Langmuir 1995,11, 3218-3222
3218
Extending the Angular Range of Neutron Reflectivity Measurements from Planar Lipid Bilayers: Application to a Model Biological Membrane S. Krueger,*?tJ. F. Anher,?$$S. K. Satija,? C. F. Majkrzak,?D. Gurley,§ and M. Colombinis Reactor Radiation Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, and Department of Zoology, University of Maryland, College Park, Maryland 20742 Received March 21, 1994. I n Final Form: May 15, 1995@ A novel experimental setup has been used to measure the specularly-reflected neutron intensity from a model biological membrane containing components from the outer membrane of Neurospora crassa mitochondria. The specular reflectivity from a single bilayer membrane, formed by the fusion of vesicles onto a phospholipid monolayer supported on a flat substrate, was measured in both DzO and HzO solvents. were measured for wavevector transfers out In D20 solvent, reflected neutron intensities down to to 0.25A-l. A symmetric model for the neutron scattering length density profile perpendicular to the bilayer surface was constructed based on fits to the DzO data. The overall bilayer thickness was found to be 43 f 2 A. The individual lipid head group and hydrocarbon tail layer thicknesses were 7.5 f 1.4 and 28.0 f 2.8 A, respectively. "he fitted results are consistent with the H2O data. The integrity of the model membrane bilayer was confirmed by comparing its measured reflectivity to that obtained from a single lipid bilayer consisting of soybean phospholipids (asolectin)which was deposited on a flat substrate by the Langmuir-Blodgett technique.
Introduction Model membrane systems are important for the study of the physical properties of biological membranes as well as structural changes in response to surface interactions or stimuli such as electrical potentials. Single phospholipid bilayers supported on a flat substrate surface offer advantages over vesicle suspensions for this purpose since they are unilamellar and only a single, well-defined surface is exposed to the aqueous medium. The neutron reflectometry technique, a n important tool in the study of surfaces and interfaces,l provides an excellent mechanism for probing the structure ofsuch supported planar systems. Since neutron interactions with different isotopes of the same element can vary greatly, most notably in the case of hydrogen and deuterium, neutrons can provide a distinct advantage over X-rays when deuterium can be substituted for hydrogen in the bilayer or the solvent. In addition, the sensitivity of neutrons to the locations of hydrogen atoms makes them excellent probes of the interface between the bilayer surface and the solvent. Specular reflection of neutrons has been used to probe the structure of dimyristoylphosphatidlycholine(DMPC) bilayers adsorbed onto planar substrates2and to measure changes in the thickness of single dipalmitoylphosphatidlycholine (DPPC) bilayers on quartz surfaces aRer incorporation of ch~lesterol.~ In addition, several experiments have focused on the study of streptavidin binding to biotinylated lipid monolayers, which are either formed a t the aidwater interface4r5or adsorbed onto solid sub-
approximately 50 A thickness which is uniform over a macroscopic area, i.e., on the order of 80 cm2. If multiple layers and/or nonuniform layers exist, much ofthe system cannot interact freelywith the solvent. Therefore, changes in the membrane structure a t the surface do not occur in all layers, making them difficult to detect. Planar supported bilayers can be formed by the direct fusion technique, whereby vesicles are fused directly to planar quartz substrate^,^^^ and by the monolayer-fusion technique, whereby vesicles are fused to phospholipid monolayers supported on quartz substrate^.^ Fluorescence microscopy studiedohave found that proteins incorporated into model membranes using the direct fusion technique are not laterally mobile.' The monolayer-fusiontechnique may better preserve some protein structuresgsince vesicles fuse to hydrophobic tail groups of the supported monolayer rather than to the hydrophilic quartz substrate, making it less likely that membrane proteins would irreversibly interact with the substrate surface. In this work, the monolayer-fusion technique has been used to form a single bilayer model biological membrane containing components from the outer mitochondrial membrane (OMM)of Neurospora crassa mitochondria on a planar substrate. A novel experimental setup has been used to measure the specularly reflected neutrons down to in reflected intensity, thereby extending the angular range and the spatial resolution. This is especially important since the model outer mitochondrial membrane (MOMM) is formed from vesicles purified from mitochon-
The success of a reflectivity measurement depends partly upon the ability to form a single lipid bilayer of
(4) LOsche, M.; Piepenstock, M.; Diederich, A.; Griinewald, T.; Kjaer, K.; Vaknin, D. Biophys. J. 1993,65,2160. (5) Vaknin, D.; Als-Nielsen, J.;Piepenstock, M.; LOsche, M. Biophys. J. 1991,60,1545. (6) Schmidt, A.; Spinke, J.;Bayerl, T. M.; Knoll, W. Biophys. J. 1992, 63. .- , 118.5. - - - -. (7) Brian,A. A.; McConnell, H. M. Proc. Natl. Acad. Sci. U.S.A. 1984, 81,6159. (8)McConnell, H.M.; Watts, T. H.; Weis, R. M.; Brian, A. A. Bzochim. Biophys. Acta 1986,864, 95. (9) Kalb, E.; Frey, S.; Tamm, L. K. Biochim. Biophys. Acta 1992, 1103, 307. (10)Thompson, N.L; Palmer, A. G., III; Wright, L. L.; Scarborough, P. E. Comm. Mol. Cell. Biophys. 1988,5 (2),109.
National Institute of Standards and Technology. address: MURR, Research Park, S. Providence, Columbia, MO 65211. 5 University of Maryland. Abstract published in Advance A C S Abstracts, J u l y 15,1995. (1)Majkrzak, C. F.; Felcher, G. P. Mater. Res. SOC.BUZZ. 1990, 15, 65. (2)Johnson, S.J.;Bayerl, T. M.; McDermott, D. C.; Adam, G. W.; Rennie, A. R.; Thomas, R. K.; Sackmann, E. Biophys. J.1991,59,289. (3) Reinl, H.; Brumm, T.; Bayerl, T. M. Biophys. J. 1992,61, 1025. +
* Present @
0743-746319512411-3218$09.00/0 0 1995 American Chemical Society
Langmuir, Vol. 21, No. 8, 1995 3219
Neutron Reflectivity Measurements from Bilayers
dria and it was not possible- to selectively deuterate hydrogen atoms in the lipid or protein moieties in order to enhance the contrast of the different membrane regions. The integrity of the MOMM bilayer formed by the monolayer-hsion method was tested by comparing the measured reflectivity to that obtained from a bilayer formed from soybean phospholipids (asolectin) using the Langmuir-Blodgett technique.ll Measurements were made in both D20 and H20 solvents in order to confirm the consistency of the results. The sensitivity of the measurements and their implications for further experiments are discussed.
p-type S l l i c o n W a f e r n- type SI 1 icon W a f e r Llpid B l l a y e r on Surface
DzO Solution
Experimental Section Soybean phospholipids (asolectin)were purified according to the method ofKagawa and Racker.12 Phospholipid bilayers were formed by first coating one single-crystal p-type silicon plate with a monolayer of phospholipid using the Langmuir-Blodgett technique." Single crystal silicon is much more transparent to neutrons than the solution so it serves as the incident medium. Phospholipid (1%phospholipid, 0.2%cholesterol) dissolved in hexane was added dropwise to the surface of water in a large tank, forming a monolayer at the water surface as the hexane evaporated. Lateral pressure was maintained with an excess of octanol on the water surface which was kept separated from the phospholipid by a loop of suture. The p-type silicon plate was raised through the interface at a rate of approximately 2.5 c m l min, transferring a phospholipid monolayer to the silicon plate with the phosphate head groups interacting with the hydrophilic silicon oxide layer at the surface of the plate. A second n-type silicon plate was placed in a flat bowl containing D2O salt solution (0.1M KC1, 0.5 mM MES, pH 5.8) and the same phospholipid dissolved in hexane was added dropwise to the water surface to form a monolayer. To form the phospholipid bilayer, the p-type silicon plate containing the adsorbed monolayer was lowered, with its face parallel to the water surface, through the surface until it rested upon a Teflon gasket (25-50 pm thick) that had been secured on the outer edge of the n-type silicon plate with a thin layer ofhigh vacuum siliconegrease. Note that the bilayer is accessible to the solvent which remains between the two plates due to the thin Teflon gasket. The entire assembly was clamped between aluminum plates for measurement in the neutron beam. Outer mitochondrial membrane (OMM)vesicles fromA? crussu were prepared by the method 0fManne1la.l~A monolayer-fusion technique9 was used in order to form a bilayer containing components ofthe OMM. First, the p-type siliconplate was coated with a monolayer of soybean phospholipid as described above. The n-type plate was placed in a bowl containing DzO or H2O salt solution. The second monolayer was formed by, first, slowly pumping vesicles of OMM solution (1mM MES, pH 5.8), which had been sonicated in an ice water bath for 30 s in a 300-W sonicator (Laboratory Supplies Co., Hicksville, NY), just under the water surface.14 The injection of vesicles onto the water surface ensures that a high concentration of vesicles is available at the surface in a relatively short time (-20 min). Then the p-type silicon plate, containing the soybean phospholipid monolayer, was lowered through the water surface as described above. The model outer mitochondrial membrane (MOMM) bilayer which forms at the surface of the p-type silicon plate consists of a soybean phospholipid monolayer and a second phospholipid monolayer containing components of the OMM. However, the exact composition of the MOMM formed using this method is not known. Neutron reflectivity measurements were performed at the BT7 spectrometer at the National Institute of Standards and Technology. l5 Filtered, monochromatic (2.367 A) neutrons are collimated by two slits, defined by absorbingmasks, located before the sample position. Neutrons specularly reflected from the sample, which is oriented in a vertical geometry, are detected by (11)Langmuir, I.; Blodgett, K. B. Phys. Reu. 1937,51, 964. (12)Kagawa, Y.;Racker, E. J. B i d . Chem. 1971,246,5477. (13)Mannella, C.A. J. Cell. Biol. 1982,94,680. (14)Schindler, H.; Rosenbusch, J. P. Proc. Nutl. Acud. Sci. U.S.A. 1978,75,3751. (15)Majkrzak, C. F.Physica B 1991, 173, 75.
T e f l o n Gasket Path o f Neutron
Figure 1. Lipid bilayer and model outer mitochondrial membrane sample geometry. The bilayer is adsorbed to the surface of a p-type silicon plate containing a thin silicon oxide layer. It is accessible to the solution, which is held between the two silicon plates by a thin Teflon gasket. The neutron beam enters and exits through the p-type silicon plate.
a)
b) water
Nbw
2
Nbi
Nb2 Nbi
Figure 2. (a) Schematic drawing of a lipid bilayer supported on a planar substrate and accessible to aqueous solvent. (b) Corresponding layer profile with average neutron scattering length density, Nb, for each layer. a highly ('90%) efficient 3He detector. The sample geometry is shown in Figure 1 without the outer aluminum clamp assembly. Note that the neutron beam enters and exits through the p-type silicon plate, reflecting from the bilayer adsorbed at the surface. The lipid bilayer can be modeled as series of layers as shown in Figure 2, with an average neutron scattering length density (Nb) for each layer representing an equivalent component, Le., lipid head group, hydrocarbon tail, etc., of the bilayer or membrane. The incident beam of wavevector ki strikes the surface at the angle, 8,and the reflected beam of wavevector kf exits at the same angle. The reflectivityis measured as a fhction of wavevector transfer, Q = Ikf- kil in the direction normal to the bilayer surface (2). The intensity of specularly reflected neutrons from both soybean phospholipids and the MOMM in D2O solution was measured for wavevector transfers up to Q = 0.25 8-l. Beyond this value, the signal became noisy due to incoherent scattering from the D2O solution. The reflectivity curves were corrected for background, slit opening size and finite sample size and then converted to a log&eflectivity) vs Q scale. The thin (25-50pm) water layer which existed between the two silicon plates allowed for a greatly-reduced incoherent background, making measurements out to this high Q value possible. However, since t h e
water layer is so thin, reflections from the surface of the back (n-type) silicon plate make a significant contribution to the total reflectivity. The program TMLAYER,16which explicitly takes the reflection from the solvenun-type silicon surface into account, was used to generate model reflectivity curves for comparison to the data. The reflectivity was also measured for MOMM in HzO ~~
(16)Ankner, J. F.;Majkrzak, C. F. Proceedings of the 1992 SPIE Conference; SPIE Proceedings Series; SPIE: Bellingham, WA 1992; Vol. 1738,pp 260-269.
Krueger et al. Table 1. Best Fit Scattering Length Density Profile for DQO
-1
r,
0 1 2
Si SiO,
3
SiO, Si
2.1a 3.1a 6.0b 3.l0 2.1a
D2O
4
10f 1 (2.0f 0.5) 105
lo* 1
This value of Nb was k e d during the fittingprocedure. Error A-2 for layer 2.
on Nb is 0.4 x
I
0.00
I
I
I
0.10
0.05
I
I
0.15
I
I
0.20
'
0.25
9 (A-') Figure 3. Measured neutron reflectivity on a log scale as a function of Q from D2O between two uncoated silicon plates separated by a thin Teflon gasket. The solid line represents the best fit curve generated with the program TMLAYER. solution using the same sample geometry. However, the much larger incoherent scattering from the HzO solution resulted in noisy data beyond Q = 0.15 A-1. '
Results and Discussion The intensity of the specular reflection of neutrons was measured from a DzO blank, a soybean phospholipid (asolectin)bilayer in D20, and a model outer mitochondrial membrane (MOMM) in both DzO and HzO. Best fits to the data were determined for each sample by obtaining model intensity curves using the program TMLAYER. Reasonable starting Nb values were used for each layer and each resultant model curve was examined, mainly via the x2 criterion, in order to determine which models best fit the experimental curves. Since the reflectivity was measured over several decades of intensity values, the errors on the data points a t high Q values are much greater than those at the lower Q values. Therefore, a lower x2 value did not always indicate the best fit through all of the data points throughout the entire Q range measured. Thus, the x2 test was always checked against a plot of the data versus the fitted curve to be sure the model curve actually did fit the data points for all Q values. Typical x2 values were in the 4.0-8.0range. The D 2 0 blank was fit first in order to confirm the thickness of the silicon-oxide layer a t the surface of the silicon plates and to verify that the fitting program was correctly taking into account reflections from the n-type silicon plate. The thickness of the oxide layers on the silicon plates used has been known to be 10-15 A. Thus, values between 10 and 15 A were tested when fitting the data. The data and resultant best fit line are plotted in Figure 3 on a log&eflectivity) versus Q scale. The back (n-type)siliconplate serves to increase the totalreflectivity for Q > 0.1 k l . This increase could not be accounted for even if no roughness of the oxide layers was assumed. Any additional roughness would only serve to decrease the total reflectivity in this Q range. In fact, the reflectivity curve could only be fit once the reflections from the D20/ n-type silicon interface were explicitly taken into account. The best fit parameters obtained with TMLAYER for the scattering length density profile are listed in Table 1.The values for the oxide layer thickness agreed with capacitance measurements and good fits could be obtained with fairly small ( 5 2 A) values of the root mean square Gaussian roughness parameter. In order to confirm that a soybean phospholipid bilayer had formed on the surface of the p-type silicon plate using
0 00
1
1
0.05
I
I
I
0.10
1
0.15
I
I
0.20
I
I 0.25
Q @-'I F'iguw 4. Measured neutron reflectivity on a log scale as a function of Q from soybean phospholipid in DzO (data points) and the best fit curve (solid line) generated with the program TMLAYER,assuming a symmetric scattering length density profile. The best fit curve for the DaO between two uncoated plates (dashed line) from Figure 3 is included for comparison.
the Langmuir-Blodgett technique, the data from soybean phospholipids in D20 are compared, on a loglo(reflectivity1 vs Q scale, to the fitted curve for the D20 blank in Figure 4. Note that the intensity dro s off faster for the phospholipid bilayer for Q I0.05 -l but begins to show a shoulder beyond this value before dropping again to a distinct minimum a t Q 0.2 A-l. At this resolution, the overall thickness of the bilayer can be obtained with a sensitivity on the order of 1-2 A. On the other hand, structural details within the bilayer cannot be distinguished with the same sensitivity. Therefore, it is not possible to distinguish between a symmetric and a n asymmetric scattering length density profile under the present experimental conditions. Thus, a symmetric density profile, in which the thickness and scattering length density were constrained to be the same for both of the lipid head group (LHG)layers, was assumed. This not only resulted in fewer fitting parameters but also seemed more reasonable given the resolution of the data. The soybean phospholipid bilayer data are compared with its best fit curve in Figure 4 and the resultant symmetric neutron scattering length density profile parameters are listed in Table 2. The parameters for the oxide layers were taken to be the same as those found from the DzO blank sample. The fitted curve itself is sensitive to changes of 1.0A on the LHG layer thickness and 2.0 A on the CHz layer thickness around the region of the minimum a t Q 0.2 A-l. The best fits to the data occurred if a water layer approximately 5 A thick, with the same scattering length density as the large water layer between the plates, was assumed to be present a t the surface of the silicon oxide layer. A similar water layer was also found in the studies of DMPC bilayem2 The minimum Q value a t which the reflectivity first falls below 1.0 (or log&eflectivity) falls below 0.0)
-
-
w
Langmuir, Vol. 11, No. 8, 1995 3221
Neutron Reflectivity Measurements from Bilayers Table 2. Best Fit Symmetric Scattering Length Density Profile for Soybean Phospholipid Bilayer in DaO layer no. 0 1 2 3 4 5
6 7 8 a
type Si SiO, D2O
LHGd CH2 LHG DzO SiO, Si
~ b a s (10-6 b A-2)
2.1c 3.lC 5.6 4.0 0.50 4.0 5.6 3.1c 2.lC
d
11
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
1
I
I
c7
(A)
10f 1 5*1 7 f 1 29 f 2 7 f 1 (2.5 f 0.5) x 10 f 1
I
-11
lo6
Error on Nb is 0.4 x loW6A-2 for layers 2,3,5, and 6 and 0.07 for layer 4. b Nb values were constrained to be equivalent
x 10-6
for layers 2 and 6 and for layers 3 and 5. e This value of Nb was fxed during the fitting procedure. LHG, lipid head group. ,
corresponds to the critical angle. The fitted value of Nb for the D2O layer is sensitive to this critical Q value and is a measure of the amount of D20 actually present in the solvent. The Nb value for 100% D2O is 6.4 x which is the same as the fitted value found for the D2O blank (Table 11,within experimental error (5%). However, the fitted value for the D20 layer in the soybean phospholipid sample (Table 2)is lower than would be expected for 100% D2O solvent. Therefore, a contribution to Nb must be coming from hydrogen in the water layer, since Nb is negative for hydrogen and thus could lower the overall Nb value of the solvent. Approximately 10 f 2% of hydrogen contamination in solution would be required to lower Nb to the level of the fitted value in Table 2. It is possible to estimate the total coverage of soybean phospholipid bilayer on the p-type silicon plate by comparing the best fit Nb values for the CH2 layers to those calculated assuming that no water penetrates into these layers. Thus, any contribution to Nb due to water must be occurring because the bilayer does not cover the entire surface of the plate, allowing patchy regions of water, which are assumed to be small compared to the neutron coherence length ( > 1000A), to influence the value ofNb. Similarly, it is possible to estimate the approximate amount of DzO in the LHG layers by comparing the best fit values for Nb to those calculated assuming no penetration into the head group layers, taking the total coverage of phospholipid on the silicon plate into account. The calculated values for Nb assuming full coverage of the silicon late and no hydration in the LHG layers are 1.1 x A-2 for the for the LHG layer and -0.31 x CH2 layer. By comparing these values to those obtained in Table 2,it is estimated to within f 5 %that the soybean phospholipid bilayer covers 88%of the silicon plate and that there is 48% hydration in the LHG layers. The soybean phospholipid bilayer was measured in order to test our experimental geometry by characterizing a single lipid bilayer in D20, formed using a well-established technique. Once it was established that a single bilayer could be measured very well under these conditions, it became feasible to use a monolayer-fusion technique to form MOMM bilayers. The data for the MOMM in D20 are shown along with the best fit model curves on a loglo(reflectivity)vs Q scale in Figure 5, with the corresponding scattering length density profile parameters listed in Table 3. The constraints on the LHG and D20 layer parameters are the same as those used for the soybean phospholipid bilayer. Note that the overall bilayer thickness of 43 f 2 A does not change. In addition, the LHG and CH2 layer thicknesses are approximately the same for both samples and the best fits to the data occurred if a water layer approximately 5 A thick was assumed to be present at the surface of the silicon oxide layer. The fitted Nb value for the D2O layer, which is determined by the location of the
-7
t
layer no. 0 1 2 3
4 5 6
7 8
typea
Nbbc (low6A-2)
Si
2.ld
SiO, D2O "LHGe "CH2" "LHG" D2O SiO, Si
3. Id 6.0 3.8 0.8 3.8 6.0 3.1d 2.ld
d
(A)
10 f 1 5%1 8 f l 27 f 2 8 f 1 (5.0 k 0.5) x 1 0 %1
lo6
a The exact composition of layers 3,4, and 5 is unknown. Error on Nb is 0.4 x A-2 for layers 2, 3, 5, and 6 and 0.07 x for layer 4. e Nb values were constrained to be equivalent for layers 2 and 6 and for layers 3 and 5. This value of Nb was fixed during the fitting procedure. e LHG, lipid head group
critical angle, is the same as the fitted value found for the D2O blank (Table l),within experimental error (5%). One major component of the OMM is VDAC (voltagedependent anion-selective channel), a 30 kDa protein that forms large-diameter ion channels17J8and represents 50% of the total membrane protein by weight. The totalamount of protein present in a single bilayer MOMM is near or below the detection limit even for the most sensitive protein assays that could be used for this purpose. Measurement of the protein concentration is further complicated by the presence of excess OMM vesicles in the subphase. However, the most VDAC that could be incorporated in the MOMM, even in the most ideal case, is estimated to be 12.5% by weight, based upon the known amount of protein in the vesicles. (The OMM is 50% protein by weight and V 2 of this total protein is VDAC. The model OMM sample contains one OMM monolayer with, a t most, 25% VDAC by weight and one soybean phospholipid monolayer with no VDAC. Thus, the maximum VDAC concentration in the MOMM sample is 12.5% by weight.) Ideally, the neutron reflectometry technique would serve as a sensitive protein assay. However, since the protein does not protrude significantly from the bilayer in this case,18its presence cannot be detected by a change in bilayer thickness. (17)Colombini, M. Nature (London) 1979,279, 643. (18)Mannella,C. A.; Forte,M.; Colombini, M.J. Bioenmg. Biomembr. 1992,24, 7.
Krueger et al.
3222 Langmuir, Vol. 11, No. 8, 1995
the binding of the polyanion, with a diameter of approximately 30 would increase the roughness of the bilayerlwater interface by a measurable amount. Measurements with a n excess (25 pglmL) of the polyanion produced a null result. While this does not confirm the amount of VDAC in the MOMM, it does indicate that, even if 12.5 wt % of VDAC was incorporated into the bilayer, the amount of protein was not sufficient to be detected under the current experimental conditions. Improvements to the instrument, which should allow measurements down to low8in reflected intensity, are currently underway and may provide the additional sensitivity needed to resolve finer structural details in the lipid bilayers.
A,
i
-1R
- 71
0.00
1
I
0.05
I
I
I
0.10
I
0.15
I
I
I
0.20
I
Conclusions 0.25
Q Figure 6. Measured neutron reflectivity on a log scale as a function of Q from model outer mitochondrial membrane in H2O. The solid line is the calculated reflectivity using the parameters from the best fit curve for the D2O data, adjusting the scattering length density to reflect H20, rather than DzO, in the solvent.
Since the exact compositions of the LHG and CH2 layers are not known, the amount ofhydration in the LHG layers and the total coverage cannot be estimated based solely upon the fitted values of Nb in the two layers. However, ifthe maximum amount (12.5w t %) ofVDAC incorporation is assumed, the calculated scattering length densities, assuming full coverage and no hydration in the LHG, are for A-z for the LHG layers and 0.52 x 1.7 x the CH2 layer. Thus, the MOMM bilayer covers a maximum of 95% of the silicon plate and there is a maximum of 48% hydration in the LHG layers. Figure 6 illustrates the data from the MOMM in H2O on a logl&eflectivity) vs Q scale. The solid line in Figure 6 is the calculated reflectivity using the best fit parameters obtained for the MOMM in D20, except that the Nb value for H20 has been substituted for that of D20 in the water layers. The lipid head group hydration was assumed to be 48% and total coverage ofthe silicon plate was assumed to be 95%,as estimated for the MOMM in D2O. The data are noisy beyond Q = 0.15 A-l due to the larger incoherent scattering cross section of hydrogen. Therefore, the measured H2O data are not as sensitive as the D2O data to the scattering length density profile parameters. While the data are consistent with that from the MOMM bilayer in D20, they do not confirm the fitted parameters for the individual layers. Clearly, a n independent determination of the amount of protein incorporated in the MOMM would allow for a much more accurate determination of surface coverage and LHG hydration based on the D2O data. Since a separate technique with the sensitivity to measure the amount of protein in the actual sample used for the reflectivity was not available, a n attempt was made to confirm the existence of VDAC by measuring the MOMM in D2O in the presence of Konig’s polyanion, a 10 kDa protein which binds to W A C and closes the ~ h a n n e 1 s . l ~ If a sufficient amount ofVDAC was present in the MOMM, (19)Colombini,M.; Yeung, C. L.; Tung, J.;Konig,T. Biochim.Biophys. Acta 1987,905, 279.
A planar single bilayer model biological membrane containing components from the outer mitochondrial membrane of Neurospora crassa mitochondria has been measured using specular reflection of neutrons. Reflected intensities down to were measured in D20 solvent for wavevector transfers out to 0.25 A-1 by employing a novel experimental setup which allowed for only a thin solvent layer between two silicon substrates. A comparison of the measured reflectivities from the model outer mitochondrial membrane (MOMM)bilayer, formed by the fusion of vesicles onto a phospholipid monolayer supported on a planar substrate, and from a single lipid bilayer consisting of soybean phospholipids (asolectin), deposited on a flat substrate by the Langmuir-Blodgett technique, revealed that single bilayers were formed in both cases. The measurements were sensitive to the total thickness of the bilayer to within 1-2 Assuming a symmetric model for the neutron scattering length density profile, the total bilayer thickness was found to be the same for both the phos holipid and MOMM samples, with a value of 43 f 2 Within this model, small differences between the two samples were found in the fitted thicknesses of the lipid head group (LHG) and hydrocarbon tail (CH2)layers. However, since the values are the same to within statistical error, they were averaged together to yield a n average thickness of 7.5 f 1.4 for the LHG layers and 28.0 f 2.8 for the CHZlayer. The results for both samples also suggest that a water layer approximately 5 f 1 A thick exists between the silicon oxide layer of the substrate and the LHG layer. In order to more accurately model structural details within the bilayers, and to confirm the existence of VDAC in the MOMM, the Q range of the measurements must be extended even further and/or a suitable, selectivelydeuterated replacement for the soybean phospholipid must be found.
A.
f
A
A
Acknowledgment. This work is from a dissertation submitted to the Graduate School,University of Maryland, by David Gurley in partial fulfillment of the requirements for the Masters degree in Zoology. This work was supported in part by the Office of Naval Research under Contract Number N0014-90-5-1024 and in part by the National Science Foundation under agreement DMR9122444. Inquiries regarding the availability of the TMLAYER program should be addressed to John Ankner. LA940249S
