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Order and dynamics in the lamellar La and in the hexagonal H,, phase Dioleoylphosphatidylethanolamine studied with angle-resolved fluorescence depolarization

Herman van Langen, Charles A. Schrama, Gijs van Ginkel, Gerard Ranke, and Yehudi K. Levine Department of Molecular Biophysics, Buys Ballot Laboratory, University of Utrecht, 3508 TA Utrecht, The Netherlands

ABSTRACT Fluorescence depolariza- H,, phase at 450C from samples pre- TMA-DPH molecules in the hexagonal tion techniques are used to determine pared in the lamellar La phase at 70C. H1, phase are comparable with those in the molecular order and reorientational The interpretation of angle-resolved the lamellar La phase. Furthermore, the dynamics of the probe molecule TMA- fluorescence depolarization experi- lateral diffusion of the probe molecule DPH embedded in the lamellar La and ments on these phases, within the on the surface of the /water cylin- the hexagonal H,, phases of lipid/water framework of the rotational diffusion der in the hexagonal phase is found to mixtures. The thermotropically induced model, yields the order parameters be considerably slower than the re- La - H,, phase transition of the lipid (P2) and (P4), and the diffusion con- orientational motion. DOPE is used to obtain macroscopi- stants for the reorientational motions. cally aligned samples in the hexagonal The reorientational motion rates of the

INTRODUCTION larger than the cross-section of the hydrocarbon chains, the molecule has the overall shape of an inverted cone. The biological membrane is a flexible and dynamic Such cones are most easily packed in a micellar structure structure and undergoes many conformational changes to in water. The third possibility, where the cross-section of accommodate its various functions. The membrane the lipid headgroup is smaller than that of the hydrocar- are thought to play a vital role in these processes. Lipid bon chains, gives rise to a conical shape. These lipids pack polymorphism, the ability of lipids to organize in different preferentially in the hexagonal HI, phase on dispersion in macroscopic structures, may well be involved in these water. structural changes. This concept of membrane structure The environmental conditions are also expected to is based on a large number of model studies in which the influence the overall shape of the lipid molecules so that lipid phase behavior was investigated. It has been shown structural transitions between the different macroscopic that at physiological temperatures, variations in the phases can be induced by changes in hydration and experimental conditions such as ion concentration (e.g. temperature. Cylindrically shaped hydrated lipids which Ca2"), pH, degree of hydration of the lipid headgroups, are organized in planar bilayers can form a hexagonal and insertion of proteins into the lipid matrix can mark- structure at elevated temperatures. The temperature edly affect the membrane structure and lipid phase increase appears to change the balance between the transitions (Reiss-Husson, 1967; Luzzati et al., 1968; cross-sections of the headgroup and hydrocarbon regions. Ranck et al., 1977; Cullis and de Kruijff, 1978; Cullis et A larger cross-section for the hydrocarbon region result- al., 1980; van Venetie and Verkleij, 1981; Rilfors et al., ing from temperature-induced gauche defects in the acyl 1984; de Kruijff et al., 1985a and b; Gruner, 1985; Crowe chains of the lipid molecule is now expected. This leads to et al., 1988). more cone-shaped lipid molecules, which will organize in These effects can be rationalized in terms of the elegant the hexagonal phase. molecular shape concept proposed by Israelachvili and The bilayer-to-nonbilayer phase transitions have been Mitchell (1975), for reviews see refs. (Rilfors et al., 1984; widely investigated; in particular the thermotropically de Kruijff et al., 1985a; Israelachvili et al., 1980). In this induced phase transition from the (La) to concept it is assumed that the overall form of the lipid the inverted hexagonal phase (HI,) in phosphatidyletha- molecules, which is influenced by a number of structural nolamines has been the subject of many studies (Cullis and thermodynamic factors, determines the structure of and de Kruijff, 1978; Cullis et al., 1980; van Venetie and the hydrated lipid matrix. For example lipids with the Verkleij, 1981; Caffrey, 1985; Tate and Gruner, 1987; overall shape of a cylinder pack most efficiently in a Bentz et al., 1987). The La phase is a , 40 A in planar bilayer structure on dispersion in water. On the thickness and separated by a water layer of 18 A (Ranck other hand, if the cross-section of the lipid headgroup is et al., 1977; Cullis and de Kruijff, 1978; de Kruijff et al.,

Biophys. J. e Biophysical Society 0006-3495/89/05/937/10 $2.00 937 Volume 55 May 1989 937-947 1985a; Blaurock, 1982). The hexagonal HI, phase con- HI, phase (Cullis et al., 1980). In the La phase the bilayer sists of lipid/water tubes 104-105 A in length with diame- planes are parallel to the glass plates, while in the HI, ter of -70 A and a central water core of 30 A in diameter phase the lipid/water tubes possess this orientation (Mely (Reiss-Husson, 1967; Gruner, 1985; Caffrey, 1985). On a et al., 1975). Nevertheless, the long axes of the tubes are microscopic scale the La, -- HI, phase transition requires uniformly distributed (uniaxial symmetry) around the a considerable rearrangement of lipid molecules (Kirk et normal to the glass plates. The fluorescent probe 1- al., 1984). Calculations on the formation of a precursor in [4-trimethyl ammonium phenyl]-6-phenyl- 1,3,5-hexa- the transition, the inverted micellar intermediate (IMI), triene (TMA-DPH) is used to monitor the behavior of the have recently been presented (Siegel, 1986a and b). This orientational order and the reorientational dynamics of precursor may be of biological interest for the description the lipid molecules in the system. of processes related to membrane fusion and exocythosis. A number of experimental techniques have been used to obtain structural information about the La and H11 phases. However none of these techniques provide a THEORETICAL DESCRIPTION detailed picture of molecular order and reorientational dynamics of the lipid molecules in the hexagonal struc- Sample Geometry ture. X-Ray (Reiss-Husson, 1967; Luzzati et al., 1968; In the hexagonal HI, phase the lipid molecules are Gruner, 1985; Caffrey, 1985; Blaurock, 1982) and neu- ordered with their polar headgroups anchored at the long tron diffraction techniques (Biildt et al., 1978) have been central water core of the cylinder, Fig. 1. On the other used to characterize the lattice constants, while differen- hand, the nonpolar lipid tails are directed away from this tial scanning calorimetry (DSC) experiments have central water core. These lipid/water hexagons form a yielded phase transition temperatures and thermody- hexagonal lattice. On a microscopic scale these cylinders namic properties of the transition (Ellens et al., 1986; are organized in bundles while on a macroscopic scale the Brown et al., 1986; Gasset et al., 1988). On the other bundles are randomly oriented with their long axes paral- hand, nuclear magnetic resonance (NMR) (Cullis and de lel to the glass plates of the thin sample (Mely et al., 1975; Kruijff, 1978; Cullis et al., 1980; de Kruijff et al., 1985a; Boggs and Hsia, 1973). The sample possesses a uniaxial Sjolund et al., 1987; Marsh and Seddon, 1982) and symmetry about the normal to its surface. electron microscopy (van Venetie and Verkleij, 1981) The excitation light pulse, with polarization direction have only been used to identify the phase of the samples. ei, is incident on the sample at an angle 0 relative to the Electron spin resonance (ESR) spectra were also found to normal to the sample surface as depicted in Fig. 2. The reflect the phase structure, but their detailed interpreta- director of the sample, the normal to its plane, defines the tion in terms of molecular behavior is not straightforward laboratory z-axis. The x- and y-axis are chosen so as to (Hardman, 1982; Lasic and Hauser, 1984). Finally we form a right-handed coordinate frame. The fluorescence note that the phase transitions can also be monitored light, polarized along the direction ef, is observed along an through the changes in the fluorescence intensity (Hong axis making an angle so with the director, Fig. 2. et al., 1988). We have therefore undertaken a fluorescence depolar- ization study of the molecular order and reorientational dynamics of systems in the macro- hexagonal phase using r-7- scopically ordered samples. The method has been previ- ously applied by us to planar multibilayer systems (van Langen et al., 1987a; Deinum et al., 1988; Mulders et al., 1986) and detailed information about the molecular properties in these systems was obtained from the inter- pretation of angle-resolved fluorescence depolarization D ,. (AFD) experiments. Here we describe a modification of our previous work which is applicable to the study of hexagonal phases. To justify the theoretical description of the experiment FIGURE 1 The sample structure of the hexagonal HI, phase. (A) a we have used the thermotropic L HI, phase transition cone-shaped lipid molecule. (B) A molecular dispersion in water; tubes of the lipid dioleoylphospatidylethanolamine (DOPE). It are formed having the cross-section of a hexagon. (C) in three dimen- has been shown by NMR techniques that a sample sions these tubes are arranged in a hexagonal lattice forming bundles. oriented macroscopically between glass plates in the (D) On macroscopic scale the long axes of these bundles are aligned lamellar La phase retains the alignment in the hexagonal parallel to the glass plates of the sample.

93893 BiophysicalipysclJunaJournal Volume 55 My18May 1989 A B (Rose, 1957), as SAMPLE = {2P2[cos (ei * ,)] + 11/3 z z - = [2D2 Cei ) + 1]/3 (2a) e. y ~~~~~~e. Cef * -,)2 = {2P2[cos (Cef --t)] + 11/3 LiiLiid;~~- 01 = [2DOO(jf- Pt) + 1]/3 (2b)

where P2(x) = (3x2 1)/2, the Legendre polynomial of I~ y rank 2. On inserting Eqs. 2 in Eq. 1 we obtain

I x (P "I Iif(t) = (1 + 2D 2(Qo) + 2Do2(gt) + 4Do2(Qo)D2(Q9t))F(t)/9. (3) FIGURE 2 The experimental geometry (left). The x-y is the sample plane, and z-axis, the director, is perpendicular to the sample plane. (A) Here Q0 and Q, denote the Euler angles of the absorption Light pulse, polarization along ei, impinges on the sample under an angle and emission dipoles with respect to the polarization o and the emission, polarization ef, is observed under an angle 4. These angles are defined with respect to the director of the sample, z. (B) The directions of the light beams. The Wigner formalism is Euler angles. The Euler angles (spj, 0i, 0) of the polarization direction e; used to carry out the orientational transformations which of the incident beam. are necessary to separate the geometrical factors and the intrinsic molecular effects. This is done by the repeated use of the closure relation (Zannoni et al., 1983; Merz- Basic relation bacher, 1961; Rose, 1957) for Wigner functions. The of fluorescence depolarization transformation from the laboratory frame to the absorp- tion dipole in the molecular frame requires four successive Fluorescence from a molecule is a two-photon process; steps. These transformations from the laboratory frame to first a photon is absorbed and the molecule undergoes a the absorption dipole moment in the molecular frame at transition to an excited electronic state. The molecule t = 0 and the corresponding steps to the emission dipole then relaxes in a short period of time (fs-ps) to the first moment at time t can now be easily separated from the excited state from where fluorescence will occur. The orientation of the electric field vectors in the laboratory characteristic time duration of the fluorescence process is frame. nanoseconds. We shall assume that the process of fluores- cence depolarization involves pure dipole transitions. For this case the time dependent intensity Iif(t) at time t after Orientational transformations excitation with a 5-pulse is given by (Zannoni et al., The transformation from the laboratory frame to the 1983). orientation of the transition dipole fixed in the molecular frame is carried out in four steps. For the absorption Jif(t) = (Cei *0)2ef * -)2)() 1 dipole we have (a) the transformation from the sample Here the subscripts i and f denote the initial and final director to the axis of the water/lipid cylinder, denoted by state and e; and ef define the states of the polarizers used QSC = (v;, 7r/2, 0); (b) the transformation from the axis of in the experimental geometry. F(t) is the isotropic fluo- the cylinder to the local director on this cylinder, denoted rescence decay function and it describes the probability by QCD = (-e1, -ir/2, 0); (c) the transformation from the that the probe is still excited at time t after the excitation local director to the molecular frame of the probe mole- at t = 0. 10 and -t denote respectively the orientations of cule, denoted by QDM(O) = (at, fli, 0); and (d) the trans- the absorption dipole ,u at time t = 0 and the emission formation from the molecular frame to the transition dipole i- at time t with respect to the laboratory frame. dipole of the molecule, denoted by QMT = (a,,, flM o). The quadratic terms in Eq. 1 originate from first order Furthermore the polarization direction of the incident perturbation theory (Merzbacher, 1961). The brackets electric field relative to the director (z-axis) of the sample (. ) denote an ensemble average over all the probe is denoted by QLS = (soj, Oi, 0). Similarly the transforma- motions up to time t. Eq. 1 is the basic relation in the tions from the laboratory frame to the orientation of the theory of fluorescence depolarization from reorienta- emission dipole in the molecular frame are described by tional processes. The Wigner matrices (Rose, 1957) pro- the four transformations QSC = (Df, 7r/2, 0), QCD vide a powerful formalism for carrying out the orienta- (-cf, -ir/2, 0), QDM(t) = (af, Of, 0), and M = tional transformations implicit in Eq. 1. The dot products (a,, 3,, 0). The polarization direction of the fluorescence in Eq. 1 can be rewritten in Wigner functions (D' [Q]) relative to the z-axis is denoted by QLS = (50f, Of, 0). As a

van Langen et al. Order and Dynamics in L,, and H, Phases 939 result, Eq. 3 is written as dent terms:

1if(t)= E (D2((Qo) D(0)) = E D2* (QLS) D2 (Qs) mnpqsuvw mnpqsuvw (D2(ls)(rD2n(QSC D2D2*(SC)(v) * (1 + 2DMO(QLS)DmX2SC)Dnp(1CD)Dp[ODM(0)]DqO 2 (D2 (QCD) D2V (QC'D)) + 2D2(1LS)D (QC)D2* (QCD)DV* [QDM(t) ]DWO (QMT) 2 (DpqWDM(O)] Dvw*[DM() + 4D2 (QLS)D2(QLS)D (nSC)D2( QSC)D2P(QCD)D2* (QCD) Dwo (MT) (9) * DW(QDM)DV M)D (Qmr)D (QMT) )F(t)/9 (4) (DqO(IMT) Each of these factors will not be considered in detail: (a)

The first contains the Euler angles (pj, 0, 0) and Symmetry considerations ((PF, Of, 0) describing the experimental geometry as depicted in Fig. 2. (b) The second factor reduces the The average in Eq. 4 requires the evaluation of at least 2 58 = 390,625 terms. Fortunately the symmetry properties (Dmn(ri, 7r/2, 0) Dmu2*(rf, 7r/2, 0) )b., (10) of the hexagonal HI, phase and the use of cylindrically symmetric probe molecules will reduce this number dras- because the axis of the water cylinder does not reorient on tically. the time scale of the fluorescence process. (c) The third As the orientational transformations are independent, describes the time correlation between the orientation of the averages of the products appearing in Eq. 4 are simply the local director of the water cylinder at t = 0 and t = t. the products of the averages. The local director can only undergo lateral diffusion on the surface of the water cylinder. On utilizing the uniaxial 20(QO) Z 2D*O(QLS) (2 n(QSC)) symmetry of the cylinder and using the explicit Euler mnpq angles we find (Dnp(QcD) ) (Dq[QDM(O)I )DD(Q%) (Sa) (Dnp(QCD) Duv (QCD) ) = Dnp(O, -ir/2 0) D U(V -7r/2,0) (exp (11) suvw [i(nej-UEf)]j). *(DUV*(QCDM)DVW* [QDM(t)] )D 2*(QMT (5b) The lateral displacement on the cylinder surface can be described as a diffusion process for which we have The uniaxial symmetry in the sample, cylinder, and the (Reichl, 1980) director frames is now used to simplify Eqs. 5 further because for each phase (Zannoni et al., 1983; Luckhurst (exp [i(ne, -uf ) ] ) = exp (- n2DIatt)6nu. (12) et al., 1974; Nordio and Segre, 1979), Here the lateral diffusion of the probe on the cylinder (Dmn(Q))) = (Don(Q))5m0. (6) surface is described by a single parameter, Dlat. (d) The fourth factor describes the orientational correlation func- Thus we obtain tion with respect to the local director. It correlates the ] orientation between the long axis of the probe molecule at (D O(QSC)) (D O(QCD)) (D 2 [QDM(O) ) bmO'nO/5pO (7a) t = 0 and t = t. For the cylindrically symmetric probe (D2O*(USC) ) (D2O*(QCD) ) (D2o*[QDM(t)] )6SO6UO6VO (7b) molecules used here and on taking the local phase to be and Eqs. 5 reduce to uniaxial, we have (Luckhurst et al. 1974; Nordio and Segre, 1979) 2 =D 2 2 (QO ) DO(QLSDo ) E (DDq)2O D(gMT)DO (D[pq [QDM(O)] DDvw [QDM(t 4 q =(Dpq[QM()]Dpq*[ DM )]) PV qw(13) 1 2 (8a) 4 Doo(O)SA, (e) The last factor contains all the information about the orientations of the transition dipoles in the molecular 1 D2(Q)) = D'(Q,) (Dow )Dw(MT) frame and cannot be reduced any further. For the sake of brevity we shall henceforth use the following notation: (8b) =4DOL2D (gLS)S,. HP = HP(t) = E (Dpq[QDM(O)]Dpq DM(t)]) q The evaluation of the averages of the correla'tion prod- 2 Dqo(a, 0) D2*(a,, f5,, 0)6pv6qw (14) uct appearing in Eq. 4 is more elaborate. A rearrange- ment of the factors in this product leads to five indepen- For cylindrically symmetric probe molecules Hp(t) =

940 Biophysical Journal Volume 55 May 1989 H_p(t), p = 0, 1, 2 (Zannoni et al., 1983; Luckhurst et al., defined 1974; Nordio and Segre, 1979). The average of the product is now written as A [F(t)S,/18]dt = SJ,/18 (17a)

(D D D 0) D (p,f, EO, 0) A,= [F(t)S,/18]dt S,/18 (17b) mnpw *DMD(O, v/2, 0) DMDO r/2, O)DDP(O -w/2, ) Ao - fO (F(t) [2(Ho + 312) + 3(3Ho + 4H, + H2) D2p (O, -r/2, 0) exp (-Dh,tn2t) Hp. (15) - exp (-4D,,tt)]/721 dt (17c) On carrying out the summations and using the explicit expressions for the Wigner rotation matrix elements with Al = fI {F(t) [4(H, + H2) exp (-Dhtt) the notation spif = (pi - ,pf, we finally obtain + (3Ho + 4H, + H2) exp (-4D,,tt)I/481 dt (17d)

Iif(t) F(t) (1/9 + P2(cos 0) Ss + P2(cos f) S, A2 = | {F(t) [6(Ho + 3H2) + 16(HI + H2) exp (-D,1,t)

+ P2(cos 0,)P2(cos Of) [2(Ho + 312) + (3Ho + 4H, + H2) exp (-4DIatt) ]/1921 dt. (17e) + 3(3Ho + 4H, + H2) exp (-4Dktt)]/72 A light wave in an uniaxial medium has + sin (20i) sin (20f) cos (vif) [4(HI + H2) exp (-Dhtt) propagating two independent directions of the electric field. The first, + (3Ho + 4H, + H2) exp (-4D,tt)]/48 the ordinary (o) ray, has its electric field polarized + sin2 (0i) sin2 (of) cos (2rpif) [6(Ho + 3H2) perpendicular to the plane of incidence and hence to the + 16(HI + H2) exp (-Dlktt) axis of symmetry for all angles of incidence. Under our + (3Ho + 4H, + H2) exp (-4D,Jtt)]/192j (16) experimental conditions the light is x-axis polarized so that 0i = Of = ir/2 and vp = Sof = 0 (Fig. 2). The second, the It is important to realize that in Eq. 16 the geometrical extraordinary (e) ray, is polarized in the plane of inci- factors (0i, Of, (pif) and the molecular properties [F(t), Sp, dence, in our case the horizontal zy-plane, and is defined - - = S?, Ho, HI, H2, Diat] are completely separated. by the angles 0i = 7r/2 0, Of = 'ir/2 p, 'P; 7r/2, and pf = 7r/2 (Fig. 2). We can now make use of four combina- tions of the polarization directions of the excitation and Determination of the emission beams: oo, oe, eo, ee. The corresponding fluores- fluorescence decay cence intensities are: I,,I,,, and IC,, where the subscripts denote the polarization of the incident and The fluorescence decay function F(t) can be determined fluorescence light ray, respectively. Eq. 16 thus yields for in a time-resolved experiment. An experimental geometry the fluorescence intensities has to be found for which every contribution of the correlation functions Hp(t), p = 0, 1, 2 to the time- l/79 - A/2 - A,/2 + A0/4 + A2 (18a) dependent intensity defined in Eq. 16 disappears. It can = 9 - A4/2 - (1 - 3 sin2 p)A,/2 be shown that this condition is fulfilled on setting = cc 36.30, Of = 900, sp = 900, and spf = 450. However, in + (1 - 3 sin2')AO/4 - (1 - sin2 p)A2 (18b) practice these angles need to be corrected for refraction = '/ - (1 - 3 sin2 - A,/2 effects at the sample interface. For a lipid with a refrac- 0)A/2 tive index of 1.50 the following experimental geometry is + (1 - 3 sin22)AO/4 - (1 - sin2 0)A2 (18c) used. Excite with light polarized horizontally under an Iee= lb - (1 - 3 sin2 0)A4/2 - (1 - 3 sin2 p)A,/2 angle 0 = 600 and detect the fluorescence light along the normal to the sample surface (the director). The analyzer + (1 - 3 sin2 0) (1 - 3 sin2 p)Ao/4 - sin (20) must be set at 450 with respect to the horiziontal plane. *sin (2%o)AI + (1 sin20) (1 sin2 p)A2. (18d) The observed signal is now proportional to F(t). To remove the dependence of unknown experimental quantities as the illuminated volume and the intensity of Steady-state experiments the exciting light, polarization ratios are determined. In angle-resolved experiments the polarization ratios Ro = In angle-resolved steady-state experiments, the samples I,,,/II. and R, = II/I,, are observed as a function of the are illuminated continuously with linear polarized light. angles 0 and sp. We find for the angle-dependent polariza- Therefore, instead of time-dependent intensities, their tion ratios Ro and R, for the ordinary and the extraordi- time integrals are measured. The following quantities are nary experiment, respectively.

van Langen et al. Order and Dynamics in L.L, and NH, Phases 941 RJ(, sp) = [1 - RI + (R, + R2) sin2 (p)]/[l + RI] (19) functions Ho(t), HI(t), and H2(t) for the proble molecules considered here are given by R,,(0,) = [1 - R, + (R, + RO sin2 (0)] /[1 + R, + (R2- R,) sin2 ((p) Hp(t) = (D20(QDM) Do(QDMM)) P2(COS p3) + (R3- R,) sin2 (0) + R4 sin (20) sin (2wp) = Gp(t) P2(cosf3,,j, p = 0,1,2 (23) + R5 sin2 (0) sin2 (p)] (20) The functions Gp(t) correlate the orientation of the long axis at the time of absorption and at the time of emission. Here the relations between the parameters RI-R5 Because we have assumed a stochastic motional behavior, describing the angle dependence of the observed polariza- these molecular orientations are completely correlated at tion ratios and the five quantities describing the time t = 0, but this correlation is totally lost at t = cc. It can be integrated intensities are shown that the limiting values of the correlation functions at t = 0 and t = cc can be obtained in terms of the order R, = 9A2/N (21a) parameters (P2) and (P4) in a model independent way (Zannoni et al., 1983; Fisz, 1985; Fisz, 1987). R2 = (27A,/2 - 27Ao/4)/N (21b) = (24a) R3 = (27A,,/2 - 27Ao/4)/N (21c) GO(0) 1/5 + 2(P2)/7 + 18(P4)/35 = - (24b) R4 = 9A,I/N (21d) G,(O) 1/5 + (P2)/7 12(P4)/35 G2(0) = - 2(P2)/7 + 3(P4)/35 (24c) R5 = (81AO/4 + 9A2)/N (21e) 1/5 G0(o0) = (P2)2 (24d) with N= 1 - 9(AJ, + A,)/2 + 9Ao/4. (21f) GI(o) = G2(m) = 0- (24e) It should be noted here that Eqs. 19 and 20 are analogous to those derived for the description of angle- The time decay of these functions, however, must be dependent fluorescence depolarization measurements on evaluated in terms of a motional model. We shall here use macroscopically ordered planar multibilayer systems the rotational diffusion model (Luckhurst et al., 1974; (van Langen et al., 1987a; Deinum et al., 1988; Mulders Nordio and Segre, 1979) in which the probe molecules are et al., 1986; van der Meer et al., 1982; van Langen et al., assumed to undergo small step orientational diffusion 1988a). However the physical significance of the parame- subject to an angle-dependent ordering potential U(Q) ters RI-R5 is different than the ones given in Eqs. 21. which is taken to be of the form

U(N ) = X2 P2 (cosf3) + X4 P4 (cosM. (25) INTERPRETATION The rotational diffusion in this potential is described by the diffusion constants perpendicular and parallel to the Hexagonal phase long axis, D1 and DII, respectively. Because the absorp- The quantities A,,, A,, AO, Al, and A2 contain all the infor- tion dipole coincides with the long axis of the probe mation about the molecular behavior in the system. For molecule the experiments are only sensitive to the diffu- the probe molecule TMA-DPH the absorption dipole sion constant D1. The decay of Gp(t) is obtained from a coincides with the long axis of the probe molecule while numerical solution of the diffusion equation (Nordio and the emission dipole is tilted away by an angle A,,,, (van Segre, 1979) and it has been shown that Gp(t) can be Langen et al., 1987a and b, 1988a and b; Deinum et al., expressed as an infinite sum of exponentials. 1988; Mulders et al., 1986; Johansson and Lindblom, 1986; van Langen et al., 1989). On using Eq. 8, A,, and A, Gp(t) = E bpm exp (-apm D1t), p = 0, 1,2. (26) can be simplified to m The preexponential factors bpm and the decay coefficients A,, = (P2)/18 (22a) apm are complex functions of the parameters X2 and X4. It is A, = (P2 ) P2 (cos#,,,)/ 18. (22b) interesting to note that in most practical cases the slowest decay term dominates the decay of Gp(t) (Nordio and We shall now show that the dynamic parameters as Segre, 1979). Consequently this monoexponential form of well as the order parameter (P4 ) = '/8(35 cos4 3 - Gp(t) was used in our numerical calculations. 30 cos2 ,3 + 3), where : denotes the angle between the The fluorescence decay function of TMA-DPH mole- long axis of the probe molecule and the local director, are cules embedded in lipid systems is often found to be contained in AO, Al, A2. The orientational correlation biexponential. It is now important to note that the intensi-

942 942~~~~~~BiophysicalBipyia~Journal~ora~ ~ VolumeVlm 555My18May 1989 ties observed in steady-state experiments are determined parameters, X2, X4, D1( TF) and f3I, are contained in RI-R5 by the time integrals of the decay terms, Eqs. 17. It is easy (van Langen et al., 1987a; Deinum et al., 1988; Mulders to show that in the time integral of the multiexponential et al., 1986; van der Meer et al., 1982; van Ginkel et al., decay, each decay component is weighted by its decay 1986). time. Consequently the slowest decay components make a large contribution to the integral and in our case it is in fact dominated by them. Thus, the steady-state experi- Evaluation of the model ment is much more sensitive to the decay component of parameters the intrinsic fluorescence with the longest lifetime. We Values for the model parameters were obtained from a have previously shown in multibilayer systems that taking direct nonlinear least squares fit of the measured depolar- the fluorescence decay of TMA-DPH molecules to be ization ratios at 56 different combinations of the angles 0 monoexponential with the average fluorescence lifetime and p, to Eq. 20. The five parameters RI-R5 appearing in (rF) as decay time, introduces only small changes in the Eq. 20 were evaluated in terms of the model parameters physical parameters (Deinum et al., 1988; Mulders et al., using Eqs. 17 and 21-27. The ZXSSQ routine of the 1986; van Ginkel et al., 1986). Therefore we assume that IMSL library was used in the search for the minimum in the intrinsic decay function F(t) has the simple form x2. The errors were determined from the Hessian matrix produced by the ZXSSQ routine. No significant correla- F(t) = (1/(TF)) exp (-t/(rF)) (27) tions between the model parameters were found. This By following this approach the time integrals appearing in procedure has been described by us previously (Deinum et the expressions for AO, Al, and A2 are readily performed. al., 1988; van Gurp et al., 1988; van Langen et al., 1987a; It is important to note that both the diffusion constant Mulders et al., 1986) and shown to be reliable and D1 for the reorientational dynamics and Dlat for the lateral numerically stable. We note that the parameters RI-R5 diffusion of the probe molecule on the water cylinder can can also be determined with this procedure. be obtained. These will be expressed as the products D1 (TF) and D1at(rF ). The orientational ordering is described by the orienta- MATERIALS AND METHODS tional distribution functionf(13). This function describes Sample preparations the probability of finding a probe oriented with an angle d with respect to the local director of the system. It is shown The lipid DOPE was the kind gift of Prof. B. de Kruijff (Institute of that this function takes the form (van Ginkel et al., 1986; Molecular Biology, University of Utrecht) and used without further purification. The fluorescent probe TMA-DPH was purchased from van Gurp et al., 1988) Molecular Probes Inc., Eugene, OR, and used as received. The probe was dissolved in absolute ethanol and stored under nitrogen atmosphere f(,B) = No exp [-U(3)], (28) in the dark at 40C. Macroscopically ordered lipid systems between thin (175 um) microscope coverglasses were prepared as follows. 10 mg of where is a normalization constant. From this No distribu- lipids were dissolved in a chloroform ethanol solution and the probe tion function the order parameters (P2) and (P4) are solution, 5.10-' M, was added to obtain a probe-to-lipid ratio of 1:250 on calculated. The number density of probe molecules a molecular basis. The solvents were evaporated by a flow of nitrogen, oriented at ,B is given byf(3) sin (13) and is normalized to and the dried lipid probe mixture was kept overnight under vacuum. The unity. lipids were equilibrated for 48 h at 50C in a humidified (96% relative The model parameters describing the angle-resolved humidity) nitrogen atmosphere over a saturated K2SO4 salt solution in an exiccator. The samples were prepared in the lamellar phase at 5oC. 2 depolarization measurements are X2, X4, D±(7F), Dlat(TF ) mg of the hydrated mixture was placed on a microscope glass plate 24 x and #,,,. These five model parameters are contained in the 24 mm, using a spatula. A second plate, 20 x 20 mm, was pressed on the experimentally observed values for R1-R5. mixture and a macroscopically aligned multibilayer system was obtained by gently rubbing the two glass slides against each other. The edges of this sandwich were sealed with a two-component epoxy resin Oriented planar and the quality of alignment was checked with a polarizing microscope multibilayers equipped with a first order red plate. Only homogeneously colored In planar multibilayers the orientational order is charac- samples were used further. A round spot, diameter 4 mm, was masked terized by the order parameters (P2) and (P4). Now A is off with black tape and used in the experiments. During the preparative steps the samples were kept as much as possible in dark at 50C. the angle between the macroscopic director, the normal to the bilayer surface, and the long axis of the probe molecule. Furthermore only the diffusion constant D1 Steady-state experiments the determines decay of the correlation functions Eq. 26, A home-built fluorimeter was used to perform the angle-resolved as the experiments are not sensitive to lateral diffusion of fluorescence depolarization experiments. Excitation light, wavelength the probe molecule over the bilayer plane. Here only four 360 + 5 nm, is selected by a monochromator (Bausch and Lomb,

van Langen et al. Order and Dynamics in L.,L,, and H*Hl Phases 943 Rochester, NY) from a 450 W Xenon arc lamp. This beam is polarized The polarization ratios 1 /R, at 56 different combina- a the horizontal by Glan-Thompson prism in zy-plane (Fig. 2) before tions of the angles 0 and X were measured in the extraordi- incidence on a rotatable, temperature-controlled sample holder. A sheet nary experiment where the incident excitation light is polarizer and filter combination (a GG 395 cut off filter and a 447 ± 13 nm interference filter, both from Schott, Mainz, FRG) define the horizontally polarized. The angle dependence of the wavelength of the fluorescence light. A Peltier cooled photomultiplier polarization ratios obtained from these two phases is (RCA-31034, Lancaster, PA) is used to detect the fluorescence light. clearly seen to be markedly different (Fig. 3). This is also The light intensity is defined by the number of photons counted in a reflected in the values of presented in Table 1. preset period of time. An Apple II microcomputer (Cupertino, CA) is RI-R5, These parameters were extracted by fitting the experi- used for the data acquisition. In the extraordinary experiments per- formed here the intensity ratio, I/II, 1/R,, is determined for 56 mental ratios to Eq. 20 with a nonlinear least squares different combinations of 0 and p. Control experiments with blank procedure (Deinum et al., 1988; van Gurp et al., 1988; samples have shown that the background intensity is <1% of the van Langen et al., 1987a; Mulders et al., 1986). The best fluorescence signal. The experimental error in the observed ratio is in theoretical fit to the observations from the lamellar and the order of 2%. hexagonal phase of DOPE are also shown in Fig. 3. For a sample in the lamellar L. phase, the temperature was kept at 70C and for the same sample in the hexagonal HI, phase the temperature Numerical simulations of the polarization ratios have, was 450C. No coexistence of the phases is expected at these tempera- however, shown that the RI-R5 values for the hexagonal tures (Cullis and de Kruijff, 1978; Cullis et al., 1980). The refractive phase are much more sensitive to random errors than index of the samples was measured with an Abbe-refractometer and those for the lamellar one. values between 1.48 and 1.50 were found. The angles in the sample are To interpret the experimental results in terms of the determined by using Snell's law on taking the refractive index of the sample equal to 1.5. A slight birefringence of -0.02 is observed. Optical physical parameters describing the orientational order corrections due to birefringence are smaller than the experimental error and dynamics, the rotational diffusion model is used. In and were neglected. It has been shown theoretically (van Ginkel et al., general, this model requires the determination of two 1986; van Gurp et al., 1988) that there is no need to correct for the diffusion constants and an angle-dependent potential. We effects of multiple reflections within the sample and transmission losses note that the correlation functions describing the at the sample/air interfaces. This was verified experimentally with Gp(t) refractive index matching methods. The sample was mounted in a rotational dynamics (Eq. 26) are now obtained from a cuvette filled with a clear liquid with the same refractive index. The geometry of the cuvette constrains the exciting and emitting light beams to be perpendicular. The experimental polarization ratios were found to be the same as those obtained from corresponding angles with the sample in air. These latter experiments suffer from the disadvantage that the angles 0 and p cannot be varied independently.

RESULTS AND DISCUSSION UJ A .e -20

Macroscopically aligned samples of lipid systems either in - 0.0.O --80-30 the lamellar La or in the hexagonal HI, phase appear to be -.-50-so 0 -30 -20 -10 0 10 20 30 40 50 60 optically homogeneous under the polarizing microscope D and thus are particularly amenable for studies with IN AIR fluorescence depolarization techniques. We have shown previously how angle-resolved fluorescence depolariza- r B HEXAGOONL tion techniques can be used with aligned samples for the determination of the molecular orientational order and co dynamics of the fluorescent probe molecules (van Langen et al., 1987a; Deinum et al., 1988; Mulders et al., 1986). In these experiments the sample is excited with light polarized in the horizontal zy-plane (Fig. 2), the extraor- dinary experiment, and the angle-dependence of the polarization ratios, I, /4, = 1 /R,, is measured. - o e-0 The observed fluorescence intensity was found to be -60-50-5 0 -30 -20 -10 0 10 20 30 '0 50 80 considerably higher in the hexagonal phase than in the 40 IN AIR lamellar phase. This is due to the fact that the probe molecules in the lamellar phase are preferentially FIGURE 3 The angle p dependence of 56 experimentally observed oriented with their long axis perpendicular to the sample polarization ratios I /RC for different angles of incidence 0 together with plane and are therefore not excited efficiently by the their best fit. (A) The lamellar L. phase at 70C and (B) the hexagonal incident beam. H,, phase at 450C of TMA-DPH in DOPE.

944 Biophysical Journal Volume 55 May 1989 TABLE 1 Summary of recovered values for the both phases, are shown in Fig. 4. The probe molecules are parameters R1-R6 found to be oriented preferentially along the normal to the Lamellar La phase Hexagonal H,, phase lipid/water interface. Whereas the function for the lamel- lar phase shows a monotonic decrease towards 13 = ± 900, Temperature (°C) 7 45 a somewhat bimodal orientational distribution is found in No. of experiments 3 2 the hexagonal phase. Nevertheless in the hexagonal phase RI 0.34 (0.02) 0.36 (0.02) the distribution around 13 = 0° is considerably narrower R2 1.1 (0.10) -0.22 (0.05) R3 1.7 (0.10) -0.11 (0.03) than that of the lamellar phase. The population of probes R4I 0.95 (0.10) 0.44 (0.02) oriented at 13 = 900 may be an artefact due to the R5 13.0 (1.5) 2.60 (0.20) assumptions used in the analysis. We have here approxi- mated a small surface element of the curved cylinder by a Errors are given in parentheses. planar surface. Nevertheless, they may well be the result of the edge effects of finite cylinders or effects of cylinders numerical solution of the diffusion equation. The poten- which are not perfectly aligned parallel to the glass plates tial U(fl), given in Eq. 25, has been used in the analysis of of the sample. It is interesting to note in this connection the experimental data of both phases. This potential has that we have observed bimodal distributions for TMA- been chosen as the pair of coefficients (X2, X4) yields DPH molecules in highly curved small unilamellar lipid unique values for the order parameters (P2) and (P4 ) vesicles (van Langen et al., 1989). (Mulders et al., 1986; van Ginkel et al., 1986; van Gurp et The results for the hexagonal phase reveal that the al., 1988). Its validity, however, is open to question as we process of lateral diffusion over the cylindrical water tube do not expect the probe molecule TMA-DPH to be is at least 10 times slower than the process of the oriented in the hexagonal phase at angles 1 > 900 or ,B < reorientational motion. Experiments on vesicle systems -900 relative to the local director. This, because the with other techniques support this observation (Houslay molecules are anchored with their polar headgroup at the and Stanley, 1982). We can now estimate the value of the aqueous interface (Prendergast et al., 1981). Neverthe- diffusion coefficients for the reorientational motion on less, we have shown previously in the case of vesicle using the average fluorescent lifetime of 3.4 ± 0.2 ns systems, where the same arguments apply, that the sim- obtained from a time-resolved experiment at 200C (data plest potential satisfying this restriction, U(,B) = XI cos (13) + X2 P2 [cos (13)], fails to describe the experiments (van Langen et al., 1988b). Unfortunately a more extended and realistic potential cannot be used as it °- A t - LfRMELLfAR requires more parameters in its description than can be I ,/\ --- HEXAGONRL determined with this technique. The behavior of the molecules in the lamellar phase is described by four model parameters X2, X4, D1(TF), 13,, L*I I I whereas five parameters X2, X4, D1(TF), Diat(TF), ,B,, are required for the description of the behavior in the hex- agonal phase. The extracted values obtained from a direct - 0 -- I 0 30 6 o fit to the experimental data as described above, are shown BETH in Table 2 together with corresponding order parameters (P2 ) and (P4). The orientational distribution function f(13) and the number density function f(13) sin (13) for °:r , ,-LRAELLAR z /1% 2 II| ---HEXRAAL TABLE 2 Model parameters and order parameters II iII a(1nI-- x Lamellar L. phase Hexagonal H,, phase cc I - I I _- X2 -2.4 (0.2) -1.2 (0.2) L X4 -0.63 (0.1) -3.9 (0.4) -60 -I0 -0 _oo so D,(TF) 0.08 (0.01) 0.11 (0.01) BETA DIa,(TF) Not obtainable 0.01 (0.005) fl, 150 (2) 200 (2) (P2) 0.60 (0.01) 0.74 (0.02) FIGURE 4 (A) The orientational distribution function f(f). (B) The (P4) 0.30 (0.02) 0.66 (0.03) number density functionf(#t) sin (,8) for TMA-DPH in the lamellar La phase at 70C and the hexagonal Errors are given in parentheses. H,, phase at 450C.

van Langen et al. Order and Dynamics in L.La and H, Phases 945 not shown). On assuming that the average lifetime does Caffrey, M. 1985. Kinetics and mechanism of the lamellar gel/lamellar not change drastically with temperature, we conclude liquid-crystal and lamellar/inverted hexagonal phase transition in phosphatidylethanolamine: a real-time x-ray diffraction study using that the diffusion coefficient D1 in the hexagonal phase is synchrotron radiation. . 24:4826-4844. comparable with that in the lamellar phase. Crowe, J. H., L. M. Crowe, J. F. Carpenter, A. S. Rudolph, C. A. The experiments yielded significant values for the Wistrom, B. J. Spargo, and T. J. Anchordoguy. 1988. Interactions of angle f,B between the transition dipoles of TMA-DPH. sugars with membranes. Biochim. Biophys. Acta. 947:367-384. Similar values for the angle were also found in various Cullis, P. R., and B. de Kruijff. 1978. The polymorphic phase behaviour other lipid vesicle systems (van Langen et al., 1987b and of phosphatidylethanolamines of natural and synthetic origin. A 31P 1988b; van Langen et al., 1989) or planar multibilayer NMR study. Biochim. Biophys. Acta. 513:31-42. systems (Deinum et al., 1988; Mulders et al., 1986) and it Cullis, P. R., B. de Kruijff, M. J. Hope, R. Nayar, and S. L. Schmid. 1980. and membrane transport. Can. J. Biochem. was found to depend on environmental conditions (van 58:1091-1100. Langen et al., 1987a). Deinum, G., H. van Langen, G. van Ginkel, and Y. K. Levine. 1988. The values of the order parameters and the diffusion Molecular order and dynamics in planar lipid bilayers: effects of constant obtained in the lamellar phase of DOPE are unsaturation and sterols. Biochemistry. 27:852-860. similar to those reported in multibilayer systems of vari- de Kruijff, B., P. R. Cullis, A. J. Verkleij, M. J. Hope, C. J. A. van ous unsaturated phosphatidylcholine (PC) lipids Echteld, and T. F. Taraschi. 1985a. and mem- (Deinum et al., 1988). In spite of the temperature differ- brane function. In Enzymes of Biological Membranes. A. Martonosi, ences in the experiments, the diffusion constants in DOPE editor. Plenum Publishing Corp., New York. 131-204. de Kruijff, B., P. R. Cullis, A. J. Verkleij, M. J. Hope, C. J. A. van and DOPC are the same, whereas the order parameters Echteld, T. F. Taraschi, P. van Hoogevest, J. A. Killian, A. Rietveld, found in DOPE are somewhat higher as the ones reported and A. T. M. van der Steen. 1985b. Modulation of lipid polymorphism for DOPC (Deinum et al., 1988). by lipid-protein interactions. In Progress in Protein-Lipid Interac- We have here demonstrated the potential of steady- tions. A. Watts and J. J. H. H. M. de Pont, editors. Elsevier Science, state fluorescence depolarization experiments in studies Amsterdam. 89-142. of lipid systems arranged in the hexagonal phase. Work is Ellens, H., J. Bentz, and F. C. Szoka. 1986. Destabilization of phospha- tidylethanolamine at the hexagonal phase transition tem- currently in progress to obtain more detailed information perature. Biochemistry. 25:285-294. about molecular order and dynamics from angle- and Fisz, J. J. 1985. Fluorescence depolarization in macroscopically ordered time-resolved experiments on lipid systems in the hex- uniaxial molecular samples. Chem. Phys. 99:177-191. agonal phase. Fisz, J. J. 1987. Symmetry simplifications in the description of molecu- lar order and reorientational dynamics in uniaxial molecular systems. The investigations were made possible by the financial support given to I. Symmetry constraints on the joint probability distribution function. Dr. van Langen by the Dutch Foundation for Biophysics under the Chem. Phys. 114:165-185. auspices of the Netherlands Organization for Scientific Research. Gasset, M., J. A. Killian, H. Tournois, and B. de Kruijff. 1988. Influence of cholesterol on gramicidin-induced H,, phase formation in phosphatidylcholine model membranes. Biochim. Biophys. Acta. Receivedfor publication 27 September 1988 and in finalform 939:79-88. 30 November 1988. Gruner, S. M. 1985. Intrinsic curvature hypothesis for biomembrane lipid composition: a role for nonbilayer lipids. Proc. Natl. Acad. Sci. USA. 82:3665-3669. Hardman, P. D. 1982. Spin-label characterization of the lamellar- REFERENCES to-hexagonal (H,,) phase transition in egg phosphatidylethanolamine aqueous dispersions. Eur. J. Biochem. 124:95-101. Bentz, J., H. Ellens, and F. C. Szoka. 1987. Destabilization of phospha- Hong, K., P. A. Baldwin, T. M. Allen, and D. Papahadjopoulos. 1988. tidylethanolamine-containing liposomes: hexagonal phase and asym- Fluorometric detection of the bilayer-to-hexagonal phase transition in metric membranes. Biochemistry. 26:2105-2116. liposomes. Biochemistry. 27:3947-3955. Blaurock, A. E. 1982. Evidence of bilayer structure and of membrane Houslay, M. D., and K. K. Stanley. 1982. Dynamics of Biological interactions from x-ray diffraction analysis. Biochim. Biophys. Acta. Membranes. John Wiley & Sons Ltd., Chichester, UK. 650:167-207. Israelachvili, J. N., and D. J. Mitchell. 1975. A model for the packing of Boggs, J. M., and J. C. Hsia. 1973. Orientation and motion of lipids in bilayer membranes. Biochim. Biophys. Acta. 389:13-19. amphiphilic spin labels in hexagonal lipid phases. Proc. Natl. Acad. Israelachvili, J. N., S. Marcelja, and R. G. Horn. 1980. Physical Sci. USA. 70:1406-1409. principles of membrane organization. Q. Rev. Biophys. 13:121-200. Brown, P. M., J. Steers, S. W. Hui, P. L. Yeagle, and J. R. Silvius. 1986. G. Role of head group structure in the phase behaviour of amino Kirk, L., S. M. Gruner, and D. L. Stein. 1984. A thermodynamic model of to phospholipids. 2. Lamellar and nonlamellar phases of unsaturated the lamellar inverse hexagonal phase transition of lipid phosphatidylethanolamine analogues. Biochemistry. 25:4259-4267. membrane-water systems. Biochemistry. 23:1093-1102. Bildt, G. , H. U. Gally, A. Seelig, J. Seelig, and G. Zaccai. 1978. Lasic, D. D., and H. Hauser. 1984. Detection of hexagonal phases by Neutron diffraction studies on selectively deuterated ESR spin labeling. Mol. Cryst. Liq. Cryst. 113:59-75. bilayers. Nature (Lond.). 271:182-184. Luckhurst, G. R., M. Setaka, and C. Zannoni. 1974. An electron

946 Biophysical Journal Volume 55 May 1989 resonance investigation of molecular motion in the smectic A meso- Sjolund, M., G. Lindblom, L. Rilfors, and G. Arvidson. 1987. Hydro- phase of a . Mol. Phys. 28:49-68. phobic molecules in lecithin-water systems. I. Formation of reversed Luzzati, V., T. Gulik-Krzywicki, and A. Tardieu. 1968. Polymorphism hexagonal phases at high and low water contents. Biophys. J. of lecithins. Nature (Lond.). 218:1031-1034. 52:145-153. Marsh, D., and J. M. Seddon. 1982. Gel-to-inverted hexagonal (LO-HI,) Tate, M. W., and S. M. Gruner. 1987. Lipid polymorphism of mixtures phase transitions in phosphatidylethanolamines and fatty acid- of dioleoylphosphatidylethanolamine and saturated and monounsatu- phosphatidylcholine mixtures, demonstrated by 3'P-NMR spectros- rated phosphatidylcholines of various chain lengths. Biochemistry. copy and x-ray diffraction. Biochim. Biophys. Acta. 690:117-123. 26:231-236. Mely, B., J. Charvolin, and P. Keller. 1975. Disorder of lipid chains as a van der Meer, B. W., R. P. H. Kooyman, and Y. K. Levine. 1982. A function of their lateral packing in lyotropic liquid crystals. Chem. theory of fluorescence depolarization in macroscopically ordered Phys. Lipids. 15:161-173. membrane systems. Chem. Phys. 66:39-50. Merzbacher, E. 1961. Quantum Mechanics. John Wiley & Sons, New van Ginkel, G., L. J. Korstanje, H. van Langen, and Y. K. Levine. 1986. York. The correlation between molecular orientational order and reorienta- Mulders, F., H. van Langen, G. van Ginkel, and Y. K. Levine. 1986. The tional dynamics of probe molecules in lipid multibilayers. Faraday static and dynamic behaviour of fluorescent probe molecules in lipid Discuss. Chem. Soc. 81:49-61. bilayers. Biochim. Biophys. Acta. 859:209-218. van Gurp, M., H. van Langen, G. van Ginkel, and Y. K. Levine. 1988. Nordio, P. L., and U. Segre. 1979. The Molecular Physics of Liquid Angle-resolved techniques in studies of organic molecules in ordered Crystals. G. R. Luckhurst and G. W. Gray, editors. Academic Press, systems using polarized light. In Polarized Spectroscopy of Ordered London. 411-426. Systems. B. Samori' and E. W. Thulstrup, editors. Kluwer Academic Publishers Group, Dordrecht, Netherlands. 455-489. Prendergast, F. G., R. P. Haugland, and P. J. Callahan. 1981. 1- [4(Trimethylamino)phenyl]-6-phenylhexa- 1,3,5-triene: synthesis, van Langen, H., D. Engelen, G. van Ginkel, and Y. K. Levine. 1987a. fluorescence properties, and use as a fluorescence probe of lipid Headgroup hydration in egg-lecithin multibilayers affects the behav- bilayers. Biochemistry. 20:7333-7338. iour of DPH probes. Chem. Phys. Lett. 138:99-104. Ranck, J. L., T. Keira, and V. Luzzati. 1977. A novel packing of the van Langen, H., Y. K. Levine, M. Ameloot, and H. Pottel. 1987b. hydrocarbon chains in lipids. The low temperature phases of dipalmi- Ambiguities in the interpretation of time-resolved fluorescence an- toyl phosphatidyl-glycerol. Biochim. Biophys. Acta. 488:432-441. isotropy measurements on lipid vesicle systems. Chem. Phys. Lett. 140:394-400. Reichl, L. E. 1980. A Modern Course in Statistical Physics. University of Texas Press, Austin. 167. van Langen, H., G. van Ginkel, and Y. K. Levine. 1988a. Fluorescence Reiss-Husson, F. 1967. Structure des phases liquide-crystallines de depolarization studies of molecular dynamics using pulsed synchro- diff6rents phospholipides, monoglyc6rides, sphingolipides, anhydres tron radiation: applications to lipid vesicles and oriented multibi- layers. In Time-Resolved Laser Spectroscopy in Biochemistry. J. R. ou en pr6sence d'eau. J. Mol. Biol. 25:363-382. Lakowicz, editor. Society of Photo-Optical Instrumentation Engi- Rilfors, L., G. Lindblom, A. Wieslander, and A. Christiansson. 1984. neers, Bellingham, WA. 377-388. Lipid bilayer stability in biological membranes. In Membrane Fluidi- ty. Vol. 12. M. Kates and B. Manson, editors. Plenum Publishing van Langen, H., G. van Ginkel, and Y. K. Levine. 1988b. A comparison Corp., New York. 205-245. of the molecular dynamics of fluorescent probes in curved lipid vesicles and planar multibilayers. Liquid Crystals. 3:1301-1317. Rose, M. E. 1957. Elementary Theory of Angular Momentum. John Wiley & Sons, New York. van Langen, H., G. van Ginkel, D. Shaw, and Y. K. Levine. 1989. The Siegel, D. P. 1986a. Inverted micellar intermediates and the transitions fidelity of response by TMA-DPH in time-resolved fluorescence between lamellar, cubic, and inverted hexagonal lipid phases. I. anisotropy measurements on lipid vesicles. Eur. Biophys. J. In press. Mechanism of the L. - HI, phase transitions. Biophys. J. 49:1155- van Venetie, R., and A. J. Verkleij. 1981. Analysis of the hexagonal II 1170. phase and its relations to lipidic particles and the lamellar phase. A Siegel, D. P. 1986b. Inverted micellar intermediates and the transitions freeze-fracture study. Biochim. Biophys. Acta. 645:262-269. between lamellar, cubic, and inverted hexagonal lipid phases. II. Zannoni, C., A. Arcioni, and P. Cavatorta. 1983. Fluorescence depolar- Implications for membrane-membrane interactions and membrane ization in liquid crystals and membrane bilayers. Chem. Phys. Lipids. fusion. Biophys. J. 49:1171-1183. 32:179-250.

van Langen et al. Order and Dynamics in L<, and H, Phases 947