The structure of complexes between ...

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The structure of complexes between phosphatidylethanolamine and glucosylceramide: a matrix for membrane rafts Peter J. Quinn

Department of Biochemistry, King’s College London, 150 Stamford Street, London SE1 9NH, United Kingdom. 1.

Analysis of binary mixtures of GlcCer with DLPE and DMPE. The results of a heating scan between 10o and 90oC of the binary mixture with DLPE are presented in Fig. 1S. An overview of the scattering intensity profiles in the SAXS and WAXS regions is shown in Fig. 1S,A.

Fig. 1S. Structural transitions in an aqueous dispersion of a binary mixture of DLPE and GlcCer in molar proportions 100:23.4 recorded during a heating scan from 10o to 90oC at 2o/min. A. X-ray scattering intensity profiles in the SAXS (left) and WAXS (right) regions. B. Lamellar d-spacings; C. Amplitude of first-order lamellar Bragg peaks/FWHM; D. Scattering intensities of lamellar peaks; E. Amplitude of WAXS peaks/FWHM; F. dspacings of WAXS peaks. Peak assignments of lamellar structures; ●, ○, DLPE; ◊, GlcCer-rich bilayers. □, total scattering intensity of first-order Bragg reflections.

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Three orders of coexisting lamellar repeat structures can be seen in the SAXS region and an analysis of the first-order Bragg reflections was performed. The sharp peak (Fig. 1S,C) corresponding to a d-spacing of about 4.65 nm (Fig. 1S,B) appearing throughout the temperature scan and which dominates the scattering intensity profile (Fig. 1S,D) is assigned as bilayers of pure DLPE. At temperatures up to 42oC this is associated with four Bragg peaks in the WAXS region, two sharp (Fig. 1S,E) with d-spacings 0.394 and 0.376 nm and the others broader and at d-spacings of 0.442 and 0.518 nm, respectively (Fig. 1S,F). These bilayer characteristics are almost identical to aqueous dispersions of pure DLPE in L 2 structure which has a bilayer repeat of 4.55 nm, WAXS peaks of similar d-spacings and relative intensities and which undergo a transition to a lamellar liquid-crystal phase at 43oC. The lamellar liquid-crystal phase is characterized by a repeat d-spacing of 4.55 nm and a single broad diffraction band centered at 0.43 nm, again with structural parameters in agreement with the pure phospholipid. Another minor lamellar peak is observed at a d-spacing of 3.981 nm which disappears upon heating above about 34oC: this is analogous to DLPE in Lβ1 structure which transforms via Lβ2 structure to Lα at this temperature. The relatively broad lamellar reflection at a d-spacing of about 6.1 nm is assigned as a GlcCer-rich bilayer structure in gel phase which transforms into a liquid-crystal phase at a temperature (74oC) close to that of pure GlcCer. The lamellar d-spacing, it should be noted, is 0.9 nm greater in the binary mixture than observed in the pure lipid and this may reflect an influence of the proximity of the pure phospholipid to the GlcCer structures. The effect of increased phospholipid chain length is seen with binary mixtures of DMPE and GlcCer in Fig. 2S. An overview of the SAXS/WAXS intensity profiles (Fig. 2S,A) indicates that there are transitions in the coexisting lamellar structures at about 50o and 70oC. A sharp decrease in one of the lamellar d-spacings from 5.76 to 5.19 nm between 48o and 50oC with a mid-point of 49.2oC (Fig. 2S,B) coincides with the lamellar gel to lamellar liquid-crystal phase transition of pure DMPE. Another minor lamellar phase (Fig. 2S,D) with a d-spacing of about 6.25nm is observed during the heating scan up to about 50oC and this is also assigned to DMPE. A transition lamellar structure is present in the temperature range 38o to 46oC indicating a transition from sub-gel to gel phase takes place prior to the main transition from gel to liquidcrystal phase. A single sharp peak in the WAXS region at a d-spacing of 0.405 nm (Fig. 2S,E & F) indicates that the phospholipid is mostly in the gel rather than the sub-gel phase. DMPE in the subgel phase transforms to gel phase before transition to fluid phase at a temperature slightly below the reported transition temperature of 56oC. A lamellar structure characterized by a broad peak located at a d-spacing of about 6 nm at 20oC progressively decreases to a d-spacing of 5.5 nm at 70oC whereupon the peak sharpens (Fig. 2S,C) and shifts to a d-spacing of about 4.9 nm. This structure is assigned as bilayers enriched in GlcCer the amount of which appears to decrease with increasing temperature (Fig. 2S,D). Heating above 70oC clearly indicates phase separation of DMPE and GlcCer to produce coexisting bilayers of pure DMPE and GlcCer at temperatures above the Tm of the GlcCer.

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Fig. 2S. Structural transitions in an aqueous dispersion of a binary mixture of DMPE and GlcCer in molar proportions 100:26.4 recorded during a heating scan from 20o to 90oC at 2o/min. A. X-ray scattering intensity profiles in the SAXS (left) and WAXS (right) regions. B. Lamellar d-spacings; C. Amplitude of first-order lamellar Bragg peaks/FWHM; D. Scattering intensities of lamellar peaks; E. d-spacings of WAXS peaks; F. Amplitude of WAXS peaks/FWHM. Peak assignments of lamellar structures; ●,■ , DMPE; ‹, GlcCer-rich bilayers; ▲, intermediate lamellar structure. „, total scattering intensity of first-order Bragg reflections.

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Determination of scattering intensities from the binary mixture GlcCer/DMPE. As seen in Fig. 2S two sharp peaks are assigned as pure DMPE at temperatures below Tm, one a lamellar gel and the other a lamellar sub-gel phase. The latter is transformed to a gel phase at temperatures approaching Tm via recognizable SAXS spacings indicative of intermediate structures. These transitions and multiple d-spacings complicate the determination of scattering intensities arising from the different structures present in the binary mixture. In

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Fig. 3S. Structural transitions in an aqueous dispersion of a binary mixture of DMPE and GlcCer in molar proportion 100:26.4 recorded after heating to 90oC, and reheating after cooling from 20o to 90oC at 2o/min. A. X-ray scattering intensity profiles of the first-order Bragg peaks. B. Lamellar d-spacings; C. Amplitude of first-order lamellar Bragg peaks/FWHM; D. Scattering intensities of lamellar peaks. Peak assignments of lamellar structures; ■, DMPE; ◊, GlcCer-rich bilayers; □, total scattering intensity of first-order Bragg reflections.

order to avoid the formation of sub-gel structures the binary mixture was first heated to 90oC, then cooled at 2o/min to 20oC and immediately reheated to 90oC. An analysis of the first-order Bragg peaks was undertaken and the results are presented in Fig. 3S. This shows that the Bragg reflections (Fig. 3S,A) can be deconvolved into two peaks of d-spacings (Fig. 3S,B) the higher orders of which (data not shown) characterize two lamellar structures. The sharper of the two peaks (Fig. 3S,C) is assigned as pure DMPE in gel phase at temperatures less than 49oC above which it is transformed into a lamellar liquid-crystal structure. In contrast to the sample equilibrated at 20oC (Fig. 2S) the abrupt change in d-spacing of the structure assigned as GlcCer coincides with the gel to liquid-crystal transition in the phospholipid rather than about 70oC consistent with the pure GlcCer. This may be explained by the creation of larger domains of GlcCer during the equilibration at 20oC which are not influenced by the phase transition of the phospholipid as are smaller domains created by prior heating to 90oC and subsequent reheating from 20oC. The relative scattering intensities of the two peaks (Fig. 3S,D) provide the data shown in Table 1 of the text. 3.

Positional specificity of unsaturated fatty acid on PE. To investigate the effect of the position of the unsaturated fatty acyl residue at the sn-1of the glycerol moiety of PE an aqueous dispersion of a binary mixture comprised of 29.3 mol GlcCer and 100 mol OPPE was examined. The overview of the SAXS/WAXS intensity profiles 4

recorded during a heating scan from 20o to 90oC is shown in Fig. 4S,A. The SAXS peaks can be deconvolved into two co-existing bilayer structures up to about 70oC above which a non-lamellar structure appears and progressively dominates the phase structure. The sharper of the two peaks (Fig. 4S,C) undergoes a structural transition in which the lamellar d-spacing decreases from 6.4 nm to 5.6 nm at a temperature of 36oC (Fig. 4S,B). The transition in the SAXS d-spacing coincides with a disappearance of a sharp peak (Fig. 4S,E) centered at 0.41 nm in the WAXS region (Fig. 4S,F) indicating that the transition is a gel to liquid-crystal phase transition. There are no published data on the phase structure or transition behavior of OPPE but based on a comparison of sharp decrease in lamellar d-spacings and gel to liquid-crystal phase transition temperatures of closely related molecular species of PE it is a reasonable assumption that the sharp Bragg peaks originate from bilayers of pure OPPE. The broad lamellar peak that contributes approximately equally to the total scattering intensity (Fig. 4S,D) and is present up to 70oC in the heating scan can be assigned as bilayers containing both GlcCer and OPPE.

Fig.4S. Structural transitions in an aqueous dispersion of a binary mixture of OPPE and GlcCer in molar proportion 100:29.3 recorded during a heating scan from 20o to 90oC at 2o/min. A. X-ray scattering intensity profiles in the SAXS (left) and WAXS (right) regions. B. Lamellar d-spacings; C. Amplitude of first-order lamellar Bragg peaks/FWHM; D. Scattering intensities of lamellar peaks; E. Amplitude of WAXS peaks/FWHM; F. d-spacings of WAXS peaks. Peak assignments of lamellar structures; ●, OPPE; ◊, GlcCer-rich bilayers; Δ, non-lamellar structure. □, total scattering intensity of first-order Bragg reflections.

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Assignment of deconvolved Bragg peaks. To check the assignments of sharp SAXS lamellar peaks in binary mixtures comprised of about 30 mol GlcCer/ 100 mol PE an examination of dispersions containing proportions of GlcCer ranging from 2.5 to 40 mol/ 100 mol POPE were examined and the relative scattering intensities of the different peaks was related to the composition of the mixture. The scattering 5

intensities of the first-order lamellar repeat spacings recorded at 37oC is shown in Fig. 5S,A and the profiles in mixtures with up to 30 mol GlcCer/ 100 mol POPE consist of two discrete peaks, one sharp and the other broad. Higher-order reflections (not shown) confirm that both are lamellar structures. The significance of this is firstly, only two repeat structures are detected in the binary mixture and therefore the composition of each structure is different. Secondly, the composition of each monolayer is complimentary to its opposing counterpart, ie. the bilayers are tightly coupled otherwise only a single broad Bragg peak would be observed. This is consistent with the temperature-dependence of the sharp peak which indicates that an order-disorder structural transition takes place in this phase at a temperature coincident with that seen in the pure phospholipid. Judging from the peak widths, which is an indication of molecular order of the unit cells, the structure of greater d-spacing at 37oC is more disordered than the structure giving rise to the smaller d-spacing. The relatively broad reflection is centered at a d-spacing of 6.10 nm and the sharp peak at 5.41 nm. The latter peak could be identified with a dispersion of pure POPE which has a d-spacing of 5.31 nm at this temperature.

Fig. 5S. Assignment of Bragg peaks in binary mixed aqueous dispersions of GlcCer and POPE in fluid structure (at 37oC). A. Scattering intensity profiles of first-order lamellar repeat spacings comprised of the indicated proportions of the two lipids. B. relationship between the relative scattering intensity of the sharp Bragg reflection and the mass fraction of GlcCer in the binary mixtures.

An additional peak at a d-spacing of 5.02 nm seen in the mixture with the greatest proportion of GlcCer coincides with the lamellar repeat spacing of pure GlcCer recorded under the same conditions. This indicates that in mixtures with proportions of GlcCer greater than 30 mol/ 100 mol POPE not all the constituents of the mixture are assembled into the two structures and that a period of equilibrium may be required for this to be achieved. To confirm the assignment of the sharp peak to bilayers of pure POPE a plot of the relative scattering intensity of the sharp peak against the mass fraction of GlcCer in the binary 6

mixtures recorded at 37oC was prepared (Fig. 5S,B). The plot shows an inverse linear relationship of the form y = 1.05 – 2.83x. Discounting the value obtained from the mixture containing 40 mol GlcCer/ 100 mol POPE where there is evidence that the sample is not at equilibrium, the intercept with the x-axis at zero scattering intensity from the sharp peak is at a mass fraction of GlcCer of 0.354. The mass fraction of POPE contributing to the broad peak is therefore 0.646 giving a stoichiometry of 2.06 mol POPE/ mol GlcCer. A similar analysis was performed under conditions where the phospholipid was in a gel structure and the results are shown in Fig. 6S.

Fig. 6S. Assignment of Bragg peaks in binary mixed aqueous dispersions of GlcCer and POPE in gel structure (at 20oC). A. Scattering intensity profiles of first-order lamellar repeat spacings comprised of the indicated proportions of the two lipids. B. relationship between the relative scattering intensity of the sharp Bragg reflection and the mass fraction of GlcCer in the binary mixtures.

Scattering intensities of the first-order Bragg reflections were recorded from mixtures showing the indicated proportions of GlcCer in POPE (Fig. 6SA) and a plot of the relative scattering intensity of the sharp peak assigned as POPE against the mass fraction of GlcCer is shown in Fig. 6SB. The plot shows an inverse linear relationship of the form y = 0.96 – 2.07x. The intercept with the x-axis at zero scattering intensity from the sharp peak is at a mass fraction of GlcCer of 0.44. The mass fraction of POPE contributing to the broad peak is therefore 0.56 giving a stoichiometry of 1.5 mol POPE/ mol GlcCer. 5.

Calculation of molecular stoichiometries.

The calculation of mass of PE in binary mixtures of PE and GlcCer from the scattering intensities of the first-order Bragg peaks assigned as pure PE has an error that depends on both the d-spacings and the difference in d-spacings between pure PE and the complex. The 7

stoichiometric values for PE and C-24 GlcCer in Table 1 were calculated on the assumption that this error is insignificant. To check this assumption a method for determining the stoichiometry of the complex which does not rely on absolute scattering intensity has been undertaken for complexes formed between POPE and C-12 GlcCer (Fig. 6) and POPE and C-24 GlcCer (Fig. 5S). A summary of the data comparing the two methods for dispersions at 31oC is presented in Table 1S. Table 1S. Comparison between two methods for calculating stoichiometry of complexes between PE and GlcCer. Method

d-space (nm)

Relative mass (~30mole%GlcCer) Relative scattering

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POPE/C12 GlcCer

d-space (nm)

POPE/C24 GlcCer

POPE 5.35

Complex 5.15

1.6

POPE 5.31

Complex 6.01

2.0

5.32

5.17

1.82

5.41

6.10

2.06

Structural order of diffracting cells.

The structural order within the diffracting unit cells judged from the peak shape parameter, scattering intensity amplitude/full width at half maximum scattering intensity, for structures formed in codispersions of GlcCer with different molecular species of PE is summarized in Table 1S. The peaks assigned to GlcCer-rich structures in codispersions with PE are broadened compared to dispersions of pure GlcCer irrespective of wether the molecular species of PE is saturated or unsaturated. The order of the GlcCer-rich structure is largely independent of whether the PE is in a gel or fluid phase.

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Table 2S. Lamellar d-spacings and peak shape parameters of GlcCer first-order bilayer Bragg reflections at temperatures below and above the gel to liquid-crystal phase transition of PE. Lipid

Tm PE o

5o d-space (nm) Amp/FWHM d-space (nm) Amp/FWHM GlcCer* 5.54 2.57*107 5.22 9.94*107 DMPE (Equlibrated 20oC) 5.92 4.48*105 5.47 4.58*105 5.20 1.02*107 DMPE (reheated) 5.95 6.73*106 POPE-GlcCer complex 6.13 4.61*105 5.61 5.38*105 POPE 6.38 1.41*107 5.56 1.41*107 *68o and 78oC

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