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* National Synchrotron Light Source, Brookhaven National Laboratory, Upton, New York 11973; and Department of Physics & Astronomy, Rice University, Houston, Texas, 77251, USA
Correspondence: Address reprint request to Dr. Huey W. Huang, Department of Physics & Astronomy, Rice University, Houston, TX 77251-1892. Tel.: 713-348-4899; Fax: 713-348-4150; E-mail: hwhuang{at}rice.edu.
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTRESULTS AND ANALYSISDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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). Membrane fusion involves the merger of two phospholipid bilayers. The widely accepted model for membrane fusion is built upon the hypothesis that there is an intermediate state in which the two contacting monolayers become continuous via an hourglass structure called a stalk (Gingell and Ginsberg, 1978; Markin et al., 1984; Siegel, 1993; 1999; Lentz et al., 2000; Brunger, 2001; Kuzmin, et al., 2001; Kozlovsky and Kozlov, 2002; Markin and Albanesi, 2002; Lentz et al., 2002). This structure has not been observed by experiment so far. However, many efforts have been made to estimate the free energy for such a state (Markin et al., 1984; Siegel, 1993, 1999; Kuzmin, et al., 2001; Kozlovsky and Kozlov, 2002; Markin and Albanesi, 2002; Lentz et al., 2002). Theoretical estimates naturally depend on many assumed parameters, such as the size and shape of the stalk, the bending rigidity and spontaneous curvature of lipid monolayer, and whether the lipid is of single or multiple components. The discovery of the stalk structure in a stable phase opens a way to experimentally measure some of these parameters. The experiment can also be expanded to include proteins or peptides in this new phase of lipid to change the parameters of the stalk and to estimate the energy of the stalk formation.
To understand this new phase of lipid, it is useful to know how it was discovered. For the past several years, we have been studying antimicrobial peptides that are capable of forming transmembrane pores in lipid bilayers. To observe the transmembrane pores, we prepared lipid samples containing peptides as parallel layers intercalated with water. With water replaced by D2O, neutron scattering with the scattering vector oriented in the plane of bilayers detected the transmembrane D2O columns. The data were consistent with in-plane scattering by randomly distributed (or freely diffusing) transmembrane pores with mutual hard-core exclusion (He et al., 1995, 1996; Ludtke et al., 1996). A subsequent out-of-plane diffraction study showed that the in-plane positions of the pores in lipid bilayers were uncorrelated between adjacent bilayers (Yang et al., 1998, 1999). This was the result from fully hydrated multilayers. However, when the hydration level was reduced to less than full, the out-of-plane diffraction profile of the in-plane scattering peak indicated that the in-plane positions of the pores became correlated between adjacent bilayers (Yang et al., 1998, 1999). When we further manipulated the temperature and hydration of the multilayer samples, we succeeded in crystallizing the transmembrane pores in the multilayers (Yang et al., 2000). In one of the crystalline phases, the pores were distributed on a regular hexagonal lattice in each bilayer, and the layers were stacked in the ABCABC... sequence (Kittel, 1971). We called this the hexagonal ABC stacking, although unlike close-packed spheres our lattice was not cubic, because the stacking distance was not geometrically related to the hexagonal size. The symmetry of the diffraction patterns was rhombohedral, space group R
. One of our recent projects was to make use of the rhombohedral crystals to construct the electron density profile of the transmembrane pores. It was during such an experiment, we discovered that, to our surprise, a pure lipid also exhibited a rhombohedral phase.
The lipid was DPhPC, a modification of the well-known dipalmitoyl phosphatidylcholine (DPPC) with four methyl groups attached to each acyl chain at the carbon atom 3, 7, 11, and 15. DPhPC is characterized by its bulky, disordered chains. Consequently the lipid has a small ratio of cross sections between the headgroup and the chains (Ahg/Ach). If DPPC monolayers have a zero spontaneous curvature, DPhPC has a negative one. In our previous studies (see Huang, 2000), DPhPC was an important lipid bilayer that was used to observe the transition of membrane-active peptides from a state of embedding in the headgroup region to a state of forming transmembrane pores, again due to its small Ahg/Ach.
DPhPC multilayers were prepared as a thin film on a silicon nitride substrate. X-ray diffraction from DPhPC multilayers was recorded as a function of temperature and the degree of hydration of the sample. The latter was controlled by the ambient relative humidity in the sample chamber (Yang et al., 2000). Three phases were identified by their diffraction patterns within the range of 20°C to 30°C and relative humidity 50%–100%: a lamellar phase, a rhombohedral (hexagonal ABC stacking) phase, and a two-dimensional hexagonal phase. The lamellar phase is the familiar fluid phase of lipid bilayers that was discussed in Hung et al (2000). The two-dimensional hexagonal phase is the inverted hexagonal (HII) phase (Gruner, 1989; Turner and Gruner, 1992). In this paper we will concentrate on the analysis of the rhombohedral phase. We will determine the phases of the diffraction patterns by extending a well-known one-dimensional swelling method to three dimensions and making use of Luzzati's pattern recognition method (Mariani et al., 1988). We will then discuss the electron density distribution in the unit cell that contains a stalk structure.
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EXPERIMENT |
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0.5 mg/cm2. The organic solvent was a trifluoroethanol (TFE)-chloroform mixture. The ratio of trifluoroethanol to chloroform was varied to give optimal membrane alignment on the substrate (Ludtke et al., 1995). The organic solvent was removed in vacuum or evaporated in open air. The deposit was then hydrated with saturated water vapor at room temperature. Under this condition, the lipid spontaneously formed a stack of hydrated bilayers parallel to the substrate (Ludtke et al., 1995; He et al., 1996).
To allow for transmission diffraction, we used silicon nitrite windows (Silson Ltd., UK) as the substrate. The thickness of the window was 100 nm, which allowed for 99% transmission of 8 keV x rays at 30° incident angle. The dimension of the window was 1.0 mm (vertical) x 3.0 mm (horizontal), and the footprint of the incident x ray in the transmission geometry was 0.5 mm (vertical) x 1.0 mm (horizontal). The window was supported by a silicon frame.
Fig. 1 shows the experimental setup that was used to measure the diffraction patterns from aligned multilayer samples, at the beamline X21 of the National Synchrotron Light Source, Brookhaven National Laboratory (Upton, NY). The x-ray beam was collimated by two sets of slits before a sample chamber, resulting in a beam size of 0.5 mm x 0.5 mm at the sample. A helium beam path between the sample chamber and a MarCCD detector (MAR USA, Evanston, IL) was used to reduce the scattering by air. A beam stop and an attenuator were glued to a kapton film covering the CCD detector. The attenuator (a stack of aluminum foils) reduced the intensity of the first few orders of reflections off the crystal planes parallel to the substrate to avoid saturating the pixels on the CCD detector.
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This compact temperature-humidity chamber, only 7 cm on each side, was mounted on a small x-z rotary stage. The position of the sample chamber was carefully aligned to ensure that the beam and the sample surface intersected at the rotational axis of the rotary stage. The vertical position of the sample was separately adjustable so that different parts of the sample could be exposed to x rays. This was used to avoid radiation damage to the sample.
Data collection
Aligned lipid multilayers are ordered in the direction perpendicular to the substrate surface but randomly oriented in the plane parallel. Thus the crystal domains in such a sample have a crystal plane parallel to the substrate, whereas the in-plane orientations of the crystal domains are isotropically distributed. Consequently, in the reciprocal space, the lattice points are distributed in a series of Bragg rings parallel to the substrate and centered around the qz axis. (q is the momentum transfer of x-ray scattering; z is normal to the plane of substrate, r or (x, y) are in-plane coordinates.) Each Bragg ring will be registered as a diffraction peak on the qz-qr plane, where qr is the in-plane radius of the Bragg ring. We used a combination of three CCD images to record a complete diffraction pattern.
Supramolecular lipid structures have a length scale of 50–300 Å, and their diffraction peaks tend to be limited within <1 Å-1. With x rays of 8 keV (
= 1.55 Å) incident at a grazing angle (almost parallel to the substrate), the Ewald sphere intercepted the majority of the Bragg rings (recorded as CCD image 1 in Fig. 2), but with two exceptions. First, the Bragg rings with qz = 0 were inaccessible by reflection, because in practice reflection always imparted a finite qz. These qz = 0 Bragg peaks were recorded by transmission diffraction at an incident angle -30° (CCD image 3 in Fig. 2). Second, at a fixed incident angle, the Ewald sphere intersected the qz axis twice: one at the origin of reciprocal space and another at the specular angle. Therefore, the diffraction pattern by a grazing incident angle did not include the complete series of the qz peaks (peaks with qr = 0). These qr = 0 peaks were measured by a 
-scan, in which the sample was rotated continuously to vary the incident angle, so that the intercept of the Ewald sphere with the qz axis moved along this axis and recorded as CCD image 2 (Fig. 2). These three CCD images were combined to give a complete diffraction pattern.
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Data reduction
The data collected above were used to obtain the amplitude of the form factor for the unit cell, F, at the positions of the Bragg peaks. In addition to the correction factors, we paid special attention to the (relative) normalization of the three CCD images.
The integrated intensity of a diffraction peak E measured in the reflection or the transmission geometry is related to the square of the form factor |F|2 by
 | (1) |
: 
. The Lorentz factor is given by
 | (2) |
is the x-ray incident angle with the substrate surface (the x-y plane), 
is the angle between the scattering vector q and the x-y plane, and 
is the angle between the in-plane (x-y) projection of q and the in-plane projection of the incident vector. The Lorentz factor is well known in x-ray crystallography (Kasper and Lonsdale, 1967), where a single crystal is rotated so that multiple diffraction peaks can be recorded. In the case of aligned lipid multilayers, the isotropic distribution of the in-plane orientation of the crystalline domains is equivalent to an in-plane rotation of a single crystal. The absorption factor is determined by the path length of the diffracted x-ray through the sample (Yang et al., 1998). For transmission diffraction, we have
 | (3) |
 | (4) |
is the angle between the substrate normal and the incident beam (0 < < 
/2). ß is the angle between the substrate normal and the scattered beam. µ is the linear absorption coefficient of the sample, and a is the sample thickness. The amount of sample that was bathed in the x ray and thus contributed to diffraction depended on the incident angle. This is taken into account by the factor
 | (5) |
. 
T is the exposure time.
For -scan, the integrated intensity of a diffraction peak is given by
 | (6) |
, and 
. The absorption factor is given by 
. The geometric factor Cgeo is the same as Eq. 5. The last factor 2/
is the normalization factor relative to the exposure time for the reflection scan, with being the angular velocity of the -scan.
One can also view the -scan as a special case of the reflection diffraction, with 
, 
, 
and the crystal rotation about the qx axis instead of the qz axis. Then all the correction factors are the same between Eq. 1 and Eq. 6.
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RESULTS AND ANALYSIS |
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. The sample was then housed in a chamber of controlled temperature and relative humidity (RH). Two other phases were identified in the range of temperature and RH shown in Fig. 3. Fig. 3 B shows the diffraction pattern of a rhombohedral lattice. Fig. 3 C shows a two-dimensional hexagonal lattice. The latter is the well-known inverted hexagonal (HII) phase. In the past, the HII phase was typically recorded by the method of powder diffraction, which produced a series of diffraction circles (Gruner, 1989; Turner and Gruner, 1992). Here the diffraction pattern shows a hexagonal lattice normal to the substrate, indicating that the HII tubes were all oriented parallel to the substrate surface. The dimensions of the unit cell in the rhombohedral phase were 65 Å (one side of the hexagon) and 44 Å (stack spacing). The unit cell in the two-dimensional hexagonal phase was 41 Å for each side of the hexagon. These dimensions varied somewhat with the degree of hydration.
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70% to 80%RH. This hydration dependence will be utilized for phase determination.
Swelling method in three dimensions
Phase determination for the diffraction of the L phase (Fig. 3 A) is often accomplished by the swelling method (Bragg and Perutz, 1952; Perutz, 1954; Blaurock, 1971). This method takes advantage of the hydration changes of the layer system that alter the stacking distance while the bilayer structure remains approximately the same (Torbet and Wilkins, 1976). If the bilayer is symmetric, the phases are real, either positive or negative. Then the form factor of the bilayer is a continuous, real function of qz that changes sign only when it goes through zero. The variation of the stacking distance covers a short range of qz at every peak position. Thus a series of diffraction patterns over a range of hydration often provide sufficient information to determine the phases as in the case of DPhPC (Fig. 4). Once the phases were determined, the diffraction patterns of the L phase were used to construct the trans-bilayer electron density profiles. These profiles of the L phase will be compared with the trans-bilayer profiles in the rhombohedral phase.
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 | (7) |
rather than the electron density itself. We have also assumed that the unit cell is centro-symmetric. The purpose of the diffraction experiment is to measure the magnitudes of the diffraction amplitudes AH, K, L and then determine their phases, so that the electron density contrast can be constructed:
 | (8) |
 | (9) |
We now separate the vectors into their in-plane and out-of-plane components, i.e., 
, 
, 
and carry out the spatial integration over z from -d/2 to d/2 (d is the stacking distance) to obtain
 | (10) |
 | (11) |
When 
, we have 
and therefore, we obtain
 | (12) |
). To normalize the diffraction amplitudes between different sets of data, we note that
 | (13) |
proportional to its stacking distance d.
Phase determination and electron density distribution in the unit cell
As mentioned above, every diffraction peak with 
is distributed on a Bragg ring around the z axis. Peaks with the same 
and qz overlapped in the same ring, so they were assumed to have the same diffraction amplitude. Fig. 5 shows the reciprocal lattice and the diffraction peaks measured on the qz-qr plane. The reciprocal lattice of rhombohedral symmetry is composed of a stack of hexagonal layers stacked up in the ABCABC... sequence. Fig. 5 shows A layers as green, B as blue and C as red. In the qz-qr plane, we showed the four columns of the observed diffraction peaks. The spacing for each color in each column is 2/d. The occupied positions on qz depend on the values of (H, K). In column a and c, the green peaks are at nqzo (n = 0, ±1, ±2,...; qzo = 2/d), and in column b and d, blue peaks occupy (n + 1/3)qzo, and red peaks occupy (n - 1/3)qzo. On the detector (Figs. 2 and 3), the blue peaks and the red peaks were mixed because domains of both +z and -z orientations were equally populated in the sample. The mixed blue and red peaks were, however, the same under the assumption of centro-symmetry. The multiplicities for the diffraction peaks at qr/qro = 0,1,
,2 are 1, 3, 6, and 3, respectively, where qro is the magnitude of the lowest in-plane component qr from the peaks on the first column off the qz axis (column b in Fig. 5).
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phase shown in Fig. 4. Nevertheless, for the majority of the lattice points we could choose the phases so that the qz-dependence of the data was consistent with that of the constructed F(qH,K,qz). The phases shown in Fig. 6 are the best choices according to the swelling method. The choices are also consistent with the minimum wavelength principle. This principle was introduced by Bragg and Perutz (Bragg and Perutz, 1952; Perutz, 1954) as an aid to the swelling method. They showed that whatever the arrangement of the scattering points in the structure, the maxima and minima of the form factor succeed each other with a minimal wave number dictated by the overall dimensions of the molecule in a corresponding direction. In our case, the minimal wave number for the qz dependence should correspond to the thickness of the layers of the rhombohedral structure, which is about that of a bilayer as compared with the L phase. Independently, the phases for the lamellar structure shown in Fig. 4 apparently satisfy the minimum wavelength principle.
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and references therein). This method recognizes the fact that the basic structural element of lipid in water is the lipid monolayer. A change of the structure of a lipid-water system is a rearrangement of the lipid monolayers (Tardieu et al., 1973). Therefore, if a lipid-water system has more than one structure, the different structures should have comparable electron density contrast histograms H(). For example, the structure of DPhPC in the L phase was known (Hung et al., 2000) and was reconstructed here, so its H() could be used as a reference. For the rhombohedral phase we computed H() for each of the eight possible choices of relative phases and compared the results with that of the L phase. For practical purposes, the method was carried out by computing the fourth moment 
()4
following the procedure of Mariani et al., 1988:
 | (14) |
Among the eight possible choices (Fig. 7), two with ()4 = 2.610 and 2.640 are closest to the value of the L phase which has ()4 = 1.750. The electron density distributions of these two choices (3 and 7 in Fig. 7) are very similar, both show a stalk structure. However, upon close inspection, we found that choice 3 produced somewhat uneven electron densities in the lipid headgroup region along the monolayers. The most reasonable choice of phases (7 of Fig. 7) gave the electron density distribution shown in Fig. 8. Other details were shown in Yang and Huang (2002).
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DISCUSSION |
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phase. On the contrary, examples of irrational results can be seen in the choice 0, 1, 2, 4, 5, and 6 in Fig. 7 where the electron density distributions are not consistent with the expected properties of lipid in water as described.
The electron density distribution shows a structure similar to the theoretically expected stalk structure (Gingell and Ginsberg, 1978; Markin et al., 1984; Siegel, 1993), namely, the two apposed monolayers apparently merged and bent into an hourglass shape. This structure has been speculated to be an intermediate state during membrane fusion. It has also been speculated that stalks are the intermediate state between the lamellar phase and the inverted hexagonal phase (Siegel, 1999). However, the stalk (rhombohedral) phase was not observed in previous studies of lamellar to inverted hexagonal transitions (Gawrisch et al, 1992). Apparently a direct transition between any two of the lamellar, stalk, and HII phases is possible.
The discovery of the putative membrane fusion intermediate state in a stable phase is significant. It not only lends a strong support to the stalk hypothesis for membrane fusion, but also opens a new experimental approach to the fusion problem. Firstly we should now try to enhance the resolution of the diffraction to improve the measurement of the structural parameters that characterize a stalk structure. This includes a possible refinement of the experimental setup and possible modifications of the contrast between the headgroup and the hydrocarbon chains by multiwavelength diffraction or other means. Secondly we can vary the lipid composition and include fusogenic peptides or other chemical variables in the system to observe their effects on the stalk structure. In the last 10 years, significant progress has been made in the elucidation of the structures of membrane fusion proteins (White, 1992; Brunger, 2001). However, how proteins induce membrane fusion remains speculative, even in the best-studied case of viral fusion proteins (White, 1992; Doms, 1993; Stegmann, 1994; Chernomordik et al., 1997). Direct studies of the intermediate state will help clarifying the free energy pathway for the lipid conformation changes during fusion. This information will in turn provide guidance to identify the functions of the protein structures.
In our previous investigations, we have shown that lipids bilayers containing peptides initially prepared in the lamellar phase often developed into a rhombohedral phase by temperature and hydration manipulation (Yang et al., 2000). The technique described here allows the peptide-induced membrane structures to be studied by x-ray and neutron diffraction. The swelling method should be helpful for the phase problem in such experiments.
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ACKNOWLEDGEMENTS |
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This work was supported by NIH Grants GM55203 and RR14812, and by the Robert A. Welch Foundation. Research carried out in part at the beamline X21 of the National Synchrotron Light Source, Brookhaven National Laboratory, which is supported by the U.S. Department of Energy, Division of Materials Sciences and Division of Chemical Sciences, under Contract No. DE-AC02-98CH10886.
Submitted on August 22, 2002; accepted for publication November 11, 2002.
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indicate changeover regions where two phases were detected. The range of the changeover region depended on the direction of hydration change and the speed of the change. The x-ray diffraction patterns were recorded by reflection on an area detector (see Data collection and 
of 
