Báo cáo khoa học: Thermal behaviour of cubic phases rich in 1-monooleoyl-rac-glycerol in the ternary system docx

These MO contents correspond to a Pn3mcubic single-phase or a Pn3m cubic phase in excess water on the binary MO/water axis of the ternary phase diagram.. Remarkably, both the Ia3d and th

Thermal behaviour of cubic phases rich in 1-monooleoyl- rac -glycerol

in the ternary system

1-monooleoyl- rac -glycerol/ n -octyl-b-D-glucoside/water

Gerd Persson1, Ha˚kan Edlund1and Go¨ran Lindblom2

1 Department of Natural and Environmental Sciences, Mid Sweden University, Sundsvall, Sweden, 2 Department of Biophysical Chemistry, Umea˚ University, Umea˚, Sweden

Using synchrotron X-ray diffraction the thermal behaviour

was studied of the cubic phases in the

1-monooleoyl-rac-glycerol (MO)/n-octyl-b-D-glucopyranoside (OG)/2H2O

system with 58 or 45 wt % MO concentration and varying

OG/2H2O contents These MO contents correspond to a

Pn3mcubic single-phase or a Pn3m cubic phase in excess

water on the binary MO/water axis of the ternary phase

diagram The cubic liquid crystalline phases are stable with

small fractions of OG, while higher OG concentrations

trigger a cubic-to-lamellar phase transition Moreover, with

increasing OG concentration the initial Pn3m structure is

completely converted to an Ia3d structure prior to the La

phase being formed Upon heating this effect is reversed,

resulting in an Ia3d-to-Pn3m phase transition For some

samples additional peaks were observed in the diffracto-grams upon heating, resulting from the metastability notoriously shown by bicontinuous cubic phases This judgement is supported by the fact that upon cooling these peaks were absent Remarkably, both the Ia3d and the Pn3m cubic structures could be in equilibrium with excess water in this ternary system A comparison is made with previous results on n-dodecyl-b-D-maltoside (DM), showing that cubic phases with OG have higher thermal and composi-tional stability than with DM

Keywords: 1-monooleoyl-rac-glycerol; n-octyl-b-D -gluco-side; monoolein-rich cubic phases; thermal behaviour

Access to the complete structure of membrane proteins is

one of the cornerstones in obtaining a better understanding

of their function in the biological cell For larger proteins the

most important method for achieving this information is

X-ray diffraction, which require high quality crystals of the

protein It is frequently possible to crystallise water-soluble

proteins, which can be inferred from the large number of

structures determined so far [1] However, it is considerably

more difficult to get suitably good crystals of membrane

proteins, mainly due to the need to remove them from

their native membrane environment, and solubilize them in

mild detergent micelles The solubilization process may lead

to denaturation of the proteins, thus destroying them

Therefore, one of the foremost current issues is concerned

with the problem of obtaining such crystals A new

approach to solve this problem was introduced by Landau

and Rosenbusch in 1996 [2] Their method includes the use

of a bicontinuous cubic liquid crystalline phase as the crystallization medium The general idea behind this approach was to introduce the proteins into an envi-ronment that mimics the native milieu [2], and the bicon-tinuous cubic phases formed by 1-monooleoyl-rac-glycerol (MO) [3,4] was utilized to meet these basic requirements The exact mechanisms involved in the crystallization process are yet to be elucidated, although an attempt

to describe the process has been published [5] It should

be noted that since the introduction of this method, only two to three membrane proteins have been successfully crystallised [2,6–8] Therefore, for the method to be generally functional it is necessary to have a detailed understanding, at the molecular level, of what is driving the protein crystallization To utilize this method fully, several crucial issues need to be solved From a colloid or surfactant chemistry point of view knowledge about the microstructure of the liquid crystalline phases involved, and information about the possible effect(s) different additives may have on the liquid crystalline phases present are very important A recent paper presented the compati-bility of a number of substances with the MO cubic phases [9], but among these additives only one was a surfactant (cetyltrimethylammonium bromide) Moreover, the most frequently used surfactant for the solubilization of the membrane proteins is n-octyl-b-D-glucopyranoside (OG) However, to our knowledge, only a few attempts to partially investigate the effect of OG on the stability of the MO cubic phases have been published [10,11], and it seems appropriate

Correspondence to G Persson, Department of Natural and

Environmental Sciences, Mid Sweden University, Holmgatan 10,

SE-851 70 Sundsvall, Sweden.

Fax: + 46 60 148802, Tel.: + 46 60 148932,

E-mail: Gerd.Persson@kep.mh.se

Abbreviations: DM, n-dodecyl-b- D -maltoside; DSC, differential

scanning calorimetry; MO, 1-monooleoyl-rac-glycerol; OG,

n-octyl-b- D -glucopyranoside; PMOS, phosphomolybdic

acid in sulphuric acid solution; SAXD, small-angle X-ray diffraction;

TLC, thin-layer chromatography.

(Received 8 July 2002, revised 27 October 2002,

accepted 11 November 2002)

to extend these studies further In a previous paper we

presented the entire phase diagram for the ternary system

MO/OG/2H2O [12], from which it can be concluded that

only a small fraction of OG is sufficient to convert the

MO-rich cubic phases to a lamellar liquid crystalline (La)

structure, and that these cubic phases are also found in

equilibrium with excess water (cf the binary

MO/water-system) In this report, the thermal behaviour of such cubic

phases rich in MO has been investigated by X-ray

diffrac-tion The study comprises both one- and two-phase regions,

including cubic phases in equilibrium with excess aqueous

solution

Materials and methods

Materials

1-Monooleoyl-rac-glycerol (MO) (> 99% purity) and

n-octyl-b-D-glucopyranoside (OG) (> 98% purity) were

purchased from Sigma Aldrich Chemie GmbH, Germany

and the substances were used without further purification

2H2O (99.9% in2H) was obtained from Cambridge Isotope

Laboratories, USA

Sample preparation

Samples were prepared by weighing the appropriate

amounts of each substance into 8-mm glass tubes, which

were sealed with removable caps The samples were

homogenized by rotation and stored in the dark at 25°C

All samples were inspected both visually and between

crossed polarizers to check homogeneity and optical

anisotropy The samples were then placed in 1.5-mm

capillaries All samples were stored in 5°C for 12 h and

then at 25°C for 4 days prior to measuring

Methods

Small-angle X-ray diffraction (SAXD) The small angle

X-ray diffraction, SAXD, experiments were performed at

the Austrian Academy SAXS station at the ELETTRA

synchrotron (Trieste, Italy) using the 8 keV beam,

corres-ponding to a wavelength of 0.15 nm The temperature was

controlled by an in-line differential scanning calorimeter

(DSC), and the temperature-scanning rate used was

1°CÆmin)1 during heating, while the cooling rate was

approximately 5°CÆmin)1 To ensure that a powder pattern

was obtained, the capillary was rotated with an angular

velocity of 1.26 radÆs)1 using a purpose-built device To

minimize the exposure time we used a sampling time of

10 sÆmin)1and the shutter was closed during the residual

50 s The space group of the structure of the cubic liquid

crystalline phase was achieved from the locations of the

peaks in the SAXD diffraction patterns [13] For the two

space groups identified in this work (Pn3m and Ia3d) the

first four Bragg reflections appear at spacing ratios of

Ö2 : Ö3 : Ö4 : Ö6 and Ö6 : Ö8 : Ö14 : Ö16, corresponding to

the Miller indices (110), (111), (200), (211) and (211), (220),

(321), (400), respectively The cubic cell lattice parameter, a,

is obtained as the slope of a plot of the reciprocal spacings

(1/dhkl) of the Bragg reflections vs m¼ (h2+ k2+ l2)1/2,

where h, k and l are the Miller indices

Thin layer chromatography (TLC) The SAXD samples were checked for purity and radiation damage by thin layer chromatography (TLC) in a mixture of hexane, diethyl ether and acetic acid in the ratio 80 : 20 : 1 (v/v/v), using a 20 cm long and 0.2 mm thick silica plate (Fluka) Spots were developed by spraying the plate with 2.5% (v/v) phospho-molybdic acid in sulphuric acid solution (PMOS), followed

by heating The appearance of two unidentified spots and another corresponding to oleic acid showed that there was some degradation of MO However, comparison with unexposed samples show that this very small degradation is not a result of the exposure to X-rays, but rather a result of the presence of water which leads to hydrolysis of MO [14]

Results

Cubic phases Cubic liquid crystalline phases with a number of different structures have been reported [13,15, and references therein] These are usually grouped into two main types, namely the discrete and bicontinuous structures The discrete structure

is also known as the micellar type, since it is constructed from micellar entities, normal or reversed, arranged in a cubic lattice In cubic phases of the bicontinuous category a surfactant bilayer is associated with an infinite minimal periodical surface resulting in a structure that is continuous with respect to both water and amphiphile These bicontin-uous phases can also be of the normal or reverse kind [16] Within the two main groups of cubic structures several spatial arrangements are possible, leading to a number of different space groups [13]

In the binary system of MO/water at 25°C two reversed bicontinuous cubic phases, belonging to the space groups Ia3dand Pn3m, are present [17,18] Upon addition of small amounts of OG the Pn3m structure is transformed via Ia3d

to an Lastructure From Fig 1 it can be inferred that, at

25°C, the molar ratios between OG and MO (calculated on the total lipid concentration) needed for inducing the Ia3d and lamellar phases are in the order of 1 : 90 and 1 : 16, respectively

Figure 2 shows partial phase diagrams, where the OG concentration is plotted against temperature at a constant

MO concentration of 57.8 wt % The compositions of the samples studied are found in Fig 1 (located on an almost

Fig 1 Section of the ternary MO/OG/ 2 H 2 O phase diagram at 25 °C, showing the location of the samples studied Pn3m, MO-rich cubic phase

of space group Pn3m; Ia3d, MO-rich cubic phase of space group Ia3d;

L a + Ia3d, lamellar + cubic two-phase area; L a , tentative boundary

of the lamellar phase [12].

straight line in the figure) We assume that a heating rate of

1°CÆmin)1is slow enough to allow for the phase-transitions

to occur, while the cooling rate is set by the equipment The

cooling was performed to determine whether the initial

structure would reappear on a reasonable time scale, since

cubic structures are prone to be meta-stable Table 1

summarizes heating scans of SAXD measurements at

different compositions (A–E) with the corresponding lattice

parameters It can be concluded that the general behaviour

upon heating, in this area of the phase diagram, is to shift

the aggregate structure towards a more curved surface in the

liquid crystalline phase (Fig 2A) Moreover, the lattice

parameters of both cubic phases tend to decrease with increasing temperature For the Ia3d phase a increases with the OG content, while for the Pn3m structure it seems to decrease compared to the structure in the binary system [4]

An additional, very strong, peak is observed for sample

A, located between (110) and (111) peaks in the Pn3m pattern in the temperature range 25–35°C (Figs 3 and 4B) Extra peaks are also observed for samples B (weak) and C (medium) that are located between (220) and (321) peaks in the Ia3d pattern at temperatures 25–30°C and 20–35 °C, respectively We have not observed any optical anisotropy

in this temperature range for these samples Upon cooling,

Fig 2 Temperature vs composition for the samples A to E as

deter-mined by SAXD (A) Heating scans at a rate of 1 °C min)1; (B) cooling

scans at a rate of approx 5 °CÆmin)1 The MO concentration is

approx 58 wt % (d) Pn3m, (black circle containing white cross)

Pn3m + additional peak, (s) Ia3d, (white circle containing black

dot), Ia3d + additional peak, (shaded circle) Ia3d + Pn3m, (shaded

triangle) Ia3d + L a

Table 1 Sample composition and the lattice parameter, a, obtained upon heating scans Heating rate 1 °CÆmin)1 To ensure that a powder pattern was obtained, the capillary was rotated with an angular velo-city of 1.26 radÆs)1.

Sample

wt % MO/OG/ 2 H 2 O

T (°C)

Pn3m (nm)

Ia3d (nm)

La (nm)

A 57.81/0.34/41.85 20 9.66

55 8.68 13.81

F 44.77/0.30/54.93 21 9.78

G 44.67/1.14/54.19 21 10.89

30 10.76

40 10.52

45 10.03

H 44.54/2.29/53.17 21 11.12 17.15

these metastable structures do not occur in these three

samples, and no unexpected peak occurs either in the

heating scans of samples D and E

The origin of the extra peaks observed is unclear and

we can only speculate at their origin One plausible explanation is that they result from another cubic

Fig 3 Diffractograms of the samples (A–E) shown in Fig 1 (a) Heating rate of 1 °CÆmin)1, (b) cooling rate of approx 5 °CÆmin)1.

structure The energy needed for the Pn3m–Ia3d phase

transition is very small [3]

A peculiarity was observed upon cooling the samples C

and D where Pn3m and Ia3d patterns coexist instead of the

Ia3dand lamellar patterns observed in the heating scans

(Fig 3 and Table 2) We have not investigated whether a lamellar structure will appear at sufficiently long waiting time All these observations are most probably caused by the notorious problem of reaching a true thermodynamic equilibrium for bicontinuous cubic phases [13]

Fig 3 (Continued).

In samples D and E a rather drastic broadening in the line

shape of the peaks indexed to the Ia3d structure occurs just

below 40°C, indicating the occurrence of a more disordered

structure Also, the lattice parameter jumps to a smaller

value in this region (cf Figure 3 and Table 1)

Finally, visual as well as microscope observations, show

differences in the optical appearance of the two cubic

phases, one being slightly turbid (Pn3m), while the other one

is completely clear (Ia3d) As a final point it could also be

mentioned that the transition enthalpies between the

different cubic structures are so small that they are very

difficult to observe by conventional DSC [3]

Cubic phases in excess water

In the binary system of MO/water only the Pn3m cubic

phase is found in equilibrium with excess water At 25°C

this structure is retained upon addition of small amounts

OG (samples F–H) The composition of the excess aqueous

solution has not been determined, but from the ternary

phase diagram it is obvious that the aqueous phase contains

very little OG, referred to hereafter as excess water [12] As

in the previous paragraph, Fig 1 shows the sample locations

in the ternary system, while the compositions and corres-ponding lattice parameters for the heating and cooling scans are summarized in Tables 1 and 2 Upon heating, the Pn3m structure is retained in sample F and G, while in sample H the structure is changed to an Ia3d space group (Figs 5 and 6) However, upon cooling the structure returned to the Pn3mstructure at 35–30°C, which is a higher transition temperature than obtained from the heating scan, again pointing to the difficulty of obtaining a true thermodynamic equilibrium for bicontinuous cubic phases

Fig 4 Diffractogram of sample A showing (A) without and (B) with the

extra peak The dots above the peaks indicate the calculated positions

obtained for the Pn3m structure based on the first peak in the

dif-fractogram.

Table 2 Sample composition and the lattice parameter, a, obtained upon cooling scans Cooling rate approx 5 °CÆmin)1 To ensure that a powder pattern was obtained, the capillary was rotated with an angular velocity of 1.26 radÆs)1.

Sample

wt % MO/OG/ 2 H 2 O

T (°C)

Pn3m (nm)

Ia3d (nm)

La (nm)

A 57.81/0.34/41.85 45 9.15

B 57.81/1.31/40.88 64 8.40

30 8.74 13.19

26 9.00 13.91

34 9.71 15.08

20 9.71 15.13

40 9.67 14.66

34 9.55 14.93

28 9.62 14.84

23 10.30 15.31

F 44.77/0.30/54.93 44 9.23

G 44.67/1.14/54.19 45 9.83

34 10.66 16.69

29 10.68 15.31

18 10.48

Similarly to the results obtained for the Ia3d cubic phase

the lattice parameters for the Pn3m cubic phase in

equilibrium with excess water increase with the OG content

Furthermore, as for the cubic phases without excess water

the lattice parameters tend to decrease with increasing

temperature Also, for the samples with excess water,

additional peaks appear at specific temperature intervals

upon heating, while they are absent when cooling The

temperature range of these extra peaks lies between 30 and

40°C for sample G and H, while sample F shows a wider

temperature range The additional peak is quite strong in

samples F and G positioned between (110) and (111) in the

Pn3mpattern, while in sample H it is very weak occurring

between (220) and (321) in the Ia3d pattern

Discussion

The aim of this work is to investigate how the structure and

thermodynamic stability of the cubic phases formed by MO

in water is affected by the presence of OG, which is

commonly utilized to solubilize or extract membrane

proteins from native membranes The OG micellar solution

containing the membrane protein may hold quite a high

concentration of the surfactant In this study we have shown

that the cubic phases in fact are very sensitive to relatively

small amounts of OG An increase in the OG concentration

results in a decrease in the absolute value of the curvature of

the lipid bilayer, building up the cubic phase structure This

conclusion is drawn from the observation that upon addition

of OG an increase in the size of the unit cell within a

one-phase area arises, and from the order of occurrence of the

phases formed The reason for this change in the curvature

can be understood in terms of a simple geometric

considera-tion of the shape of the molecules involved [19–21] MO,

forming reversed nonlamellar phases, is considered to be

wedge shaped having quite a small glycerol head group, while

the C18 hydrocarbon chain is rather bulky with its double

bond between C9 and C10 On the other hand, the highly

water soluble OG has a more conical shape with the base

located at the glucose head group and a relatively short

hydrocarbon chain with only eight carbons, yielding a

packing parameter of less than one at high water contents

[21] The hydrocarbon chain of OG is of similar length as the

distance from the head group to the double bond in MO

Thus, for an OG molecule present in the MO bilayer its head

group will be located at the bilayer interface together with the

MO head groups, while the hydrocarbon tail of OG

penetrates deeper into the bilayer It will reach approximately

down to the MO double bond The combined MO-OG

molecular entity will attain a shape of a cylinder, resulting in

a packing parameter that is closer to one Moreover, the

flexibility possessed by a bilayer containing MO only will be

reduced, resulting in a preference for an arrangement of a

flat bilayer, eventually forming an Laphase

Similarly, the effect of an increase in temperature on the

phase behaviour can be understood with this simple model

based on the molecular shape Within a region of a cubic

phase in the diagram an increase in the temperature

generally results in a decrease in the size of the unit cell

This is interpreted to be an effect of an increase in the

curvature of the lipid bilayer affected by the increased

mobility of the hydrocarbon chains together with a possible

decrease in the hydration of the glucoside head groups (cf the nonionic alkylethylenoxide surfactants [22]), i.e the lipid molecules will attain a more wedge-like shape To put it another way, with increasing temperature the bilayer gets thinner, with the minimal and the parallel surfaces of the polar/nonpolar interface approaching each other, again resulting in an increased wedge shape of the lipids Previously, Ai and Caffrey investigated the effect of a different sugar lipid, n-dodecyl-b-D-maltoside (DM), on MO cubic phases [23], and it seems useful to compare our results with their study They showed that the addition of DM converts the Pn3m cubic phase to an Laphase via an Ia3d structure For both OG and DM the order in the phases formed upon addition of the surfactants is similar However,

on a molar basis, the stability of the liquid crystalline phases is higher for additions of OG than of DM, as can be seen when comparing the fraction of each surfactant necessary to induce phase transitions At 25°C, the ratios between the sugar surfactant and MO, where an Ia3d structure was formed, is in the order of OG/MO¼ 1 : 90 and DM/MO ¼ 1 : 170, while for the lamellar structure the appropriate fractions between the surfactant and MO are OG/MO¼ 1 : 16 or DM/MO¼ 1 : 21 Note however, that these ratios refer to total concentrations and not to the actual fraction of OG or

DM in the bilayer, since there is a large difference in critical micellar concentration for these surfactants (25 mMfor OG [24]; 0.15 mMfor DM [25]) Therefore, it is fair to assume that there is somewhat less OG incorporated in the MO bilayer than these ratios indicate However, this is probably of minor importance in a comparison of the effect between the two sugar surfactants

Furthermore, the cubic Ia3d and Laphases containing

OG have a higher thermal stability than if DM is present in the phases, i.e the temperature at which the phase boundaries shifts towards higher OG concentration (Fig 2A) Thus, with OG the Ia3d phase is stable up to about 50–55°C, while for DM the Ia3d-to-Pn3m phase transition occurs at about 40°C We have not determined how OG affects the two-phase area of water/Pn3m The effect of OG and DM on the phase behaviour of the MO-system can be explained by a consideration of the effective packing parameter, resulting upon addition of the sugar surfactant The maltoside head group is a disaccharide and therefore the DM head group is larger than that of OG with

a monosaccharide head group, but because the hydrocar-bon tail is longer for DM the packing parameter is closer to one than for OG This difference is also reflected in the binary phase diagrams of DM/water [26] and OG/water [27] In the DM/water system only micelles and a lamellar phase is present, while in the OG system both hexagonal and bicontinuous cubic structures as well as micelles and a lamellar phase are formed at room temperature When OG

is present in the MO bilayer, the shorter tail will only affect the part of the MO molecule from the head group to the double bond, leaving the rest of the MO hydrocarbon chain free, while introducing DM into the bilayer will affect the packing more, as the longer tail will reach approximately to the centre of the bilayer Thus, considering only the hydrocarbon tail, addition of DM should not be very different from adding another MO molecule, but the properties of the head groups are slightly different and must also be considered A study of the effects of maltose

Fig 5 Diffractograms of the samples with excess water (F–H) shown in Fig 1 (a) Heating rate of 1 °CÆmin)1; (b) cooling rate of approx 5 °CÆmin)1.

and glucose on the Pn3m phase of the MO system shows

that both sugars are tolerated to rather high concentrations,

but the lattice parameter decreases with increasing sugar

concentration The lattice parameter decreases more rapidly

for maltose [23], indicating an effect on the hydration of the

cubic phase to a larger extent than for glucose It is thus

obvious that the longer hydrocarbon chain and the larger

head group of DM are the causes for the stronger effect on

the phase behaviour for this surfactant than for OG

Therefore, DM exhibits a larger effect at low concentrations,

and shows a stronger influence on the temperature

depend-ence on the cubic Ia3d and the lamellar phases than OG

Conclusions and final remarks

In this report, we have investigated the thermal behaviour of the MO-rich cubic phases found in the ternary phase diagram of MO/OG/2H2O It is shown that only small amounts of OG (OG : MO¼ 1 : 16) are sufficient to transform the cubic structure to a lamellar one Addition

of OG to the Pn3m cubic phase converts it to an Ia3d structure in analogy with previous results on the MO system containing a different sugar surfactant [23]

The results obtained in this study on the stability of the cubic phases in the ternary lipid system may be of importance for getting a better understanding of the crystallization process of membrane proteins In particular,

it is of great importance to realize that for the OG-solubilized proteins added to the cubic phase, the OG content is limited

to a few percent to keep a stable cubic phase If the proteins are reconstituted in the lipid bilayer, as suggested by the proposed mechanism [5], the large protein molecules will also affect the phase behaviour in such a way that a more planar lipid aggregate is created However, the effect may not be large enough to change the phase behaviour of the entire sample, since the total protein concentration is quite low, but locally the effect may be dramatic, which in turn may affect the crystallization process

Acknowledgements

We wish to thank the local contacts at the Austrian Academy SAXS station at the ELETTRA synchrotron, Trieste, Italy: H Amenitsch,

M Rappoult, M Strobl and S Bernstorff for their support during the experiments Professor Laggner is gratefully acknowledged for granting

us the beam time Mid Sweden University and The Swedish Research Council are acknowledged for financial support.

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Supplementary material

The following material is available from http://www blackwellpublishing.com/products/journals/suppmat/EJB/ EJB3361/EJB3361sm.htm

Table 1 Structures, indices and spacings obtained for some temperatures during heating