into membrane Indeed, the amount of the isotropic signal

in the31P-NMR spectra of Pam2Gro-PGro/CTII mixture (Fig 7A) showed a characteristic pH-dependence The pKa value of 6.1 of this process (Fig 7B), is close to the value of 5.8 found for His31 of CTII in dodecylPCho micelle [16] Thus, the neutralization of His31 residue results in the increase of the deteriorating effect of the toxin onto Pam2Gro-PGro bilayers

It was shown recently [36] that CTII molecule changes its disposition in dodecylPCho micelle depending on the ionization state of His31 residue By the change of the ionogenic state of the imidazole ring from a protonated state

to a deprotonated one, the molecule of CTII inserts loop 3 more deeply into the micelle The inserted volume of CTII molecule within the micelle and within the membrane increases when the imidazole group of His31 becomes neutral This is reflected in the pH-dependence of the ability

of CTII to induce an isotropic phase in the membranes of Pam2Gro-PGro

Modes of CTII interaction with the phospholipid membranes

CTII was characterized in detail in aqueous solution by NMR spectroscopy [21] This toxin has two slowly inter-converting forms called ÔmajorÕ and ÔminorÕ [21] They have

a Val7-Pro8 peptide bond in trans- or cis-configuration, respectively Both ÔmajorÕ and ÔminorÕ forms of CTII are not

Fig 5 Temperature dependence of the31P-NMR spectra of Pam 2

Gro-PGro (molar ratio of water/Pam 2 Gro-PGro is 200 : 1) in the presence

of CTII at L/P ¼ 14 : 1 Theoretical spectrum corresponding to the

experimental one taken at 38 C is shown on the right with the

decomposition bands drawn in broken lines.

Fig 6 Temperature dependence of the amount of isotropic signal (A) and of the MLV deformation (B) In (A) L/P ¼ 14 : 1 In (B) data for Pam 2 Gro-PGro (diamonds) and Pam 2 Gro-PGro/CTII mixture (L/P ¼ 14 : 1) (triangles) are shown The molar ratio of water/Pam

2-Gro-PGro in the samples was 200 : 1 The ellipsoidal shape of MLV with semiaxes a and c was assumed in the computer simulations of the data in (B) The vertical bars correspond to the error in the parameter estimation.

bound to zwitterionic membranes of Pam2Gro-PCho at the

membrane hydration levels used in the present study ( 200

water molecules per lipid molecule)

The observations of the present study agree with a

previous suggestion that CTs interact with membranes by a

combination of electrostatic and hydrophobic forces [5] In

this respect the interaction of CTII with negatively charged

membranes is similar to that of other basic peptide toxins:

thionin, purothionins, crambin, viscotoxin and

delta-haemo-lysin [37,38] The data from this suggest that the modes of

CTII interaction with the membranes can be described as

follows (Table 1)

Mode 1: Attracted to the membrane surface (Table 1).It

was suggested [39] that a positively charged peptide can be

attracted by a negatively charged membrane–water interface

due to electrostatic interaction and then partitioned

into membrane hydrophobically It is likely that CTII is immobilized by Pam2Gro-PGro in the gel state without hydrophobic partitioning 1H-NMR study of CTII interaction with MLV of Pam2Gro-PGro at 30C showed (Fig 2) that at L/P > 10 : 1 all CTII is bound to lipid At the same time31P-NMR data indicated that bilayer structure of Pam2Gro-PGro liposomes is not disturbed at 30C (Fig 5) This suggests that all lipid surface is covered by CTII molecules at L/P 10 : 1 as a further decrease of L/P results in the appearance of unbound CTII (see Fig 2, L/P ratios below 10 : 1) Assuming that Pam2Gro-PGro molecules occupy the same surface area in the gel state of the bilayer as Pam2Gro-PCho, i.e 0.48 nm2[40] the CTII molecule would have 4.8 nm2accessible surface area on the bilayer at L/P ¼ 10 : 1 The maximal cross-section of CTII molecule perpendicular to its long axis is 5 nm2and thus no more than one toxin molecule per 10 lipids can be accommodated on the surface of Pam2Gro-PGro MLV in the gel phase Thus at L/P¼ 10 : 1 the Pam2Gro-PGro liposomes in the gel phase are covered with CTII in a carpet-like fashion suggested for cecropins [41] and thionins [42] It

is of note that the membrane charge is fully compensated by the charge of CTII at these conditions

Mode 2: Partitioned (inserted) state (Table 1) In the liquid crystalline phase of Pam2Gro-PGro membrane the hydrophobic partitioning of CTII is favoured In the case

of the P-type CTs the insertion is accomplished via the tips

of three hydrophobic loops (fingers) of the molecule [14–16,43] The tightly bound water molecule in the second loop of these CTs provide W-shape to this loop [16,21,43,44] and the tips of the loops 1–3 are joined into a continuous hydrophobic column The modelling of CTII binding with negatively charged membranes suggested that the only ÔmajorÕ form of CTII is energetically favoured to insert into membranes [15] The mode of the insertion is similar to one described for CTII bound to zwitterionic dodecylPCho micelles [16] The depth of the penetration is determined by the width of the hydrophobic motif of CTII molecule ( 1 nm) and disposition of positively charged side-chains which (Fig 1, lysines 4,5,12,23,50, arginine 36) are bound to the negatively charged groups of the lipid molecules at the lipid bilayer/water interface

The liquid crystalline bilayer of Pam2Gro-PGro can accomodate a definite number of CTII molecules (L/P 20 : 1, Fig 4B) without induction of an isotropic phase The gel to liquid crystal transition is accompanied by

an 0.16 nm2increase of the surface area per lipid molecule

in Pam2Gro-PCho bilayer [40] and we assume the same value for bilayers of Pam2Gro-PGro Thus, the surface area occupied by 20 molecules of Pam2Gro-PGro upon transi-tion from gel to liquid crystalline phase increases by 0.16· 20 ¼ 3.2 nm2 This value is smaller than the molecular area of 5 nm2occupied by a single molecule of CTII on the surface of a dodecylPCho micelle [16] This suggests that insertion of CTII into Pam2Gro-PGro membrane is likely to cause expansion of the membrane Mode 3: Isotropic phase (Table 1) When concentration

of CTII in the liquid crystalline membranes of Pam2 Gro-PGro exceeds a threshold value (L/P 20 : 1), the liposomes are not able to accommodate CTII further and

Fig 7 pH-dependence of the31P-NMR spectra of Pam 2 Gro-PGro/

CTII (14 : 1) dispersions at 50 °C (A), the anisotropic component in the

spectra is shown with broken line, the isotropic signal is truncated (B)

The amount of the isotropic signal plotted vs pH and approximated

by the best nonlinear fit for the determination of pK a value.

are transformed into an isotropic phase with L/P ratio of 10.

The induction of this phase by CTs in anionic membranes

was suggested to be due to formation of stoichiometrically

well defined electrically neutral complexes of CT/

phospholipid having inverted micellar structure [18,45]

This observation seems to be valid for CTII/Pam2

Gro-PGro mixtures too

Biological implications

It is well known that CTs preferentially target and disrupt

bilayers that are rich in acidic phospholipids on the

extracellular side of the plasma membrane [46] This effect

was related to ability of CTs to induce formation of an

isotropic lipid phase [19,45] At a lower concentration, at

which cytotoxic effect is less pronounced, CTs modulate

Na+ and Ca2+ ion fluxes acting on specific membrane

channels [47] This effect can be attributed to CTs bound to

the lipid bilayer with the tips of three fingers We conclude

from this work that CTs influence the bending elastic

modulus of the membrane and, hence, the transbilayer

pressure profile originating from the stiffening of lipid

molecules in the polar but not in the acyl region of

the membrane (In this respect the influence of CTs on the

membrane stiffness is opposite to the action of the

cholesterol [48] which is ubiquitous in the membranes of

animal cells.) The membrane stiffness is important for

functioning of membrane receptors and ion channels [49]

Thus a deleterious effect of low CT doses on living cells

might be due to insertion of CTs into membranes (see mode

2, Table 1) resulted in improper functioning of the ion channels and receptors

Acknowledgements

This work was supported partially by the Ministry of Science and Technology of Russian Federation and by the Russian Foundation of Basic Research (RFBR) grants 00-04-55024, 00-15-97877, 01-04-48548.

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Table 1 Modes of CTII/Pam 2 Gro-PGro interaction.

L/P P 10 : 1

L/P P 20 : 1

L/P < 20 : 1

a

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