Neutron diffraction studies on phosphatidylcholine model membranes

J. Mol. Biol.

(1979) 134, 673-691

Neutron Diffraction Studies on Phosphatidylcholine Model Membranes I. Head Group Conformation G.B~~LDT,

H.U.

GALLY, J. SEELIG

Department of Biophysical Chemistry Biocenter of the University of Base1 Klingelbergstrasse 70, CH-4056 Basel, Switzerland AND G.ZACCAI Institti

Laue-Langevin, 156’ X Centre de Tri F-38042 Grenoble, France

(Received 13 March

1978, and in revised form 20 June 1979)

Neutron diffraction experiments on selectively deuterated lipids provide a new method of determining to a segmental resolution the mean conformation of a lipid molecule as projected along the bilayer normal, despite the high amount of disorder that exists in these bilayers. In addition, a time-averaged picture of the extent of the positional fluctuations of the individual segments in this direction can be given. This is demonstrated for a multilamellar system of bilayers of 1,2-dipalmitoyl-slz-glycero-3-phosphocholine. In this paper the head group region of the molecule is examined and this carries the zwitterionic phosphocholine group that determines the electrostatic interaction in the bilayer. Samples deuterated at four different positions in the head group region were measured as oriented samples at 6% (w/w) water content at 20°C (Lo, phase) and at 10% (w/w) at 70°C (L, phase) and as unsonicated dispersions with 25% (w/w) water at 28% (Lp, phase) and 50°C (L, phase). From the oriented samples, reflections up to ten orders, and from the powder type samples only four orders, were collected. The derived structure factors for the deuterated segments were fitted assuming a Gaussian distribution of the segments along the bilayer normal. The mean label position was determined for each label under different conditions of water content and temperature with a precision of better than & 1 angstrom in most cases. The data clearly show that the average orientation of the zwitterionic phosphocholine group is almost parallel to the membrane surface in the gel state (Lo,) as well as in the liquid crystalline state (La). It is interesting to note that in a recent dielectric investigation on this multilamellar system at 25% (w/w) water content the same mean orientation of the dipole was found (Shepherd & Biildt, 1978).

1. Introduction The lipid bilayer has many interesting physical properties that have been studied extensively, especially since it was accepted to be an important component of biological membranes. Its molecular structure and organization have been elucidated by X-ray diffraction studies on unoriented dispersions (Ranck et al., 1974, and references 673 0022-2836/79/320673-19

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Press Inc. (London)

Ltd.

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therein) and on oriented multilayers (e.g. see Levine & Wilkins, 1971; Lesslauer et (~1,. 1972; Janiak et al., 1976; Torbet & Wilkins, 1976). Recently, the problem has been tackled by neutron scattering (Zaccai et al., 1975 ; Worcester, 1976; Worcester & Franks, 1976) ; while the single crystal structure of 1,2-dilauroyl-nr,-glycero-phosphatidy1 ethanolamine has stimulated new ideas on the detailed arrangement of lipid molecules in membranes (Hitchcock et al., 1974). Systematic X-ray studies on unoriented dispersions have shown the existence of a variety of thermodynamic phases for lipid water systems. each phase with welldefined structural characteristics (Luzzat,i, 1968: Tardieu d nl., 1973; Ranck et al.. 1974). The nature of the phases and the temperature ranges in which they exist depend on the chemical composit,ion of the lipid a,nd the water content of the mixture. In the case of phospholipids, there are two main lamellar phases: a gel phase, below t, and a liquid crystal phase, above t,.. The transition tema transition temperature perature depends on the chemical structure of the two hydrocarbon chains bonded to the glycerol backbone and the structure of the headgroup toget,her with the extent of hydration. For 1,2-dipalmitoyl-sr~-glycero-3-phosphocholine fully hydrated t, = 42°C (Chapman et al.. 1967). The quantitative understanding of the building forces of lipid bilayers requires a better knowledge of the forces acting in the head group region of phospholipids in membranes. One obvious point that causes difficulties in understanding the static and dynamic properties of the head group region arises from the fact that this region is chemically more complex than the chain region, and this leads to a variety of possible conformations:. ln addition to investigating the electrostatic forces between the charged or zwitterionic head groups, t,he dielect,ric properties of the water penetrating into this region must be known. Until now. however, only a 6 A resolution water distribution has been determined from neutron diffraction experiments (Worcester, 1976 ; Btildt et al. 1976.1978). At present, two phospholipid head groups, the phosphoethanolamine and t#he phosphocholine groups, have been investigated in more detail. By means of X-rays a high-resolution crystal structure of 1,2-dilauroyl-nL-glycero-phosphoethanolamine has been obtained, showing the phosphoethanolamine dipole oriented parallel to t,he surface of the membrane (Hitchcock et al.: 1974). The crystal did not contain water but did cont’ain one molecule of acetic acid. A recent study of phosphorus-31 and deuterium nuclear magnetic resonance measurements on bilayers above the phase transition temperature (liquid crystalline state) is consistent with this structure (Seelig & Gally, 1976). For the phosphocholine head group structural interpretations have been report’etl using a variety of methods. The sharp double pea,ks in the head group region in X-ra) profiles of 5 A resolution can give an indication for a choline dipole parallel to the bilayer surface (Cain et al., 1972; Torbet & Wilkins, 1976). The analysis of the water distribution as determined by neutron diffraction on egg lecithin/cholesterol/water multilayers (Worcester & Franks, 1976) gave additional support for this conformation. Hauser et al. (1976) and Barsukov et aZ. (1976) h ave reported different xtruct,ures for the phosphocholine dipole using lH and 31P nuclear magnetic resonance in combination with paramagnetic probes. The first group postulated a lipid structure with the head group perpendicular to the bilayer surface, the second group found it parallel as seen by the X-ray experiments mentioned above,. Recent experimenDs with deuterium and phosphorus-31 nuclear magnetic resonance on unsonicated 1,2-dipalmitoyl-srL-glycero-3-phosphocholine bilayers gave strong evidence that: at, least

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in the liquid crystalline state, the choline dipole is oriented parallel to the bilayer surface (Seelig et al., 1977). Furthermore, the deuterium nuclear magnetic resonance studies demonstrate that the phosphocholine is not completely rigid, but characterized by a limited conformational flexibility. With the advent of neutron scattering, an independent set of structural observations became available for the lipid bilayer (Zaccai et al., 1975; Worcester, 1976; Worcester & Franks, 1976; Biildt et al., 1976,1978). This is because atomic scattering cross-sections for neutrons are quite different from those for X-rays. Neutrons are scattered by nuclei, each isotope having a different scattering cross-section. The advantages of neutron scattering for the study of biological structures derive mainly from the large difference in the scattering lengths of hydrogen and deuterium: -0.37 x 10 - l2 cm and 0.67 x lo-l2 cm, respectively. Thus, the water distribution in the lipid bilayer was determined by H20/2H20 exchange. Using this distribution as an isomorphous replacement site helped to phase and scale the structure factors of the neutron scattering density profile. Also, by specifically deuterating part of a molecule, it is possible to locate it in the structure. Such an approach was used by Worcester & Franks (1976) to analyze cholesterol/phosphatidylcholine bilayers. By selectively deuterating part of the cholesterol molecule, the average position of the steroid frame in the membrane was determined. Neutron scattering experiments with lipid molecules deuterated at certain segments is a direct and sensitive method for tracing the structure along the bilayer normal, since the deuterated segment gives an intense peak in the neutron density profile and can thus easily be located in the membrane. In this and the accompanying paper we have undertaken a systematic structural investigation of the conformation of the 1,2dipalmitoyl-sn-glycero-3-phosphocholine molecule in the gel phase (Lb,) and the liquid crystalline phase (La). This paper describes the results for the head group region, whereas the accompanying paper deals with the hydrocarbon chains. Two parameters are of interest in these studies ; the mean position of a deuterated segment 1 determining the mean conformation of the lipid along the bilayer normal and the width of the distribution of each segment along this direction giving a time-averaged picture of the fluctuations in space. For studying the head group conformation, four samples were deuterated, each in a different position at the Cy, C/l and Ca segment in the phosphocholine dipole and the GC-3 position in the glycerol backbone. For comparison, one sample was prepared that was deuterated at the beginning of one chain in the C-2(1) position (see Table 2 for the nomenclature of the labels). The samples were investigated at different water content and temperatures. The low water content samples were oriented on quartz slides and studied at 20°C (Lp, phase) and 6% (w/w) water and at 70°C (L, phase) with 10% (w / w ) water. Under these conditions, with relatively well-ordered multilayer stacks, it was possible to achieve profiles of 6 A resolution. At higher water content, three different phases were found and characterized by X-ray diffraction work (Tardieu et aE., 1973; Janiak et al., 1976). In order to elucidate the lipid structure in two of these phases, our higher water content experiments (25% (w / w ) water) were performed at 28°C (LB, phase) and 50°C (L, phase) on unsonicated dispersions. The nomenclature of the phases used is that of Luzzati (1968) ; see also Tardieu et al. (1973). In these dispersions the multilayer stacks were less well-ordered, so that these samples were prepared and investigated as crystallographic powder type samples. Preliminary accounts of this work have been published (Biildt et al., 1978).

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2. Materials and Methods (a) Materials The synthesis of the samples deuterated at various positions in the Ilead group described in detail by Gally et al. (1975) and by Seelig & Seelig (1975). (b) Sample

has beeu

preparation

The samples were prepared in 2 different ways, as oriented samples on glass slides and powder type samples sealed in a call with quartz windows. Oriented multilayers were prepared by spreading a briefly sonicated lipid dispersion in water on qua& slides and allowing it to evaporate slowly. The slide was then equilibrated against an atmosphere of constant relative humidity above a saturated solution of LiCl. Exchange of H,O by D,Ot was achieved by making the saturated salt solutions in H,O/D,O mixtures. Levine (1970) includes a table of water content and corresponding relative humidity, and Tardieu et al. (1973) have related water content to t,he spacing, d, of the hilayers. With H,O/D,O exchange, using the neutron scattering density profile on an absolute scale, it was possible to estimate directly the number of water molecules per lipid molecule (Zaccai et al., 1975; Worcester, 1976). The value found for multilayers equilibrated at 150/b relative humidity (over a saturated solution of LiCl at 20°C) was ~2.5 water molecules per lipid molecule, which agrees well with Levine (1970) (just under 6”/b (w/w) water content). The spacing, d = 57.4&0.3 A, agrees ,well wit.11 Tardinu et al. (1973) who gave cl = 57.7 A for that, water content. Broad mosaic spreads were measured for t,hose samplos ; typical values for slides with ~30 mg of lipid and a surface area of 2.5 ‘x: 6 cm2 lay between 15” and 30” full width at half-height. Highly oriented multilayers were recently prepared by partly following a procedure given by Powers & Persham (1977). Multilayers oriented on quartz slides as described above were heated up to 120°C for 4 h and the temperature was then slowly lowered with a cooling rate of about 10 deg. C/h. This annealing procedure gave mosaic spreads of about 1” (Fig. 1) and an enhancement in the intensity of the reflections of up to 20. We did not treat the surface of the quartz slides with any surfact,ant, in order to prevent disturbances in the bilayer structure in contrast to t,he above authors. These samples are called highly oriented in the text. Generally, before and after each measurement the integrity of the samples was checked by thin-layer chromatograplly. Unoriented powder samples were prepared as described by Janiak et al. (1976). The lipid and water mixture (25% (w/w) water content,) was sealed in cells with quartz windows and Teflon spacers defining a thickness of 1 mm, perpendicular to t,he beam. It was confirmed by X-ray diffraction from powder type samples that all phases st,udied here gave diffraction patterns similar to those reported by Janiak et al. (1976). (c) D@action

eqveriments

Lamellar diffraction for oriented samples at low water cont,eut was analyzed in the gel highphase Lo, and in the liquid crystalline phase L, in order to he able to establish resolution profiles in both phases. These samples were examined on the 4-circle diffractometer, the D16 instrument at Institut, Laue-Langovin (ILL) and gave useful diffraction out to 3 < 0.18 A-l (s = 2 sin0/h). A beam from a neutrort guide was reflected by a pyrolitic graphite monochromator set for 4.64 pi neutrons with a. spread of 2%. Higher order wavelength contamination was removed by a beryllium filter, which scattered out’ all wavelengths shorter than 3.95 A. ,4 single 3He neutron cormter could be scanned in the horizontal plane and also in a vertical plane. The sample was held with the lamellar scattering vector in the horizontal plane. Collimation wa,s by slits placed before and after the sample. In most cases, the available beam divergence of 0.75” (full width at halfmaximum) was allowed in the incident beam. Reflections were separated by more than 4’ in 28 and there was no overlap between them. Strong reflections could be measured in minutes, whereas weaker reflections were scanned for several hours to get good statistics. In order to perform the experiment, in a reasonable length of time, high-resolution (5.6 A) t Abbreviations

used:

D, deuterium;

DPPC,

1,2-dipalmitoyl-in-glycero-3-phosphoohol~e.

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8 (deg) FIQ. 1. Mosaic spreads of a DPPC sample with:6% (w / w ) water content at 20°C. The detector was maintained at a fixed angle of 20 = 4.59’, the position of the first order (d = 57.4 b, h = 4.6 A). Neutron counts are measured for different angles 0 of the vertically mounted slide with respect to the primary beam (w-scan). The curves are not corrected, so the dip at 0 = 0” is produced by absorption of the slide in the primary beam and the dip at 0 = 4.59” by the absorption of the slide in the secondary beam. Thus the neutron counts qualitatively show the distribution of the multilayer stacks which have various angles with respect to the quartz slide. The distribution has its maximum at 0 = 2.30’ for multilayer stacks parallel to the slide. Open circles show the experiment when the slide was not annealed (broad mosaic spread), and filled circles were measured with the same sample after the annealing procedure (narrow mosaic spread of 7 - 3’ full width at half-height). In this case an enhancement in intensity by a factor of 15 was obtained.

data up to 10 orders were collected for a few samples, and only the first strong orders for the others, corresponding to -15 A resolution. Temperature control of the oriented multilayers was achieved by circulating water from a thermostat around a closed aluminium cylinder containing the slide in the atmosphere above a saturated salt solution. Oriented multilayers were equilibrated for at least 1 day at the appropriate relative humidity before diffraction was measured. In the Lo, phase a saturated solution of LiCl maintained a relative humidity of 15% in the cylinder at the measuring temperature of 20°C. Under these conditions the water content of the sample is 6% (w/w). at 70°C In the liquid crystalline phase La, the highly oriented samples were examined at saturated relative humidity, which was achieved by a container of pure water in the aluminium cylinder. The water content of the sample under these conditions was 10% (w/w), as was determined gravimetrically. The phase transition temperature was found at about 55°C from observing a large decrease in lamellar spacing from 62 A to 50.8 k. Powder samples at high water content (25% (w/w)) were investigated in order to obtain mean conformations of the lipids under more physiological conditions. They gave lamellar diffraction in Debye-Scherrer rings, but reflections corresponding to s > 0.07 A-l were too weak to observe in a reasonable time. These samples were examined on the Dll instrument at ILL with a multidetector that allowed the complete diffraction rings to be

678

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observed simultaneously (Ibel, 1976). Sample-to-detector distance was 86 cm, so that the maximum s value observable was 0.08 k-l for a wavelength of 4.8 A. The wavelength was determined by a velocity selector with a spread of 8%. A 0.8 cm diamet0r circular slit was used at the sample. A typical data accumulation time for 1 spectrum was 20 min. (d) Treatment of the data (i) Oriented

multilayers

For the oriented samples, integrated intensities were measured from 8---28 scans as described by Zaccai et al. (1975). Structure factors were derived from integrated intensities after corrections that are summarized by the following equation: P(h)

= I(h) a(h) P(h) w(h) L(h),

where P(h) is the structure factor of order h, I(h) is the measured integrated intensity. a(h) is an attenuation correction, P(h) is a correction for when t’he projection of the sample for th0 perpendicular to the beam was larger than the incident beam, o(h) is a corr0ction extent of the reflection in the vertical direction and L(h) is the Lorentz factor. Wherever possible, the correction factors were determined experimentally. The projection factor was determined by scanning a piece of vanadium of the same dimensions as the sampl0s. Vanadium has a large incoherent cross-section for neutrons, which gives rise to isotropic scattering. The attenuation correction was determined by measuring the attenuation of a neutron beam by the sample to obtain its attenuation coefficient and applying the correction for a plate sample (International Tables for X-ray Crystallography). The vertical slit correction was determined by scanning a set of reflections in the vertical as well as in the horizontal plane (using the tilting counter). Integrated intensities in two dimensions were compared with integrated intensities in only the horizontal plane to yield the correction factor. The Lorentz factor was equal to sin28 (26’ is the scattering angle). (ii) Powder samples For the powder samples, the data were corrected for detector responsu by using a pure water sample and mixtures of H,O/D,O, which give isotropic scattering. 360” averages were then taken round the diffraction ring. The path-length in the sample did not change appreciably over the angular range, so that an attenuation correction was not applied. The attenuation of the beam by each sample was measured, nevertheless, to provide a measure of the amount of scattering material. A Lorentz factor of (sin28) sine related intensities averaged ov0r the Debye-Scherrer rings t,o th0 square of the structure factors.

Phase

(0)

The DPPC bilayer profile Fourier summation :

is one-dimensional

2

czwignwbtmt

and centrosymmetric.

It is given

by th0

hrnar

p(z) = ;i hz, F(h) ~0~272z h/d, where the sign of P(h) is either + 1 or - 1, d is the lamellar spacing and p(z) is tho ldimensional scattering length density. The strong reflections were phased by the isomorphous replacement method, using H,O/D,O exchange (Zaccai et al., 1975). Even though in principle one isomorphous replacement site is sufficient to determine completely the phases of a centrosymmetric structure, additional conditions were necessary to phase some of the weaker reflections. (1) A plot of the structure factors against D,O content of the water layer must, be linear for a centrosymmetric structure. Thus these plots can determine a phase change going from Ha 0 to D&l. (2) The difference structure factors of a sample in Hz0 and D,O for all samples with the same water content and temperature must be the same. (3) The difference structure factors of 2 samples in Hz0 labelled at different positions must be the same as those in D,O. (4) The label position themselves can be used to assign phases. Some structure factors

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are determined using the water layer as isomorphous replacement site. From these a label position can be calculated by methods described in section (f), below, to a certain degree of accuracy. The unknown phases are then not allowed to move the positions out of these limits. Despite the above conditions, in some cases of very weak reflections it was not possible to find the phases. It was then verified that they introduced only negligible differences in t,he profile, and they were usually omitted rather than given an arbitrary sign. (f) Determination

of label positim of the mean label positions for the oriented sanaples

(i) Procedures for the calculation

The easiest way to find the label positions in the direction of the bilayer normal from the structure factors of 2 samples, 1 deuterated at a certain position and 1 undeuterated, would be from the difference profile in real space. This procedure has the disadvantages that the Fourier series truncation errors would affect directly the distribution of the label, since the resolution in membranes is generally low, and because very weak orders with uncertain amplitudes and phases cannot be suppressed. A way to avoid these problems is to find a model for the label distribution and to fit the parameters to the difference sets of the observed structure factors in reciprocal space. From high-resolution profiles a Gaussian label distribution seems to be a good approximation with the advantage of having only a small number of parameters to be fitted. But one should note that this distribution might not be suitable in all cases. It was used in the following form :

t?(x)= where v is the measured from factors are Wh)

-+(B”p

-

r+)2

l/e half-width of the distribution 1 of the 2 centres of symmetry

= j-r,

+ exp -

r+)‘),

and z0 the mean position of the label in the membrane. The related structure

t, g(x) cos( 2nxh/d)dz = 2 tN exp - ( vnh/d)2 cos( 2nz,h/d),

(3)

where tN is the scattering length of the label at position N. The precision to which the parameters z. and Y can be obtained from a fit of the calculated .8’>(h) to the observed structure factors depends on their influence on the orders. The parameter z,, in the argument of the cosine of eqn (3) is largely influenced by the first orders, whereas Y in the Gaussian decay function normally needs several orders more to be determined with reasonable accuracy. Considerable uncertainties can be introduced by the scaling procedure and differences in the lamellar spacings. In order to answer some of the questions concerning the conformation of the lipid molecules in the membrane, the data at least must provide an accuracy of better than 1.5 A in z 0. The following procedures show how this accuracy can be achieved even for data of 15 to 20 A resolution. (1) The most direct method to determine the label positions is as follows. Two oriented specimens of bilayers are prepared, specimen N deuterated at segment N and specimen M undeuterated. Diffraction patterns of both slides are measured with a water layer of H20, D,O or a mixture of both. Thus, 4 sets of structure factors are derived :

In practice, the sets with index N and M belong to slightly different lamellar spacings. If one assumes that this arises from the different amounts of water between the bilayers without a change in the bilayer structure, the effect can be corrected by the sampling theorem for the so-called “minus fluid model” (Worthington et al., 1973):

P(h/d) = 2

it’

F (h’/D) sin(nDh/d

-

nh’)/(nDh/d

- nh’).

(4)

We found this to be a good approximation when the changes in the lamellar spacing are small (about 1 A). In order to make this correction, the not measurable zero order F(h’ = O/D) must be

680

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estimated. It can be calculated from the sum of the scattering lengths in the unit cell after subtraction of the scattering length density of the fluid layer. The difficulty is to scale this sum with respect to the observed st,ructure factors. If t’he number of water molecules per lipid is known from other methods, t’his can be achieved by using the structure factors of the water layer:

In the next step the structure factors that have been metasured from the different specimens M and N must be scaled with respect t’o each ot,her, because the lattice fact,ors for different preparations could be different,. The structure factors with the same index M or N but derived from measurements with a different content of D,O in the water layer are on bhe same intensity scale. The change from H,O to D,O did not alter the properties of the lamellar lattice, because it was made by changing only t,he water containing atmosphere from H,O to DzO for about 24 11. The scaling fact,or for different slides is then obtained from:

(6) Thus

which

2 sets of structure

enter

factors

into the following

for the label

least-squares

distribution

are established

fit for t,hc pararnet,ers

“r,, and v :

gh is a weighting factor by which it is possible to suppress the orders that are uncertain in amplitude or phase. The scaling factor S, can also be determined from the fit (eqn (8)). The factor AN couples the calculated structure factors F”,(h) with the observed F:(h) value. (2) The following procedure is more suitable if a series of structure factors for samples, each labelled in a different position, has been determined. In order to avoid the difficulties in the scaling procedure, all sets of structure factors are normalized in the same way, e.g. :

Then

the least-squares

fit should

minimize

the following T&Jh)

z gh T~ko@) -

expression

--}- M’:;.(h)

; IT,,,(h)

+ AF',(h)I

:

2

.

(10)

T&,(h) is the normalized structure factor of the undeuterated bilayer with a certain water layer indicated by the index W. The factor A couples the calculated structure factors of the label distribution with the observed T$,,<.(h) value. The import’ant advantage of this procedure is that this scaling factor A is a constant for all samples labelled at different positions. It is determined once from a very precise high-resolution set of data. by the extent of deuteration of t,he The parameter t, in F>(h) (eqn (3)) is determined segment in the structure and is known from the synt,hesis. Thus only x0 and v must be fitted. If there are small differences in the lamellar spacing, in a first approximation using equation (lo), x0 and v are obtained from the uncorrected st’ructure factors. In t8ha next step these values are used to estimate T,(h = 0) by:

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where the t, terms are the scattering lengths of the nuclei in the lipid molecules, t, is the scattering length of the label, and t, is the scattering length of the water that corresponds to the volume of the lipid molecule in the bilayer according to the “minus fluid model”. Inserting T,(h = 0) into equation (4), the corrected structure factors are calculated, and by means of equation (10) an improvement in the value of z,, was obtained. This procedure was very valuable where only the first strong structure factors had been measured. (ii) Procedures water

for

calculating

the mean label position

for powder

type samples

of 25

(w/u:)

content

For the powder samples the lattices of the individual samples were found to be of about the same quality, so that the scaling factor was only dependent on the dimensions of the scattering volume. Without correcting for differences in the spacings, a first approximation for z0 was calculated according to equation (8). Pg,,,(h = 0) was deduced from ‘i,w(h

= 0) = ~ATV(1 ti + tN - t,),

with AN as determined from the first fit (eqn (S)), thus giving the corrected structure factors via the sampling theorem of equation (4). In a second approximation by equation (S), improved values of z,, and v were calculated.

3. Results (a) The Lp phase To demonstrate the sensitivity of the method, Figure 2 shows the measured neutron counts for the oriented samples (mosaic spread -20”) at low water content (S%, w/w) and 20°C. Two plots for the deuterated chain segments C-4 and C-14 are added in order to see the quite strong changes in the reflections when the labelled position is moved from the head group to the chain end through the molecule. The nomenclature of the chain labels is such that the C=O carbons at the beginning of the chains are C-l and the terminal methyl group carbons are C-16. Table 2A summarizes the mean positions x0 for the different deuterated segments in the DPPC head group in the Lo, phase. The distances are measured from the centre of the bilayer. The first column shows the measured mean positions at low water content (6%, w/w) and 20°C (lamellar spacing 57.4 A) for samples with broad mosaic group spread of r] N 20”. The positions of the three segments of the phosphocholine are quite close together, which proves that this group on average is oriented nearly parallel to the bilayer surface. This is also illustrated by the third column of Table 2A, where the distances of these segments are measured in a space-filling model with the head group parallel and perpendicular to the bilayer surface. The model was positioned in the following way. At first, the C-5 and C-15 segments were fixed at those positions as determined from the very accurate higher resolution pattern of the chain samples (accompanying paper). In order to be in agreement with the X-ray data, a tilt angle of 17” was given to the chains (Tardieu et al., 1973). A comparison of these numbers with the observed distances in column 1 gives rather clear evidence for considering the average orientation of the phosphocholine group to be nearly parallel to the bilayer surface. Figure 3 shows a neutron diffraction profile (6 A resolution) of undeuterated DPPC with 6% (w/w) water and 20°C (full line) and the water distribution from the difference of the structure factors in D,O and Ha0 (dotted line). The vertical lines drawn on this Figure give a general view of all positions investigated at low water content in this and the accompanying paper. The DPPC profile shows a strong peak and a shoulder

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FIG. 2. Neutron counts ver8’sus Bragg-angle 0 showing the sensitivity of the method by the changes in the ratios of the reflections for the various deuterated segments. The number of hydrogens exchanged by deuterons in each lipid molecule is indicated in parentheses. Log plots of the first 4 orders are given as observed by a 0-28 scan of an oriented sample (with broad mosaic spread) containing 6% (w/w) Hz0 at 20°C (Lg phme). The counting time for 1 point was about 40 s.

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TABLE 1 Experimental structure factors of the lame&w rejections from highly oriented samples in H,O (mosaic spread -1”) A. Lo, phase:

h

DPPC

8 9 10

-26018 -19*2 150*4 -23s*5 57*3 8&l -45*3 --71*6 117*10 -126+17

6% (w/w)

HzO, 2O”C, d = 57.4 A CP -384110 111+3 10054 -244&3 7314 OS1 O&l -115jlO 1271-13 -106jES

C-2(1) -282*9 -75+3 253&4 -275&5 -21*2 82*3 -42&3 -146&12 159f16 -110+16

B. L, phase: 10% (w/w) H,O, d = 50.8 A DPPC CP -17!3*10 -85*3 159*6 --121&6 -17*3 71-3

8zt-2 -34+4

7O”C,

-306*18 -1752 160&7 -16457 1052 -19&2 21&3 -30+4

in the head group region. The position of the phosphocholine group is indicated by the vertical line of the labels Ca, C/3 and Gy, demonstrating that the shoulder in the profile is due to this group, whereas the inner peak must be assigned to the glycerol backbone region. Comparing the water distribution with this profile, we see that the water penetrates to the middle of the inner head group peak, i.e. to the glycerol backbone. The last column of Table 2A shows the results for the powder samples at 25% (w/w) water and 28°C (Lo, phase). The distances for the label positions are slightly decreased compared to those at low water content. This can be understood by the increase in tilt angle of the chains from 17” to 32”, as calculated from X-ray data (Tardieu et al., 1973; Janiak et al., 1976). The three head group labels Ca, Cfl and Cy are close together, which proves that the mean orientation of the dipole is nearly parallel to the bilayer plane as was seen at 6% (w/w) water content. So far we have discussed the results concerning the mean positions of the deuterated segments that determine the mean conformation of the head group region along the bilayer normal. With the highly oriented samples it was easy to obtain good resolution (10 orders) and thus be able to look at the local fluctuations along the bilayer normal. Table 1A contains the structure factors of three such samples : undeuterated DPPC, a sample deuterated in the C/l position and a sample deuterated in one of the chains in the C-2(1) position. In Figures 4 and 5 the profiles are calculated for one half of the elementary cell. Curve 1 shows the profile of the deuterated sample, curve 2 of the undeuterated. The difference profile (deuterated undeuterated) in each Figure (curve 3) gives a first idea of the position and width of the label, which are of course affected by the Fourier truncation errors. A fit according to equation (8) to the difference of the structure factors determines the parameters z0 and V. The scaling factor S, was also adjusted in these fits. The results are shown in Table 2A in the second column. The C-2(l)-segment was found at 18.4 A, measured from the hydrocarbon centre of the bilayer with a width of v = I a3A. If one assumes the molecule to be rigid in the gel phase, this width must be interpreted as the effect of the fluctuations of the whole molecule in the direction of

25.150.6 24.8kO.7 24.5kO.7 23.111.0

I

The following nornenclatr~re (:C-3: --r-O~~-Cr)2--CH-(:H,~~~-O--

CCX GC”-3

N 20”)

6%

(w/w)

H,O,

20°C,

1.3+0.6

l&4&0*6

xva* uaod for the label positiorls: CH, -; C-2( 1) : -1’ -0

:b6+0.6

I

23.5

-0 ~-CH,---(‘I),

d = 54.1 i%

; (~g: - -1’ m-C- -CD,

21.810.6 21.2+ 1.0 21.0* 1.0 17.411.5

H,O, 5O’C, powder 20 (4

24.4 25.3 24.0

~--N(CH,,),;

29.6 28.0 27.2

57.4 I% q, values determined from a space-filling model head parallel to head perpendicular surface surface

(ly : --.-~(CD,), CH CH,mpO~

2~5~4, (w/a)

3.0t0.6

25.3*0.6

rl :

of the results in the head group region of DPPC

Highly oriented (mosaic spread N I”) % (4 v (4

(w/w) H,O, 7O”C, d m: 50.8 -4 oriented (mosaic spread w 1”) J”o @) lJ (4

21.:%,0.6

10% highly

B. The L, phase

W CR CC-3 C-2( 1)

CY

Oriented (mosaic spread Gl(4

A. The Lg’ phase

Xummary

TABLE 2

to

CZ: --1’.

-(.b

CJ),-.-CH,--N(CH,),;

24.4hO.6 24.1,-tl.O 23.611.0 21.6+1*5

x0 (-4

25% (w/w) H,O, 28OC, d = 62.5 d powder

LECITHIN

HEAD

GROUP

I

c-15

STRUCTURE

I

I

I IO

I 20

IN

BILAYERS

685

I

c-14

I 0

I I ) 30 e/2

a

FIG. 3. General

view of,the mean label paper for samples in the Lg phase with 6% diffraction profile of undeuterated DPPC profile of same resolution, determined from The vertical lines indicate the various label

I

-40 0

positions determined in this and the accompanying (w/w) water measured at 20°C. ( ) Neutron of 6 A resolution; (. . . . . . . .) water distribution the difference of structure factors in D,O and H,O. positions.

I

I

IO

20

a FIG. 4. Scattering

I

1 d/2

0

length density profiles for one half of the elementary cell from highly oriented samples with 6% (w/w) Ha0 measured at 20°C (LB’ phase). Curve 1 (-.--.-e) from a sample deuterated at the C-2(1) p osition; curve 2 (-----) from an undeuterated sample; curve 3 (. . . . .) difference profile (curve 1 - curve 2) showing the observed profile of the label; curve 4 (-----) the profile of the label determined from the same number of orders as in curves 1 and 2, which were calculated from the parameters of the fit (eqn (E)), r. = 18.4 A, Y = 1.3 A; curve 5 ( ) shape of the label distribution as calculated by eqn (11).

686

G. BifLDT,

-40 0

H.

U. GALLY,

J. SEELIG

I IO

I 20

AND

G. ZACCAI

I

I I 7-T d,‘2 --

a Pm. 5. Scattering length density profiles for one half of the elementary cell under the same conditions as for Fig. 4. Curve 1 (-.-.-.) from a sample deuterated at the C/3 position. The meaning of the other curves is as for Fig. 4.

the bilayer normal. From these parameters ten structure factors are calculated by equation (3) and the profile (curve 4) is shown in Figure 4. A comparison of this curve with the difference profile (dotted lines) shows in real space a direct measure of the quality of the fit made in reciprocal space. The curves are in excellent agreement. To obtain a direct view of the label distribution, which is not affected by truncation errors, curve 5 was calculated by PC4 = z(exp

- r+)‘),

(11)

according to the parameters x0 and v obtained in the fit with A, and t, defined by equations (3) and (8). It is now interesting to compare these results with the C/? segment in Figure 5. The least-squares fit in reciprocal space gave a mean position of 25.3 A and a width v = 3-O A. It should first be noted here that curve 5 shows the distribution of only one label and does not give the overlapping distribution with the label of the neighbouring cell as in curves 3 and 4. At first glance the half-width at l/e height of v == 3-O A for the C/3 segment seems to suggest a high degree of fluctuations around the mean position in the bilayer plane. But if the connecting vector of the two deuterons of this segment is oriented perpendicular to the bilayer surface with a length of about 2.2 A, this would give, together with the mean fluctuation of the whole molecule along the bilayer normal of 1.3 A, as seen from the C-2(1) segment, a value of Y = 2.4 A. Thus without assuming much variation in the angle between the phosphocholine group and the plane of the bilayer, a width of v = 3-O A can be explained. (b) The L, phase The powder type samples with 25% (w / w ) water were measured at 50°C on the small-angle camera Dll (ILL). Only up to four reflections were observed. Figure 6

LECITHIN

I

HEAD

GROUP

nl

STRUCTURE

I

IN

I

BILAYERS

687

I

I

I

h-2

h=3 DppC

a*”.. . .“..I . .....” ... .... .*----.....“..

-*.. .. .^ ..... ... .

.-._I- D...... -

. . .. ....” . .... .. .“.----*

.” . ..^...... .._..

-1....“..... . . ...+*

.... . ..._

*....----.,

.... ...............................

I 0

. . ....

I 0.02

.....

.. .

......

.....

--._..

. .. . ... ..... .....” .. ..“...“..M.” ..--..

. .. . . . . .. .. .. -*-.**-

I..

... ..._...”

.

.....

*”

.. .

..........

I

I

0.04

......

. ...

.”

. .. .

I

..“.....

.

. .-*

cp

GC-3 (2)

c-4

(4)

.. ...-. c- 14 (4)

I

I

0.06

C t/l-‘\ Fm. 6. Neutron counts (linear scale) of the first 3 orders VW~US 8 (a = 2 sine/X) for powder-type samples containing 25% (w/w) H,O et 50°C (La phase) after averaging over Debye-Scherrer rings on the 2.dimensional detector of the Dll instrument. The counting time for each spectrum was about 30 min. The number of hydrogens exchanged by deuterons in each lipid molecule is indicated in parentheses.

688

G.

BiiLDT,

H.

U.

GALLY,

J.

SEELIG

ANI)

U.

ZACCAJ

shows by the first three orders that also under these conditions drastic variations in the relative height of the reflections are seen for samples deuterated at different positions. These reflections are already averaged over Debye-Scherrer rings, and this has enhanced the signal to noise ratio considerably. The same ratios in t,he reflection of C/l and Ca indicate that their projections on the Mayer normal are at the same position. As is clear from equation (3), these low-resolution spectra determine only the mean positions of the individual segments. In t,he last, column of Ta,ble 2B the distances are listed as measured from the centrt: of the hydrocarbon layer. A large decrease in the lamellar spacing and in the dismnces of hea.d group labels from t#he bilayer centre is found as produced by the chain melting, but again the three labelled positions in the head group are very close together, which proves that also in the L,z phase the mean orientation of the dipole is parallel to the bilayer plane. In order to elucidate the fluctuations of the dipole out of t,he plane of the bilayer. a highly oriented sample deuterated in the C/? position was measured at saturated humidity at 70°C w&h a water content of 100;, ( w/w). Table 1H gives eight orders for an undeuterated sample and a sample deuterated at the C/I position. Figure 7 shows

FIG. 7. Scattering length density profiles for one half of thr elementary samples with 10% (w/w) Hz0 measured at 70°C (La phesr). Curve 1 (---.-.-.) from a sample deuterated at the Cg position. curves is as for Fig. 4.

cell from Thr

meaning

highly

oriented

of the other

the corresponding profiles in one half of the elementary cell. The parameters x0 and v were calculated as x0 = 21-3 d and 3.6 A from a fit to the difference of the measured structure factors according to equation (8), where the scaling factor S, for the two samples with respect to each other was adjusted in the fit. The small increase in v from 3-O in the gel phase to v = 3.6 ip in the fluid phase may indicate a slight increase in the fluctuation of the dipole out of the plane of the bilayer.

4. Discussion The aim of this work was to establish a mean (time-averaged) high-resolution conformational picture of the lipid molecules and their segmental (time-averaged)

LECITHIN

HEAD

GROUP

STRUCTURE

IN

BILAYERS

G89

fluctuations along the bilayer normal in the two important phases, the gel phase L@, and the fluid phase L,. The conformations and fluctuations in space have been determined at low water content (6 and IO%, w/w) in both phases, whereas at high water content (25%, w/w) because of limited resolution, it was only possible to establish the mean conformation in these phases. Since our high water content measurements have more physiological significance, one point of our discussion will be how far our results at low water content concerning the degree of fluctuations in space are relevant to higher water content. (a) The Lgf phase The mean positions of the deuterated segments in Table 2A show the average orientation of the phosphocholine to be almost in the plane of the bilayer at low and high water content. Figure 3 shows that at 6% (w/w) water a considerable proportion of the water molecules penetrates between the lipid head groups. At 25% (w/w) water more molecules will go into this region and will reduce the electrostatic interactions between the positively charged choline group and the neighbouring phosphate which are close together when the dipoles are packed in the plane of the bilayer. Thus the area per lipid increases from 42.7 A2 at 6% (w/w) water to 48.6 8” at 25% (w/w) (Tardieu et al., 1973). Consequently, the hydrocarbon chains increase in tilt angle from 17” to 32”, in order to compensate for the increase in area and still stay in van der Waals’ contact with each other. This is seen in the slightly decreased values of the mean label position at 25% (w/w) water. Our results at 6% (w/w) water give no clear answer as to how much the dipole can fluctuate around its mean orientation, since (as pointed out in the section above) the width measured for the Cg label is the sum of the projections of the C-D, bonds on the bilayer normal and the fluctuation of the whole molecule in this direction. A possible further increase in the width of the Cfi label at 25% (w/w) water could not bedetected because of limited resolution under these conditions. But in the L, phase, where the area per molecule is further increased, we found only a slight increase in the width of the C/3 label to Y = 3.6 A. From this result one may interpolate that the width of the C@label at 25% (w/w) water in the Lp, phase is not much increased compared to the result of 6% (w/w water. (b) The L, phase As is seen from Table 2B, the mean positions of the deuterated segments at low and high water content agree with each other and show also in this phase the orientation of the dipole parallel to the bilayer surface. From our results at 25% (w/w) water in both phases Lb, and L, we can now calculate the area per lipid molecule at 25% (w/w) water in the fluid phase. Taking into account the volume increase of 3.9% (Nagle, 1973) going from one phase to the other at maximum hydration and the reported values of the area per head group in the Lo, phase at 25% (w/w) water (48 A2, Chapman et al., 1967; and 48.6 AZ, Tardieu et al., 1973) we have determined an area per head group in the L, phase of 57 A”. For instance, taking the ($3 label the area per head group is simply obtained by (24.1 x48.3 + 24.1 x48.3 x0-039)/ 21.2 = 57.1 A2 (compare Table 2). This value is consistent with that of 58 AZ given by Chapman et al. (1967) for DPPC and does not differ much from the value of 60 A2 estimated by Engelman (1971) for palmitate-enriched membranes of Acholes&wuz laidlawii.

690

G. BtiLDT,

H.

U.

GALLY,

J. SEELIG

AND

G. ZACCAI

5. Conclusions In the gel phase Lb, and in the liquid crystalline phase La the label positions measured in the phosphocholine group demonstrate clearly that the mean orientation of this dipole is nearly parallel to the bilayer surface. Recently this result has been confirmed by high frequency dielectric measurements (Shepherd & Biildt, 1978) on multilayers of DPPC at 25’3, (w / w ) water content placed between condenser plates. The smaller the sample thickness, the better was the orientation of the individual multilayer stacks parallel to the plates (as measured by neutron diffraction) and the permittivity values were reduced correspondingly. Thus, considering the evidence for the head group orientation of the phosphoethanolamine dipole (as mentioned in the Introduction) in conjunction with the present work, it might appear to be a, general aspect of the zwitterionic head groups to stay, on average, nearly parallel to the bilayer area if no ions are present. A series of investigations by Yeagle et al. (1975,1976,1977) studying the 31P {lH} nuclear Overhauser effect on bilayers of egg-yolk lecithin, sphingomyelin and phosphaticlylethanolamine with and without cholesterol strongly support these ideas. Experiments with divalent and trivalent cations in the fluid layers are now in progress to see whether any conformational change results from their binding effects. We thank Dr V. Luzzati for his valuable comments during this study, Dr H. Stuhrmann and Dr K. Ibel for helping us to start this work, Mr S. Wilson and Dr K. Gobrecht for their assistance with the neutron experiments and Drs D. Schneider and H. Berger for their aid in the X-ray measurements. We are also indebted to Dr J. C. W. Shepherd for many helpful discussions. This work was supported by a European Molecular Biology Organisation travel grant (to G.B.) and by grant 3.008.76 from the Swiss National Science Foundation (to J.S.).

REFERENCES Barsukov, L. I., Shapiro, Y. E., Viktorov, A. V., Volkova, V. l., Bystrov, V. F. bi Bergelson, L. C. (1976). Biorg. Khim. 2, 1404-1416. Biildt, G., Seelig, A., Seelig, J. & Zaccai, G. (1976). Proceedings of the Corbference on Neutron #c&e&g, Gatlinburg, vol. 1, p. 109, National Technical Information Service, Springfield. Biildt, G., Gally, H. U., Seelig, A., Seelig, J. & Zaccai, G. (1978). Nature (London), 271, 182-184. Cain, J., Santillan, G. & Blasie, J. K. (1972). Membrane Research, Academic Press, London. Chapman, D., Williams, R. M. & Ladbrooke, B. D. (1967). Chem. Phys. Lipids, 1,445-475. Engelman, D. M. (1971). J. Mol. Biol. 58, 153-165. Gally, H. U., Niederberger, W. & Seelig, J. (1975). Biochemislry, 14, 3647-3652. Hauser, H., Phillips, M. C., Levine, B. A. & Williams, R. J. P. (1976). Nature (London), 261, 390-394. Hitchoock, P. B., Mmon, R., Thomas, K. M. & Shipley, G. G. (1974). Proc. Nat. Acad. Sci., U.S.A. 71, 303&3040. Ibel, K. (1976). J. Appl. Crystallogr. 9, 296-309. International Tables for X-ray Crystallography (1967) p. 291, The Kynoch Press, Birmingham. Janiak, M. J., Small, D. M. & Shipley, G. G. (1976). Biochemistry, 15, 4575-4580. Lesslauer, W., Cain, J. E. & Blasie, J. K. (1972). proc. Nat. Acad. Sci., U.S.A. 69, 1499-1503. Levine, Y. K. (1970). Ph.D. thesis, University of London. Levine, Y. K. & Wilkins, M. H. F. (1971). Nature New Biol. 230, 69-72.

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Luzzati, V. (1968). Biological Membranes (Chapman, D., ed.), Academic Press, London. Nagle, J. F. (1973). Proc. Nat. Ad. Sk, U.S.A. 70, 3443-3444. Pinto da Silva, P. (1971). J. Micro.sc. 12, 185-192. Powers, L. & Persham, P. S. (1977). Biophys. J. 20, 137-152. Ranck, J. L., Mateu, L., Sadler, D. M., Tardieu, A., Gulik-Kryzwicki, T. & Luzzati, V. (1974). J. Mol. Biol. 85, 249-277. Seelig, A. & Seelig, J. (1975). Biochim. Biophys. Acta, 406, l-5. Seelig, J. & Gally, H. U. (1976). Biochemktry, 15, 5199-5204. Seelig, J., Gally, H. U. & Wohlgemuth, R. (1977). Biochim. Biophye. Acta, 467, 109-119. Shepherd, J. C. W. & Bfildt, G. (1978). B&him. Biophys. Acta, 514, 83-94. Tardieu, A. (1972). Ph.D. thesis, University of Paris. Tardieu, A., Luzzati, V. & Reman, F. C. (1973). J. Mol. Biol. 75, 711-733. Torbet, J. & Wilkins, M. H. F. (1976). J. Thewet. Biol. 62, 447-458. Worcester, D. L. (1976). Biological Membranes (Chapman, D. & Wallach, D. F. H., eds), Academic Press, London. Worcester, D. L. & Franks, N. P. (1976). J. Mol. Biol. 100, 359-378. Worthington, C. R., King, G. I. & McIntosh, T. J. (1973). Biophye. J. 13, 480. Yeagle, P. L., Hutton, W. C., Huang, C. & Martin, R. B. (1975). Proc. Nat. Acud. Sci., U.S.A. 72, 3477-3481. Yeagle, P. L., Hutton, W. C., Huang, C. & Martin, R. B. (1976). Biochemistry, 15, 2121-2124. Yeagle, P. L., Hutton, W. C., Huang, C. & Martin, R. B. (1977). Biochemistry, 16, 4344-4349. Zaccai, G., Blasie, J. K. & Schoenborn, B. P. (1975). Proc. Nat. Acad. Sci., U.S.A. 72, 376-380.