J. Phys. Chem. 1995,99, 17456-17460
17456
Thermotropic Phase Transition of Phosphatidylinositol 4,5-Bis(phosphate) Aggregates in Aqueous Solution Mitsuhiro Hirai,*v? Toshiharu TakizawaJ Sadato Yabuki: Toshihiro Hirai,' and Kouhei Hayashi' Department of Physics, Gunma University, Maebashi 371, Japan, and Faculty of Textile Science and Technology, Shinshu University, Ueda 386, Japan Received: May 30, 1995; In Final Form: August 11, I995@
Thermotropic phase transition and stability of phosphatidylinositol 4,5-bis(phosphate) (PIP2) aggregates solubilized in buffer solution were studied by using synchrotron radiation X-ray small-angle scattering, and the modeling analysis using a double-shell-ellipsoid fitting to the experimental scattering data was carried out. At pH 6.7 the PIP2 molecules aggregated to form ellipsoidal micelles with an identical size. The modeling analysis shows that the hydrophilic region composed of the PIP2 polar heads is highly hydrated. The elevation of temperature from 10 to 50 "C induced a moderate shrinkage of the micellar dimension and a systematic change of the intramicellar scattering density distribution, which is considered to occur mostly in the hydrophilic portion of the micelles. In contrast to the above case, at pH 5.4 the PIP2 solution showed a disordered lamellar phase at 10 "C and changed drastically to a micellar phase by elevating temperature. The present result suggests a high sensitivity of the interaction between water and PIP2 polar heads to the variation pH and temperature.
Introduction Phosphatidylinositol 4,5-bis(phosphate) (PIP2) is one of the acidic glycerophospholipids and most abundant in the cell membranes of brains and kidneys, which exhibits high solubility in water. At the beginning inositol lipids including PIP2 were studied in connection with nerve excitation's2 and recently have been attracting strong interests as the source of the second messenger in cellular signal transd~ction.~-~ While a number of studies to clarify significant physiological functions relating to inositol lipids have been done, the physicochemical properties and behaviors of PIP2 is little known, in contrast to other phospholipids. We have studied the structures and functions of micellar and lamellar systems by using X-ray and neutron scattering methods, calorimetry, and NMR spectroscopy. We have treated PIP2/ water other phospholipids,I0*'I glycolipid/protein/ water systems,I2 surfactant/protein/water, or organic solvent systems.13*14In our previous report using differential scanning calorimetry: we found that PIPdwater system showed a quite unusual endothermal transition indicated by a rather broad endothermal peak and a remarkable hysteresis in comparison with other phospholipid^.^^^ 1,15-20 The result obtained by using NMR measurements suggests that the above endothermal transition of PIP2 is not attributable to melting of hydrocarbon chains, namely, to a so-called gel-to-liquid crystal transition,I2 we presented a thermal dehydration of model PIPz/water system based on the internal hydration hypothesis of PIP2 bilayers. To confirm such an internal hydration of PIP2 bilayers, we carried out synchrotron radiation X-ray scattering experiments at two different pH values at temperatures ranging from 10 to 50 "C.
Z(q) = Z(0) exp(-q2R;/3)
Experimental Section Sample Preparation. As we described elsewhere?' PIP2 used for the present experiments was extracted from bovine
' Gunma University.
* Shinshu University. @
brain by the Folch method.22 To separate PIP2 from other residual components such as phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylinositol Cphosphate (PIP), the crude extract was purified further by DEAE-cellulose chromatography according to the method of Hendrickson and Bal10u.~~After this purification, PIP2 was obtained as an ammonium salt, which was removed by dialysis against buffer containing EDTA and was lyophilized. Freeze-dried powder of PIP2 is known to be very hygroscopic and the PIP2 powder was dissolved in 50 mM Hepes buffer of pH 6.7 or in 50 mM phosphate buffer of pH 5.4 and was stored at 10 "C for overnight. The PIP2 concentration was adjusted to 1.6 wt %. Small-Angle X-ray Scattering Measurements and Analyses. Small-angle X-ray scattering experiments were performed by using the synchrotron radiation small-angle X-ray scattering spectrometer which is installed at BLlOC line of the 2.5 GeV storage ring in the Photon Factory of the National Laboratory for High Energy Physics, Tsukuba, Japan. The incident X-ray beam intensity was monitored using an ionization chamber placed in front of the sample cell. The scattering intensity was detected by using a one-dimensional position sensitive proportional counter. The details of the instruments were explained elsewhere.24The wavelength used was 1.49 A, and the sampleto-detector distance was 85 cm. Samples contained in sample cells were placed into a cell holder. The temperature of samples was varied from 10 to 50 "C by a thermostat. The exposure time was 150 s for each measurement. The following analyses were done on the scattering data. The beginning of the scattering curve Z(q) is known to depend on the Guinier equation in the form
Abstract published in Advance ACS Abstracts, November 1, 1995
(1)
where Z(0) designates the zero-angle scattering intensity, R, the radius of gyration, and q the magnitude of scattering vector defined by q = (4n/I)sin(8/2) (e, the scattering angle; A, the w a ~ e l e n g t h ) .By ~ ~using the Guinier plot (ln(Z(q)) vs q2)of the data sets in the small q range of 0.025-0.03 A-I, we determined
0022-3654/95/2099-17456$09.00/0 0 1995 American Chemical Society
Thermotropic Phase Transition of Phosphate Aggregates
J. Phys. Chem., Vol. 99, No. 48, 1995 17457
the values of both Z(0) and R,. The distance distribution function p(r) was obtained by Fourier inversion of the scattering intensity I ( q ) as 1 o4
.-3 C
3
c;
The p(r) function depends on the particle shape, on the intraparticle scattering density distribution and on the interparticle translational correlation.26 To reduce the Fourier truncation effect on the calculation of the p(r) function, the extrapolation of the small-angle data sets by using the least-squares method for the Guinier plot and the modification of the scattering intensity as
e
io3
h
m v 1 o2
(k is the artificial damping factor) were done. The maximum diameter D,, of the particle was estimated from the p ( r ) function satisfying the condition p(r) = 0 for r > Dmx. As the use of the Guinier approximation inevitably leads to inherent systematic distortions and similar difficulties caused by concentration or aggregation effects, the following indirect Fourier transform method (Glatter's method is useful to eliminate such artifacts for the estimation of R, and Ztota1.26) These two terms are given as 1 o2
(4) LD'""p(r)r2 d r
Figure 1. Dependence of the scattering curve on temperature ranging from 10 to 50 "C. PIP2 concentration is 1.6 wt %: (a) in 50 mh4 Hepes buffer of pH 6.7; (b) in 50 mM phosphate buffer of pH 5.4.
2LD"""pr) d r Here we used the above two different methods. Equation 4 was used for normalization of the p(r) functions. According to the convolution theory, the spherically averaged scattering function Z(q) from of a particle composed of shells with different average scattering densities is simply given as I(4) cc (A(q)A*(q))= J1[3(a,vj1(4R,)/(4R1)+ n
where ( ) means the spherical average of the scattering intensity Z(q) defined by A(q) A*(q) (the structural factor A(q)), pi is the average excess scattering density (so-called contrast) of ith shell with a shape of an ellipsoid of rotation, jl is the spherical Bessel function of the first rank.27928Ri is defined as
+
x2(y;
- 1))1'2
0.3
q /A-'
R,2 =
Ri = ri(l
0.2
0.1
0
(7)
where ri and vi are the major semiaxis and the axial ratio for the ith ellipsoidal shell, respectively. Here we assumed a simplified model, namely, a double-shell ellipsoidal structure to fit the experimental scattering data by using eq 6. The fitting parameters used are pi, ri, and vi. Although this modeling method cannot but avoid disagreement with experimental data at high angle region (4 > -n/ri) due to the simplified model representation as a set of ellipsoids, eq 6 is very useful to sort out various models and to save calculation time for fitting.
Results and Discussion Scattering Curve Analysis. Figure 1 shows the change of the scattering curves of the PIPZ/water systems at different pH
-
by elevating temperature. At pH 6.7 in Figure l a the scattering curve has an evident minimum at q 0.06 and a bellshaped hump at q 0.11 A-I, indicating a typical scattering curve from identical particles, namely from monodisperse micelles. The elevation of temperature induces the gradual shift of the position of the hump maximum from -0.11 to -0.10 A-1, suggesting that the PIP^ molecules aggregate form prolate micelles at pH 6.7 and that such a micellar shape slightly changes in the temperature range 10-50 "C, which is also shown by the distance distribution and modeling analyses in the following paragraphs. This change is mostly reversible with temperature. On the contrary, at pH 5.4 in Figure l b the scattering curve changes drastically with elevating temperature. At 10 "C the scattering curve has an evident peak localized at 0.88 8,-' and with elevating temperature the peak shape changes to a bell shape which is the same as at pH 6.7. It indicates that the structural phase transition at pH 5.4 induced by the elevation of temperature is from the lamellar phase to the micellar one. The broad peak suggests that the PIP2 aggregates form disordered lamellae. The spacing d between the bilayers in the lamellae, where d is defined by 2n/q, is evaluated to be d = 71 8, by the position of the first peak of q = 0.088 A-'. Although the second-order lamellar peak can be recognized as an evident shoulder of q 0.18 A-', other higher-order lamellar peaks are not seen. Therefore the bilayer stacking the PIP2 is considered to be rather disordered compared with @ose of other phospholipids. We can also see another low shoulder around q 0.14 at both 10 and 20 "C, and this shoulder significantly changes to a bell-shaped hump between 30 and 35 "C. The intermediate state of the transition is thought to be a mixture of the lamellar and micellar phases. Thus by elevation of the temperature, the micellar phase becomes gradually
-
-
-
Hirai et al.
17458 J. Phys. Chem., Vol. 99,No. 48, 1995
TABLE 1: Structural Parameters for 1.6 wt % PIP2 in Hepes Buffer at pH 6.7 and in Phosphate Buffer at pH 5.4 at Various Temperature temp ("C) at pH 6.7
10 20 30 35 40 45
50 10"
64.7 f 0.7 64.6 f 0.4 63.8 f 0.3 63.2 f 0.3 62.7 f 0.7 61.8 f 0.6 61.7 f 0.5 64.8 f 0.6
67.37 f 0.15 67.68 f 0.16 66.47 f 0.17 66.26 f 0.17 65.46 f 0.18 64.98 f 0.19 65.02 f 0.19 67.84 f 0.16
81.0 (f0.5) 227.2 (f0.5) 235.4 80.0 217.0 78.0 225.5 78.0 221.7 77.0 239.4 77.0 221.8 76.0 80.0 216.5
temp ("C) at pH 5.4 10 20 30 35 40 45 50 10"
69.2 f 1.0 69.7 f 1.4 74.9 f 1.3 68.5 f 0.7 68.4 f 0.6 68.2 k 0.6 67.8 f 0.7 70.6 f 0.9
-
70.10 f 0.17 69.77 f 0.17 70.43 f 0.18 70.15 f 0.19 72.30 f 0.16
90.0 (f0.5) 90.0 88.0 83.0 191.0 (f0.5) 80.0 199.2 78.0 220.8 78.0 220.4 83.0 224.2
In the first column, temperature for restoring the sample for 2 h after elevating temperature; R,8 and R:, the gyration radii estimated by the Guinier approximation and the Glatter method, respectively; in the third and fifth columns, indeterminable values by using the distance distribution analysis. 0
100
200
r /A Figure 3. Variation of the distance distribution function, p ( r ) , depending on temperature; (a) and (b) as in Figure 1. p ( r ) in (b) is displayed without normalization by the total scattering power to show the ripple profiles for low-temperature data in the same figure.
20
30
40
50
temp. /'C
Figure 2. Change of the radius of gyration depending on temperature. R, values shown are obtained by the Guinier method (see in Table 1). pH 6.7 (open circle) and pH 5.4 (open squares).
dominant and the lamellar phase disappears above 45 "C. This change is irreversible, which is in good agreement with our previous calorimetric study indicating the strong thermal hysteresis in the phase transition of the PIP2 dispersion in distilled water. In addition, although the change of the scattering curve above 35 "C shows the same tendency as that observed at pH 6.7, the change is greater than that observed at pH 6.7. Radius of Gyration and Its Change. The gyration radius R, can be estimated by the Guinier and Glatter methods using eq 1 and 5 . The R, estimation even for polydisperse systems still gives a good index of a change in aggregate structures and their state of dispersion. The R, values obtained are listed in Table 1. As shown in Figure 2, in the case of the PIP2 aqueous suspension at pH 6.7 the R, value estimated by the Guinier method decreases from 64.7 f 0.7 8, to 61.7 f 0.5 8, with elevating temperature. The R, value by the Glatter method also show the same decreasing tendency from 67.37 f 0.15 8, to
65.02 f 0.19 8, in Table 1. This decrease suggests the slight shrinkage of the micellar dimension. The above change is reversible on the temperature, which is attributable to the change of the shape and internal scattering density distribution of the PIP2 micelle as shown in the following modeling analysis. In Figure 2, in the case of pH 5.4 the Rg shows the maximum value of 74.9 & 1.3 8, at 30 "C. The thermotropic phase transition of the PIP2 aqueous solution occurs gradually, and it agrees well with the previous results in the calorimetric study which shows a rather broad endothermal peak around 30-40 "C? Thus the intermediate state of the structural phase transition at pH 5.4 can be assumed to be in the biphase, as also suggested by the change of the scattering curve. The presence of R, maximum at 30 "C is attributable to the formation of large aggregated structures in the intermediate state where the lamellae change gradually to elongated micelles. Distance Distributionand Modeling Analyses. Figure 3a,b show the distance distribution functions obtained by the Fourier inversion of the scattering curves in Figure la,b. In Figure 3a the p ( r ) profile at pH 6.7 is characterized by the two peaks at -25 and -81 8, whose positions shift to small r values by elevating temperature. Figure 4 shows the shift of the highestpeak position, pmax, of the p ( r ) function as a function of temperature, suggesting a change of the internal scattering density distribution of the micelle. In comparison with Figure 3a, in Figure 3b at pH 5.4 the profile with strong ripples changes drastically to that for a micellar structure similar to that in Figure 3a, evidently showing the structural change from lamellae to micelles. The PIP2 micellar structure can be described as a doubleshell ellipsoid with a core (hydrocarbon tail) surrounded by a
J. Phys. Chem., Vol. 99, No. 48, 1995 17459
Thermotropic Phase Transition of Phosphate Aggregates
90
lo4
v)
.-U C
?
3
X
E
0
80
I
Ip
1o2 0 70
20
30
50
40
temp.
/T
Figure 4. Plots of the highest-peak position, P,,,, as a function of temperature.
of the p ( r ) function m
.-U C
1
=I shell (phosphates' head) since the glycerol backbone of the PIP2 U molecule links to the hydrophilic head with three phosphate groups and the hydrophobic tail with two long fatty acids chains (mainly stearic acid and arachidinic acid). Such a shellv L a modeling analysis is well suited and applicable to particles having an evident internal scattering density heterogeneity with eq 6, the experimental a center ~ y m m e t r y . ~By ~ . ~using ~ 0 scattering curves and the p ( r ) functions of the PIP2 micelle at 0 100 200 pH 6.7 are simulated well as shown in Figures 5. Here the r /A outer shell radius, the inner core radius, their axial ratios and the excess scattering densities as compared to the average Figure 5. Scattering and distance distribution functions of optimized models in comparison with the experimental ones in Figure l a and scattering density of the solvent (so-called the contrasts) were Figure 3a: (a) scattering curves; (b) distance distribution functions. used as fitting parameters. The PIP2 micelle at 10 "C is an Experimental data at 10 (0)and at 50 "C (0);dotted and full lines, elongated double-shell ellipsoid of rotation with semiaxes of double-shell ellipsoid models for pH 6.7 and 5.4. 22.4 k 4 0 . 6 A for inner corelouter shell whose axial ratios are 6.2214.05. The ratio of the contrasts between those of the shell Conclusion and the core is 0.3851-0.453. The PIP2 micelle at 50 OC has In conclusion, we found that the thermotropic structural phase semi-axes of 22.3 k38.7 8, for inner corelouter shell with axial transition occurs in a PIP2 aqueous solution strongly depends ratios of 5,9013.86and the ratio of the contrasts is 0.3921-0.452. on the pH of the solvent. The polar head region of the PIP2 The R, and D,,, values of the optimized models and the molecule was confirmed to be highly hydrated by using a locations of the two peaks are also in agreement with those double-shell model. At pH 6.7 the reversible structural change experimental ones. The slight discrepancies between model and of the PIP2 ellipsoidal micelles is induced by elevating the experiment in Figure 5 is attributable to slight polydispersity temperature. The accompanying change of the internal scatin dimension of the experimental PIP2 micellar suspension. tering density distribution of the micelles can be explained to According to the well-known empirical formula for an hydroresult from the shrinkage of the hydrophilic region of the PIP2 carbon chain volume v and its extended length 1, and to the micelles, which might exclude some amount of water from this result of the partial specific volume v, of PIP2 m o l e ~ u l e , ~ ~ ~ ~ ~ region. At pH 5.4 the thermotropic structural change of the the v, I, and us values of the PIP2 molecule are 1064 A3, 24.3 PIP2 suspension is irreversible and quite different from that A, and 0.742 mL/g, respectively. Then the average excess observed at pH 6.7. In the temperature range 10-50 "C, the scattering densities of the head and tail parts of the PIP2 suspension mostly takes a disordered lamellar phase below 20 molecule are calculated to be 9.79 x and - 1.42 x 1O-Io "C, an elevation of the temperature induces a structural phase cm-2 by using the average scattering density of the solvent of transition from lamellae to micelles between 30 and 35 "C, and 9.40 x 1O-Io cm-2. The ratio of 0.3851-0.453 obtained by above 35 "C the structural change of the micelles shows the the model fitting is significantly different from that of 9.791same tendency as that observed at pH 6.7, although this change 1.42, indicating that a large amount of water molecules should is greater than that observed at pH 6.7. The previous result on hydrate the PIP2 polar head in order to reduce the average the characteristic thermogram of the PIPzIdistilled water system scattering density of the head. The change of the semi-axes PIPzIdistilled water system, showing a quite broad endothermal from 22.4 k 4 0 . 6 A to 22.3 k 3 8 . 7 A shows the shrinkage of peak and a remarkable hystere~is,~ is in good agreement with the phase behavior observed in the present experiments at pH the hydrophilic region and the slight increase of the ratio from 5.4. 0.385/-0.453 to 0.3921-0.452 suggests a more compact arrangement of the PIP2 polar heads which might result in the In general, the amounts of the two forms, charged and uncharged, of the PIP2 polar head can be considered to depend extrusion of some amount of hydrated water from of the on the pH of the solution. Namely, at pH 5.4 the head is less hydrophilic region.
'
h
17460 J. Phys. Chem., Vol. 99,No. 48, 1995 negatively charged compared with the case at pH 6.7. Such weakening of the repulsive electrostatic interaction between the polar heads would reduce the effective area per PIP2 polar head, s, resulting in the increase of the critical packing parameter (defined by v/slc)of the PIP2 molecule to cause the structural phase transition from micelles to lamellae.3' The present result implies the interaction between water and PIP2 polar head and between PIP2 polar heads show a high sensitivity to the variation of pH and temperature. Such a unique characteristic would suggest that the PIP2 molecules have some possibility to play a role as a kind of functional lipids by themselves in the cell membranes.
Acknowledgment. We thank Drs. Y. Amemiya and K. Kobayashi of the Photon Factory at the National Laboratory for High Energy Physics for their help with the small-angle scattering instrumentation. This work was performed under the approval of the Photon Factory Programme Advisory Committee (Proposal No. 92-069). References and Notes (1) Hokin, M. R; Hokin, L. E. J. Biol. Chem. 1953, 203, 967-977. (2) Bimberger, A. C.; Eliasson, S. G. Neurology 1970, 20, 356-360. (3) Michell, R. H. Biochim. Biophys. Acra 1975, 415, 81-147. (4) Sterb, H.; Irvine, R. F.; Bemdge, M. J.; Schulz, I. Nature 1983, 306, 67-69. ( 5 ) Berridge, M. J. Annu. Rev. Biochem. 1987, 56, 159-193. (6) Hill, T. D.; Dean, N. M.; Boynton, A. L. Science 1988,242, 11761178. 17) Takizawa. T.: Havashi. K.: Mitomo. H. Thermochim. Acta 1988. 123, 247-253. ( 8 ) Takizawa, T.; Mitomo, H.; Havashi, K. Thermochim. Acta 1990. 163, 133-138. (9) Takizawa, T.; Hayashi, K.; Mitomo, H. Thermochim. Acta 1991, 183, 313-321. (10) Takahashi, A.; Takizawa, T.; Nakata, Y . Chem. Phys. Lett. 1989, 163, 65-68.
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