NaC systems by SANS

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Chemical Physics 345 (2008) 173–180 www.elsevier.com/locate/chemphys

Membrane self assembly in mixed DMPC/NaC systems by SANS M.A. Kiselev a, D. Lombardo b,*, P. Lesieur c, A.M. Kisselev d, S. Borbely e, T.N. Simonova f, L.I. Barsukov f b

a FLNP, Joint Institute for Nuclear Research, Dubna 141980, Russia CNR-IPCF, Istituto per i Processi Chimico Fisici (Sez. Messina), Messina I-98123, Italy c LURE, Universite´ Paris-sud, F-91405 Orsay, France d Department of Materials Science and Engineering, Pennsylvania State University, USA e Research Institute for Solid State Physics, Budapest, Hungary f Institute of Bioorganic Chemistry, Moscow, Russia

Received 7 March 2007; accepted 25 September 2007 Available online 1 October 2007

Abstract Morphological transition in a mixed system of the dimyristoylphosphatidylcholine (DMPC)/sodium cholate (NaC) has been investigated by small-angle scattering of neutron (SANS) and X-ray (SAXS). Structural investigation, performed as a function of temperature and NaC concentration, show that the system containing 15 mM DMPC and 6 mM NaC reveals a strong dependence of SANS spectra on the temperature. The morphological transformations has been interpreted as a micelle-to-vesicle transition which is induced by the temperature variation (TI-MVT). The main features of the obtained results show that the temperature effect are far less profound in the system containing 2 or 12 mM NaC and can be assigned to small morfological changes within the same population of particles (vesicular or micellar, respectively). The main features of the structural analysis suggest that structural transformations at the TI-MVT proceed in the following sequence: globular micelles – rod-like micelles – polymer-like micelles – unilamellar vesicles. More specifically the globular and rod like micelles present an ellipsoidal cross-section rather than circular one, the former being geometrically more favourable for accommodation of bilayer-forming molecules like DMPC into the micellar structures. Ó 2007 Elsevier B.V. All rights reserved. Keywords: Phospholipid; Neutron Scattering; X-ray Scattering; Micelles; Vesicles

1. Introduction Systems formed by the mixing of lipid/detergent components are of great interest due to their wide use in membrane studies and in particular for solubilization and reconstitution processes involved in membrane proteins [1,2]. Despite successful reconstitution of a great number of membrane functions, molecular mechanisms as well as the thermodynamics of the membrane self-assembly on reconstitution are still debated [3–5]. Particularly interesting in this respect are studies the aggregates formed of bile salts (such as sodium cholate) and bilayer-forming phos*

Corresponding author. Tel.: +39 090 39762222; fax: +39 090 3974130. E-mail address: [email protected] (D. Lombardo).

0301-0104/$ - see front matter Ó 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chemphys.2007.09.034

pholipids. These systems are of general interest not only for the understanding of the assembly processes involved in the formation of the supramolecular aggregates but also as they represent efficient components for the solubilisation of poorly soluble drugs. Due to their ability to solubilize dietary lipids and fat-soluble vitamins in the gastrointestinal tract [6], bile salts have also been employed as delivery systems for application in the field of medicines and biotechnology. Interestingly, due to their peculiar molecular structure made of a rigid hydrophobic steroid core (with hydrophilic hydroxyl groups) and an carboxylic polar headgroup, bile salts such as sodium cholate show unique behaviour with respect to traditional detergents that contain, in general, a well defined polarity between the hydrophilic and hydrophobic components. A clear

M.A. Kiselev et al. / Chemical Physics 345 (2008) 173–180

understanding of the detergent action of bile salts as well as their interaction with phospholipids vesicles under different experimental conditions are then crucial for the study of the relevant parameters that regulate the morphological transitions capable to influence the solubility and functionality of the system. In this sense the micelle to vesicle transition is of special interest in this respect, since it represents an essential stage in the transformation of solubilized micellar aggregates into closed bilayer vesicles [7,8]. More specifically the temperature sensitive lipid/detergent systems, such as the DMPC/NaC herein analysed, are particularly convenient for studying individual stages of the micelle to vesicle transition since their thermotropic behavior is completely reversible and the transition can be arrested at any required step for measurements to be taken under stationary isothermic conditions. The small-angle neutron scattering (SANS) proved to be very efficient to reveal the structure of mixed micelles and vesicles [9–12]. The work of Hjelm et al. described the structure and morphology of the phosphatidylcholine/bile salt mixed micelles in terms of flexible cylindrical wormlike micelles [10] contrary to the mixed disk model popular over many years. Long et al. established the structure of rod-like micelles made of lecithin and taurodeoxycholate using the modified Guinier analysis and a core-shell model of micelles [9]. In the work of Pedersen et al., the coexistence of micelles and vesicles was interpreted with the model of flexible cylindrical micelles [11]. Moreover the study of the aggregation behavior of mixed lipid-detergent systems related to the micelle to vesicle transition has been investigated by means of different complementary experimental methods such as time-resolved fluorescence [3], cryotransmission electron microscopy [6], static and dynamic light scattering [7,13,14], gel exclusion chromatography [8], differential scanning calorimetry [13], NMR [13,14]. Previous investigation of the DMPC/NaC mixed system by turbidimetry measurements, differential scanning calorimetry, electron microscopy and 31P NMR [14–16] experiments, demonstrated that a number of various intermediates can be identified in the mixed system undergoing TI-MVT. The temperature-induced micelle to vesicle transition (TI-MVT) was also observed in different phosphatidylcholine/detergent mixed systems: (egg phosphatidylcholine/octyl glucoside [17], phosphatidylcholine/NaC [15] and in phosphatidylcholine/C12E8) under appropriate lipid/detergent ratios. The main goal of this paper is to report results obtained by SANS measurements on a DMPC-NaC mixed system competent of TI-MVT and to compare aggregative properties and thermotropic behavior with systems incapable to undergo TI-MVT in the assigned temperature interval.

was 1 mM EDTA, 75 mM NaCl, 10 mM Tris-HCl, 0.02% sodium azide. The DMPC concentration was maintained for all samples equal to 15 mM. Samples for SANS measurements were prepared in D2O (99% purity), and for SAXS measurements in H2O (Millipore, 18 MX cm). DMPC/NaC mixtures were prepared by dispersing dry lipid film with buffer solution containing appropriate amounts of NaC. The mixtures were equilibrated at 45 °C for 1 h and then kept at room temperature for at least 15 min before measurements. Due to this treatment all samples were brought to a quasi-steady state with reproducible transitional or non transitional properties determined by turbidity measurements [14,15]. Large ˚ and polydisunilamellar vesicles (mean diameter of 1250 A persity 30%) were prepared by extrusion of a heated (50– 60 °C) DMPC dispersion through Nuclepore membrane fil˚ in diameter) as described by MacDonald ters (pore 1000 A et al. [23]. On the DMPC/NaC/water phase diagram the mixed systems capable of TI-MVT are localized within phase boundaries at effective detergent/lipid ratios (Re) 0.14 < Re < 0.33 [15]. The system containing 15 mM DMPC and 6 mM NaC, termed as a transitional system, was selected for detailed investigation. For comparison two different non-transitional systems unable of TI-MVT were chosen beyond the limits of the transition area on the lipid-detergent phase diagram. These were a micellar system containing 15 mM DMPC and 12 mM NaC (Re = 0.53), and a vesicular system containing 15 mM DMPC and 2 mM NaC (Re = 0.08) or 15 mm DMPC alone (Re = 0). X-ray diffraction experiments were conducted on samples containing 15 mm DMPC and different NaC concentration (0–2 mM). A set of samples with the same Re = 0.23 but different DMPC and NaC concentra-

1.0E+2

1.0E+1

d∑ /dΩ, cm-1

174

1.0E+0

1.0E-1

1.0E-2 0.01

2. Materials Dimyristoylphosphatidylcholine, C36H72NO8P, and sodium cholate, C24H39O5Na, of 99.9% purity were obtained from Serva. The buffer solution used (pH 8.02)

0.10

q, Å-1 Fig. 1. SANS spectra of diluted DMPC/NaC systems at T = 48 °C. 15 mM DMPC/6 mM NaC system (circles), 7.5 mM DMPC/4.25 mM NaC system (squares), 3.75 mM DMPC/3.77 mM NaC system (triangles). All scattering curves are congruent.

M.A. Kiselev et al. / Chemical Physics 345 (2008) 173–180

tion (15/6, 7.5/4.25, and 3.75/3.77 mM/mM) were prepared to prove the absence of interparticle interactions and measured at T = 48 °C. The measured macroscopic cross-sections are presented on the Fig. 1. The multiplication of the experimental macroscopic cross-section measured for the 7.5 mM DMPC/4.25 mM NaC system by the factor 2 and that for 3.75 mM DMPC/3.77 mM NaC system by on the factor 4 gave essentially the same value as the macroscopic cross-section measured for the system with: 15 mM DMPC and 6 mM NaC. 3. Method SANS measurements were performed with the YuMO time-of-flight small-angle spectrometer (pulse neutron source IBR-2) at the Frank Laboratory of Neutron Physics (FLNP) [9] and the small-angle spectrometer (steady state reactor source) at Budapest Institute of Solid State Physics (ISSP). The spectra taken with the YuMO spectrometer were normalized to the scattering cross-section of vanadium, and with the ISSP instrument – to the scattering cross-section of H2O [18–20]. The results obtained with the two spectrometers were in a good agreement. The X-ray measurements were carried out with D22 spectrometer (DCI synchrotron radiation source) at LURE (France). In SANS experiments quartz cells (2 mm in thickness) were used without stirring of samples during measurements. In SAXS experiments quartz capillaries (1.5 mm in diameter) were used. Guinier approximation dr=dX ¼ ðDqV Þ2 expðq2 R2g =3Þ (where V is the volume, Dq the scattering length density relative to the solvent and q is the scattering vector) has been used to interpret the SANS spectra and obtain the radius of gyration Rg. For homogeneous spherical particles of radius pffiffiffiffiffiffiffiffi R one gets R ¼ Rg 5=3 while for ellipsoidal particles with semi-axes a, b and c one has R2g ¼ 15 ða2 þ b2 þ c2 Þ. Expression (1) is valid for q < 1/Rg. For cylindrical particles with a cylinder length L much larger than its radius, the expression for scattering cross-section is

2

  dr 2pS ðDqd l Þ ¼ exp q2 R2t dX q2

ð2Þ

For homogeneous membrane the thickness is calculated as pffiffiffiffiffi d l ¼ Rt 12. In order to distinguish the different morphology of particles the fitting of experimental spectra by the equation dr dr ¼ ð0Þ  qa exp½q2  b dX dX

ð3Þ

was used. Where a = 0 for spherical or ellipsoidal micelles, a = 1 for rod-like micelles, and a = 2 for vesicles or extended unilamellar structures. 4. Results and Discussion Three detergent concentrations has been investigated by SANS for the DMPC/NaC/water system (namely Cd = 2 mM, 6 mM and 12 mM). According to previous turbidimetry studies [14,15], at the intermediate detergent concentration, Cd = 6 mM, the ‘‘transitional system’’ evolves with temperature increase from a micellar state (below 25 °C) to a lamellar state (above 28 °C). The reference samples with Cd = 2 mM and 12 mM represent ‘‘nontransitional systems’’ from the point of view of TI-MVT and do not change their aggregation state (vesicular or micellar) during temperatures scans. In order to gain useful information about the multilamellar structures formed by the binary DMPC/water systems, a diffraction pattern of 15 mM DMPC in H2O buffer solution is presented in Fig. 2. Two diffraction peaks observed in this system fur˚ (at T = 20 °C [21,22]) nish a repeat distance of d = 65.1 A for multilamellar DMPC liposomes in the ripple Pb 0 phase. The X-ray scattering curves obtained for the ternary DMPC/NaC/water system exhibit diffraction peaks only at low detergent concentrations (up to 1 mM NaC). In 1200

ð1Þ

with the validity condition of Guinier approximation as 2p/L < q < 1/Rc. This expression is valid for flexible cylinders with persistence length much larger than the radius of the cross-section. For a homogeneous distribution of the scattering length density at the p circular cross-section of ffiffiffi the cylinder, its radius is R ¼ Rc 2, while for ellipsoidal cross-section of rod-like micelles R2c ¼ 14 ða2 þ b2 Þ, a and b being the semi-axes of an ellipsoid. For extended unilamellar structures (sheets or vesicles) with a surface area S, a thickness dl of the layer (or radius of gyration Rt) much smaller than the radii of curvature of the surface (or the lateral dimensions of the layer) the approximation, valid pffiffiffi for scattering vectors q in the domain 2p= S < q < 1=Rt , is given by the relation:

800

Intensity

 2 2 2 dr pLðDqS c Þ q Rc ¼ exp  dX q 2

175

400

0 0.00

0.05

0.10

0.15

0.20

0.25

0.3 0

q, Å-1 Fig. 2. X-ray diffraction pattern recorded from 15 mM DMPC in water (T = 20 °C). The first and the second diffraction maxima correspond to a ˚. lamellar repeat distance d = 65.1 ± 0.6 A

176

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Table 1 The NaC effect on the repeat distance d of the multilamellar structures in the DMPC/detergent/water system at T = 35 °C CNaC (mM) ˚, dA

0.00

0.25

0.50

0.75

1.00

2.00

62.8 ± 0.6

63.0 ± 0.6

64.1 ± 0.7

65.0 ± 0.7

69.9 ± 1.6

not detected

Table 1 are summarized the obtained values of the repeat distance d as a function of the detergent concentration (at T = 35 °C). Table 1 indicates that the repeat distance d increases ˚ in the system without detergent to from 62.8 ± 0.6 A ˚ in the system with 1 mM NaC and is not 69.9 ± 1.6 A observed with 2 mM NaC. This implies that at the latter concentration sodium cholate disturbs regularities in the multilayer arrangement of the mixed lipid/detergent aggregates in La phase. This circumstance is probably due to the electrostatic repulsion induced by incorporation of the negatively charged NaC molecules into adjacent bilayers. Concerning the ‘‘Non-Transitional Lamellar System’’ in Fig. 3 are reported the SANS spectra (normalized to the cross-section of vanadium) relative to the mixture of 15 mM DMPC and 2 mM sodium cholate in D2O, measured at the two temperatures of T = 20 °C and T = 45 °C. The spectrum at T = 45 °C is well described (v2 = 0.16) by Eq. (3) with a = 1.98 ± 0.02 and ˚ 2 which gives the value of membrane thickb = 116 ± 12 A ˚ ness 37.7 A. The same value was obtained by application of the Eq. (2) in the region of scattering vector ˚ 1 6 q 6 0.1 A ˚ 1. Due to the broad peak in the 0.03 A ˚ 1 the scattering curve obtained at region of q = 0.05 A T = 20 °C can not be fitted by Eq. (3). But it can be fitted by the following relation

" # 2 dr dr dr ðq  q Þ o ¼ ð0Þ  qa exp½q2  b þ ð0Þ  exp  dX dX dX2 2  r2 ð4Þ with an assumption that the scattering results either from multilamellar vesicles or from their mixture with unilamellar vesicles. The fitting by Eq. (4) with v2 = 0.16 gives reasonable value for scattering from flat particles or large vesicles (a = 2.36 ± 0.40) and estimates the position of the diffraction peak from multilamellar structures as ˚ 1, which corresponds to the repeat qo = 0.05 ± 0.007 A ˚ for DMPC membrane doped with distance d = 126 ± 12 A NaC molecules. Fig. 4 shows the Guinier representation for extruded DMPC vesicles at T = 45 °C. For comparison the curves are presented also for the non-transitional vesicular system with 2 mM NaC and for the transitional system with 6 mM NaC. The similarities of the scattering curves imply that at T = 45 °C both the non-transitional and transitional systems (with 2 and 6 mM NaC, respectively) consist of rather large unilamellar vesicles. Let now pass to the analysis of the study of the system which show the transition between globular to rod-like micelle. The obtained results for the micellar system with Cd = 12 mM are presented in Fig. 5. At T = 20 °C the macroscopic cross-section dependence on q in the region of

100.00 -4.8

-5.0

10.00

ln(q2*I), a.u.

d∑ /dΩ, cm-1

-5.3 1.00

0.10

-5.5

-5.8

-6.0 0.01 -6.3

-6.5

0.00 0.01

0.10 -1

q, Å

Fig. 3. SANS data from the mixture of 15 mM DMPC and 2 mM NaC in D2O measured at T = 20 °C (circles) and at T = 45 °C (squares, ˚ , dl = 37.7 A ˚ ). The broaded diffraction peak at T = 20 °C Rt = 10.89 A ˚ corresponds to the repeat distance of with maxima at q = 0.05 A ˚. multilamellar liposomes d = 126 ± 12 A

0.000

0.002

0.004

q2,

0.006

0.008

0.010

-1

Å

Fig. 4. Guinier plot of the scattering curves measured at T = 45 °C for extruded unilamellar vesicles prepared from DMPC alone (15 mM) ˚ ), for the mixture of 15 mM DMPC and 2mM NaC (circles, dl = 38.3 A ˚ ) and for the mixture of 15 mM DMPC with 6 mM (squares, dl = 37.7 A ˚ ). NaC (triangles, dl = 35.3 A

M.A. Kiselev et al. / Chemical Physics 345 (2008) 173–180

small scattering vectors corresponds to the law q0. Calculations of the gyration radius furnish the same value of ˚ with both the Guinier approximation Rg = 28.5 ± 0.4 A and Eq. (4). The macroscopic cross-section obtained at T = 45 °C can be fitted by the Eq. (4) with the accuracy v2 = 0.04 and the values of a = 0.63 ± 0.02 and b = ˚ 2. The power is clearly temperature depen178.7 ± 5.2 A dent: T = 20 °C, a = 0.0 ± 0.02; T = 36 °C, a = 0.36 ± 0.02; T = 40 °C, a = 0.53 ± 0.01; T = 45 °C, a = 0.63 ± 0.02. It means that with the temperature increase the scattering contribution is shifted from sphere-like micelles (a = 0) towards rod-like micelles (a = 1). A large deviation of the power of q from the value of 1.0 at all measured temperatures makes impossible exact calculations of the gyration radius for the rod-like micelles. But in any case the data obtained for the system with Cd = 12 mM at temperatures above 40 °C demonstrate clearly that the system can be considered as a mixture of sphere-like and rod-like micelles. Fit with Eq. (1) for analysis of the scattering at T = 45 °C gives a value of cylinder ˚ . This value is in good agreement radius R = 22.8 ± 0.5 A with that measured by Long et al. [3] for rod-like micelles in the egg yolk lecithin/bile salt system. By using the ˚ was obtained. core-shell model the value of R = 26.6 A ˚ difference in radii of lecithin/bile salt micelles The 3.8 A ˚ ) and DMPC/NaC micelles (22.8 A ˚ ) can be attrib(26.6 A uted to the different lengths of hydrocarbon chains in these two phospholipids. With an average number of 17 C-atoms in the fatty acid chain of egg yolk lecithin molecule and 14 ˚ can be expected C-atoms in DMPC a difference of 3.75 A ˚ per two CH2 groups. As for the globon the basis of 2.5 A ular micelles, their estimated radius of gyration ˚ is not compatible with the ideal spheriRg = 28.5 ± 0.4 A cal geometry since the radius of micelles in this case should

177

˚ ) than total length of the DMPC be much larger (36.8 A ˚ molecule (26.7 A in the fully extended conformation). Assuming an ellipsoidal model, a prolate shape (which is closer to the cylindrical geometry than the oblate one) and a minor equatorial semi-axis to be equal to the radius ˚ ) observed at higher temperaof the cylinders (22.8 ± 0.5 A ˚ for the major semiture, we obtain a value of 55.0 ± 0.5 A axis of the ellipsoid. Although the methods used in the present analysis can not supply detailed structural information we can assume that DMPC and NaC molecules are not randomly distributed within the micellar aggregates: the detergent is expected to be located in the areas of high curvature while the lipid will prefer the regions of lower curvature. Let now analyze the transition between rod-like micelle to vesicle. The transitional system with Cd = 6 mM was studied in a wide range of temperatures (20–50 °C) that covers the main phase transition temperature of pure DMPC (Tph = 23.5 °C) and a temperature interval of the TI-MVT 28–33 °C as determined by turbidity measurements. During the neutron measurements the temperature was kept constant, but between the measurements the heating or cooling rate was equal to 0.3 °C/min. The experimental protocol of that kind ensures the complete reversibility of the TI-MVT [14,15] in relation to the morphological changes observed which are reproducible upon heating and cooling scans. Fig. 6 shows the scattering curves obtained at the most representative temperature points which correlate with the pronounced morphological changes. The scattering curve obtained for the non-transitional 15 mM DMPC/12 mM NaC system at T = 20 °C is

10.0

d∑ /dΩ, a.u.

10.0

1.0

q0

1.0

d∑ /dΩ, cm-1

q-1

0.1

0.1

q-2 0.01

0.10

q, Å-1 0.0 0.01

0.10

q-4

-1

q, Å

Fig. 5. Intensity scattered by the mixture of 15 mM DMPC and 12 mM NaC in D2O. Triangles: globular micelles observed at T = 20 °C ˚ , minor semi-axis 22.8 A ˚ , major semi-axis 55.0 A ˚ ). Crosses: (Rg = 28.5 A ˚ ). rod-like micelles measured at T = 45 °C (R = 22.8 A

Fig. 6. Experimental SANS spectra of the DMPC-NaC-water system. Dots are of the 15 mM DMPC/12 mM NaC system at T = 20 °C. The other curves correspond to the mixtures of 15 mM DMPC/6 mM NaC. The micelle to vesicle transition is crossed by temperature tuning: squares T = 20 °C, triangles T = 25 °C, x’s T = 35 °C, stars T = 40 °C.

178

M.A. Kiselev et al. / Chemical Physics 345 (2008) 173–180

also presented as a reference for the sphere-like micelles. The lines corresponding to the q0, q1, q2, q4 laws are plotted on the graph in order to distinguish between scatterings from sphere-like micelles with the q0 dependence of scattering intensity at small q values, rod-like micelles with the q1 dependence of scattering intensity at small q values, and vesicles with the q2 dependence of scattering intensity at small q values. The q4 dependence of scattering intensity at large q values corresponds to the Porod law. The scattering in the transitional system is dominated by rod-like micelles in the range of 20–30 °C, the radius of gyration Rc decreasing with the temperature increase. At T = 33 °C the scattering curve begins to deviate from the ideal Guinier law for the rod-like micelles that demonstrates the onset of the structural changes in the system. At T = 35 °C the scattering curve reveals the presence of polymer-like structures in the system. The macroscopic cross-section measured for the transitional system at T = 35 °C is presented in Fig. 7. The macroscopic cross-section measured at T = 35 °C has two different dependences on scattering vector. At ˚ 1 the intensity is proporsmall q values 0.01 6 q 6 0.03 A 2.1±0.02 tional to the law q , which describes the scattering from overall dimension of polymer-like micelles [11]. In ˚ 1 the scattering intensity the q region of 0.03 6 q 6 0.1 A 1.02±0.09 is proportional to the law q Æ exp[(185 ± 15) Æ q2], which describes the scattering from the cross-section of polymer-like micelles. Fit with Eq. (1) furnish Rc = ˚ for the polymer-like micelles. At T = 40 °C 19.7 ± 0.4 A and higher temperatures unilamellar vesicles are observed which form spontaneously on heating. The scattering curve at T = 40 °C obeys well Guinier law (Eq. (2)) for unilamellar structures. The membrane thickness of the unilamellar vesicles is increasing with the temperature enhancement. Transitional system with 15 mM DMPC and 6 mM NaC in D2O are well described by the Guinier approxima-

tion. More specifically, the system at T = 20 °C are well represented with Eq. (1) for rod-like micelles. pffiffiffi Calculations of radius of rod-like micelles, R ¼ Rc 2, based on the ˚ at model of circular cross-section give R = 31.4 ± 0.5 A ˚ 20 °C and R  28 A at 30 °C. These values are distinctly larger than the maximal length of DMPC molecule (see above) and far exceed reasonable values of the DMPC molecule length calculated from the membrane thickness at corresponding temperature. For example, the length of DMPC molecule at T = 20 °C can be estimated as ˚ from data presented in Fig. 8. The inconsis21.7 ± 0.5 A tency between the radius of rod-like micelles and the length of a DMPC molecule can be reconsidered if one assumes that the rod-like micelles have an ellipsoidal cross-section rather than the circular one. In this case R2c ¼ 14 ða2 þ b2 Þ, where a and b are semi-axes of ellipsoid. If one of the semi-axes (a) is estimated as half of thickness of DMPC membranes in D2O then other semi-axis (b) can be calculated from experimental values of Rc. In such a way with ˚ the value the value of minor semi-axis a = 21.7 ± 0.5 A ˚ of major semi-axis b is equal to 38.7 ± 0.5 A. The characteristic sizes of the particles formed in the transitional system at different temperatures are presented in Table 2. It is worth noticing that the ellipsoidal cross-section of rod-like micelles is geometrically more favorable for accommodation of bilayer-forming molecules like DMPC into the structure of the rod-like micelles. The DSC measurements on the transitional system [14,15] demonstrate the presence of distinct phase transitions in the DMPC/ NaC mixed aggregated which gives an indirect support in favor of the bilayer-based structure of rod-like micelles. The assumption of non-circular cross-section of lecithin/ taurodeoxycholate mixed rod-micelles was made in [9], but no calculation of ellipsoidal semi-axes were performed.

45

Membrane thickness, Å

10.00

d∑ /dΩ, cm-1

1.00

0.10

40

0.01

35 0.00

10 0.01

0.10

q, Å-1 Fig. 7. Polymer-like micelles. Intensity scattered by the mixture of 15 mM DMPC/6 mM NaC in D2O at T = 35 °C.

20

30

40

50

60

Temperature, ºC Fig. 8. Dependence of DMPC membrane thickness on the temperature. Membrane thickness was determined by SANS experiment performed on extruded unilamellar DMPC vesicles in D2O.

M.A. Kiselev et al. / Chemical Physics 345 (2008) 173–180

179

Table 2 Geometry and size of mixed aggregates formed in the transitional system (15 mM DMPC and 6 mM NaC in D2O) T (°C)

20

25

28

30

33

35

40

50

60

Part. type ˚) Size (A

RLM

RLM

RLM

RLM

PLM

PLM

UV

UV

UV

21.7 38.7

20.8, 36.4

19.9, 35.4

19.6, 34.2

19.5 34.3

19.3 33.0 35.2

35.6

36.1

a, b or dl

RLM stands for rod-like micelles with an ellipsoidal cross-section of semi-axes a and b, PLM stands for polymer-like micelles with an ellipsoidal crosssection of semi-axes a and b and UV stands for unilamellar vesicles with bilayer thickness dl.

The membrane thickness of the DMPC/NaC vesicles formed in the transitional system in the temperature region from T = 40 °C to T = 60 °C increases slightly with temperature (see Table 2). For comparison, the bilayer thickness was measured as a function of temperature for pure unilamellar DMPC vesicles prepared by the extrusion technique. As seen from Fig. 10, in the temperature region from T = 36 °C to T = 60 °C the membrane thickness is nearly constant with a slight tendency to decrease with the tem˚ at T = 36 °C, dl = perature increase (dl = 38.5 ± 1.0 A ˚ at T = 45 °C, dl = 38.0 ± 1.0 A ˚ at T = 50 °C, 38.3 ± 1.0 A ˚ at T = 60 °C). These data show that and dl = 37.2 ± 1.0 A in the temperature domain from 40 °C to 60 °C the membrane thickness of the pure DMPC unilamellar vesicles prepared by extrusion is somewhat larger than that of unilamellar vesicles formed during TI-MVT in the transitional system containing 6 mM sodium cholate. This observation implies that NaC molecules induce some conformational disorder in the mixed bilayer, for instance, by increasing a fraction of gauche conformers in hydrocarbon chains of DMPC so that their effective length is shortened. At 60 °C the difference in membrane thickness of DMPC/NaC vesicles and pure DMPC vesicles is ˚ which is in the interval of experimental errors only 1.1 A ˚. ±1 A 5. Conclusion The transformation of ellipsoidal micelles to lamellar structures through the formation of rod-like micelles has been detected in the DMPC /sodium cholate/water mixed system by neutron and X-ray scattering. At low temperature (T = 20 °C) the system evolves with an increase in the sodium cholate concentration from vesicular (Cd = 2 mM) to rod-like micelles (Cd = 6 mM) and then to globular micelles (Cd = 12 mM). At the intermediate detergent concentration Cd = 6 mM the system becomes temperature sensitive and displays a morphological transition from rod-like micelles with ellipsoidal cross-section (observed below 33 °C) to unilamellar vesicles (observed above 35 °C). A drastic change in the morphology of particles occurs in a temperatures domain of 33–35 °C which is higher than the temperature of the main phase transition of DMPC (Tph = 23.5 °C). The particles formed as intermediates between micelles and vesicles can be interpreted as polymer-like micelles with ellipsoidal cross-section.

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