Structural analysis of membranes by means of a nuclear reaction

J. Mol. Biol. (1982) 154, 159-167

Structural

Analysis of Membranes Nuclear Reaction

by Means of a

C. WIEZOREK Institut

fiir

(Received

der Iiniversitdt Miinster 17 4400 Miinster. West Germany

Strahlenbiologie

Hittorfstr. 18 May

1981, and in revised form

1 October 1981)

A narrow nuclear resonance in low-energy proton-induced nuclear reactions in the stable isotope ‘*O has been utilized in a new technique for investigating the structure of biological membranes. This technique leads to a direct determination of the density profile of 180-labelled molecules. Results with a multilayer of egg lecithin and with erythrocyte ghosts demonstrate the applicability of the method to structural analysis.

1. Introduction The molecular structure of lipid bilayers. an important component of biological membranes, has been studied by X-ray diffraction on unoriented dispersions (e.g. see Ranck et al., 1974: Lesslauer et al.. 1972; Janiak et al., 1976; Torbet & Wilkins. 1976). Neut,ron scattering is another method giving structural information (e.g. see Zacchai et ccl., 1975: Worchester, 1976: Worchester & Franks, 1976: Biildt et al.. 1979). This paper describes a new method, which gives a density profile of a marker along a direction perpendicular to the bilayer. The stable isotope ‘a0 is used as a marker. The distribution of ‘*O is measured by means of a low-energy resonant nuclear reaction that is specific for this isot,ope: an incident proton that collides with la0 reacts with the nucleus, releasing an a-particle and the residual nucleus ’ 5N. This tvpe of nuclear reaction (named ‘sO(p, cr)“N) can be distinguished from other reactions by the energy of t,he outgoing a-particles (Q-value). The yield of these a-particles can give information about the quantit,y of marker, if the corresponding reaction probability (cross-section) is known. The probability, however, is energy-dependent. So, since the protons lose energy as they pass through the sample, the yield of the x-particles is a folded function of the energy loss of the protons, the cross-section for the nuclear reaction, and the concentration of the target nuclei along the path of the protons. A resonance in the cross-section has to be present for the distribution along this axis to be determined. The local resolution depends on the total width of the resonance and the stopping power (i.e. the rate of energy loss of protons when 0022~2836/82/01015949

$02.00/O

159

0 1982 Academic Press Inc. (London)

Ltd.

(‘. WIE:%OKEti

lfi0

traversing the sample). So the method is feasible using nuclear reactions with a \-cry narrow resonance at low incident energy (high stopping power). The low energy excit.atiott function of the t80( p, X) ’ 5N reaction (Lorenz-Wirzba et nl. 1 1979) is shown in Figure 1. The total width of the ttarrow resonance at Ir:, = 15% ke\’ used in the experiment is r I 500 eV. The order of magnitude of the energy loss per dngst,riim thickttess for protons of this energy passing through phospholipid material. which consists mainly of carbon and hydrogen with a ratio of I : I (density - I.3 g/cm3). can be est,itnated from stopping power dat.a. The Tables of’ Range and Roppittg Power of

103IO2IO’x 0* 100Lil \ “g 10-l b 10-Z10-X100

150

200

E CkeVi

FIG. 1. Excitation

function

of the l*O(p, a)15N nuclear reaction (Lorenz-Wirzba

et al.. 1979)

Chemical Elements for Charged Particles of Energy 005 to 500 me\’ from M’illiamson et nl. (1966), where the low energy ceorrected Bethe formula is used, give these values with the ratio 38 et’/A for hydrogen and 9 eV/a f or carbon. Weighting of 2 : 1 yields a stopping power of 28 eV/A. So, a double layer of 60 A thickness should cause an energy loss of about, 1.7 keV. In this rough estimation, no accouttt has been taken of the oriented quasi-crpstallitte arrangement of the stopping atoms. which increases the stopping power if chanttelling effects are avoided. Comparing this energy loss with the total width of the resonance, which has t.o be regarded as an upper litnit. the local resolution should allow an image of the lamellar structure of the probes. The interaction of the resonance and the energy loss in the probe works as follows. If the ettergy of the incident protons is close to the resonance energy tc:,. they react with marker nuclei distributed on t.he surface only. If the bombarding upon slowing down. reach the energy E, in the energy E exceeds K,, the protons, to the vicinity of a depth xO(Elo = ZCR). The a-yield Nn(E) is hence proportional tracer concentration P(x~) and, a plot of S&(E) versus bombarding energy E:

STRI’CTI’RAL

(excit.ation

function),

ANALYSIS

gives information

OF

about

161

MEMHRAXES

C(Z) :

3c a’

N&(E) cc

c(x)o(Ex)N,(E, ss0

E,, X) dE,dr,

0

where lo’ is the bombarding energy, E, is the proton energy at depth 2, o(KX) is the cross-se&on (probability) of (p, 3) react,ion at E,, and N,(E, E,, X) is the number of protons of energy E, at dept’h .r. S,(E, E,, x) is given by the high-voltage stabilit) of the accelerator (i.e. the energy spread of the proton beam) and by angular and energy straggling effects in the probe. These effects can be calculated using the stochastic theory of slowing down of particles (e.g. see Amsel, 1963; Nadai. 1967). but informat,ion may be obtained from t,he yield curve iva(E) without such a calculation. as shown by Amsel ef al. (1971) and by the following results. In this experiment, in which very thin structures (z 5 100 8) are studied. the local resolution is det’ermined mainly by the total energy resolution, because the energ? straggling amounts to about +6yb of energy loss. The total energy resolution ran be &mated by measuring the step-like excitation function of a thick uniform [ 1s0ltarpet in the vicinity of the resonance. It is defined by the energy change leading t,o an increase in the n-particle yield from 25’Y0 t,o 75:/;, of its maximum. The measured excitation function of a thick IMg’%]target is plotted in Figure 2. giving a total energy resolution of AE = 400f 100 eV. The result’s of measurements made by Lorenz-Wirzba et al. (1979) provide an estimatt, of detect,abilit,y. calculating the resonant, cross-section as follows : a re\ = 7&Ly/4r. With the total width of the 152 keV resonance r 5 500 eV, the resonance strength WY = 0.17 eV and the square of the reduced de Broglie wavelength of the protons 3; = 1.53 x 1O-24 cm’ ares 2 6 x lo-”

cm’.

E CkeV) FIG. 2. Excitation function derivation of the total energy

of the “O(p. resolution.

.x)‘%

reaction

of a thick

[Mg”O]target

measured

for

162

C. WIEZOREK

For a natural abundance of @2%, only one out of a hundred phosphatidylcholine molecules contains a, IgO nucleus localized at the phosphate head. Assuming a packing density of 2-5 x 1014 molecules per cm* arranged on a plane, corresponding t,o an area per molecule of 40 A*, one expects for a prot,on current of @6 x 10” s- ’ (1 @I) an a-yield of:

These x-particles are emitted isotropically; for our experimental set-up, with a det’ector efficiency of 276 (0.28 steradian). this would lead to the very low counting rate of 2 x 10e3 s-l So we decided to make use of 0~~~0, as a specific ‘*O marker of the polar head group region. LVe chose artificial multilayers of egg 1ecit)hin and erythrocyte ghosts, and fixed the probes with 0~~~0,. The distribution of the “0 should give some information about the region of chemical interaction of the Os’*O, and the phospholipid molecules.

2. Materials and Methods (a) Materids Egg lecithin solution (chromatography grade) was obtained from Sigma, U.S.A. (lg/lO ml in rhloroform/met,hanol, 9 : 1 v/v). Samples of oriented bimolecular layers were produced by allowing the solution to evaporate on optical flat glass microscope slides (e.g. see Levine & Wilkins, 1971). The sample areas were about 3.5 cm2. Human red cell membranes (ghosts) were prepared from fresh blood by the method of Dodge et al. (1963). Os”O, was prepared from metallic osmium heat,ed in 99% “02. For fixation, the probes were exposed to vapours of Os”O,, evaporating at room temperature.

FIG. 3. Schematic drawing of the experimental

set-up.

STRC’CTURAL

ANALYSIS

OF MEMBRANES

163

(b) Measurement procedure Measuremeuts were made usiug a 350 kV accelerator (High Voltage, Iuc., Netherlands). A schematic drawing of the experimental set-up is shown in Fig. 3. The target chamber contained the probes and the surface barrier detector for x-particle oountiug (Ortec, 177 mm’, 90” detection angle). At a distance of 25 mm, the probes were mounted iu au alumiuium holder, iusulated for beam curreut iutegratiou. Channelliug effects in the orieuted layers were avoided by usiug au angle of 60” between the surface and the beam directiou. Secondary electrous emitted from the beam-defining collimator were suppressed by a halo aperture at a potential of - 200 V. During the measurements, a reduced pressure of 5 x 10e6 mm Hg was maintained. A liquid nitrogen cooled alumiuium shield together with a cooled copper tube in the beam pipe acted as a cold trap for carbon-coutaiuiug molecules (e.g. hydrocarbons) which, beiug cracked by the beam, can build up a carbon deposit on the target surface. To prevent heatiug of the probes, the target holder was cooled to - 60°C. The proton beam was defocused homogeneously to a diameter of 9 mm with au average beam current of 1 PA. At the low a-couuting rate, a charge of 3000 &i of protons had to be accumulated for each euergy value, giviug a ruuning’time of about 50 min. After the measuremeuts, no structural chauge of the probes could be observed under the light microscope.

3. Results The excitatiori function was measured between 151.5 arrd 157 keV irr 500 eV steps. This step-width was close to the total ermrgy resolutiorr arrd the energ) stragglirig of the protons. Figure 4 shows the measured excitatioii functiotr of a lecithiri multilayer. The unbroken hue is a curve fit. by overlapping Gaussian functions on a slowI> irrcreasirrg background, calculated from the corltributiorr of the off-resonant crosssection.

E (keV)

FIG. 4. Excitation

function

of the “O(p , a)‘$N reaction of a Os’*O,-stained lecithin multilayer

164

(‘.

WIEZOKlcti

Besides the Gaussian distribution of the “0 at the head group regions, only an overall periodic lamellar architecture was assumed. As a result of the variat,ion of the positions and of the width. the best tit was obtained with the following parameters : 1’3, = k, = ti:, = if, = 15, =

rlihm = 0.8 keV.

1X?+ 154.2 1.552 156.9 157.9

ke1 keV keV key ke\

The relative positions of the peaks are in good agreement, wit,h other struct,ural neutron diffraction investigations of artificial lecithin multilayers (e.g. experiments). To further test the applicabilit,? of the new method. the second object was a probe consisting of erythrocyte ghosts. As in the first measurement. the starting energy was 1515 ke\‘. To avoid thermal destruc+on of the ghosts. the average beam current was reduced to 04 PA at a diameter of 9 mm. I)ue to the complexity of this probe compared with the pure lecit,hin target, the background is increased. Nevertheless, an oscillating minima-maxima structure can be observed in the excitation function shown in Figure 5, being well-fit by two overlapping Gauss functions. Though the statistical error does not allow a unique determination, a set of wellfitting parameters is listed below: rfeh,,, = O+Skey

z$

h’, = 152.1 ke\ E, = 154.1 keV.

60-

-----

-----

yt

40 -

152

154

156

E CkeV) FIG. 5. Excitation

function

of the “O(p,

m)15N reaction

of 0~~~0,~stained

human

erythrocyte

ghosts.

STR\‘(‘T17R~\l,

ANXLYSlS

OF MEMKRASKS

l(i6

In this case thr background is assumed to be constant with incident energy. hecause the high level is an effect of the resonant contribution of IsO distributed randomly along the beam axis. Thr S, values lying above the curve at. high prot,on ~~ncrgics may Iw due to intracellular osmiophilic compounds.

4. Discussion (‘omparing the structure of the excitation function with scattering length densit) [)rotiles of’ neutron diffra.ction experiments on undeuterat.ed lecit,hin multilayers to a local (e.g. SW t
FIG 6. 1kpt.h tnagnitic~at.ion effect, of the tiking

angle.

Ax’ = Ax sin (x, with u = 60” tilt angle d = 60 A, the thickness of the egg-phosphatidylcholine tlouble layer measured by .Y-ray and neutron diffraction experiments. The local resolution Ax depending on the t&al energy resolution AE = 400 et has heen c~alculated from the energy loss I?,~ - E,, corresponding t,o the t’hicknrsx d/sin ‘x. Referring to the energy parameters of t’he fitted curves E,, E2 and E,, it is: ft’,,i-E,r, This gives a specific

=Z((~,+(~3-~2)l%)-(~:I+(~2-~l)/~))=~.7ke\T. resolut.ion

of: 6!1.3 .4 2.7 kr\

103 A 04 kejT’

166

(‘. WIEZOREK

with : Ax = 10.3 A

Ax’ =

and

x = 60”

8.9 8.

So in our case the local resolution was about X.9 A. For well-oriented multilayers, a tilt angle of 20” would give a resolution of 3.5 A. Comparison with neut,ron diffraction also gives information about the region of the highest ‘*O concentration, which may be the region of highest OS affinity. Using the nomenclature of Biildt et rrl. (1979). t,he highest concentration is measured at the C-2(1) position or. more precisely. the region of t,he carboxylate groups. Comparing the integrated a-counting rate of about 140 x-particles in 50 minutes coming from that depth region with the counting rate from t,he natural abundance of 180 in the phosphate heads as discussed in the Intjroduction. the amount of 0~~~0, bound can be calculated as about one 0~~~0, molecule per 17 phospholipid molecules. After demonstrating the applicability of the new method in principle. some additional information should be given about, the boundary conditions. The function XP (E. E,, x.) can be regarded as a constant only for t,hin struct,ures, because the energy straggling has to be smaller than the total energy resolution, which involves the high-voltage stability and the total width of the reaction resonance. In our case the energy straggling can be calculated from the statist,ical error of the number of stopping electrons arranged on the proton pathway. There are about 250 binding electrons contributing to the st,opping power in a double layer with a statistical error of about ,Ssi,. So for 2.7 keV energy loss. the energy straggling is &- 170 eV. Two double layers would lead to + 240 eV. This thickness should not, be regarded as the upper limit but, for t’hicker structures. the energy straggling mainly determines the local resolution. On the other hand. it is an advantage of our technique to need only one double layer. in contrast to diffraction experiments where the periodic structure of a multilayer is desirable. The running time of 50 minutes for each bombarding energy can be reduced by using a detector wit,h a large detection area and bombarding the multilayer targets at small angles of tilt. Compared to a tilt angle of 60”. at an angle of 20” the proton beam would fall on an area greater by a factor of 25. So for the same thermal deposition per unit area. t)he beam current could be increased b,v the same factor. enhancing the a-yield by a factor of 6.4. The result,s of our measurements are iti good agreement with ot,her experiments using conventional techniques: thus the new method forms a powerful tool for st,udying biological membranes. Possible improvement,s are being investigated.

I thank Miss M. Volkermann for her technicaal assistance. I thank Prof. Dr R. Santa and Dr R. Cleff, Institut fiir Kernphysik rler Universit8t Miinster, for their permission to use the accelerat,or.

STRV(‘T[‘R.AI,

ASALYSIS

OF MEMBRANES

167

REFERENCES Amsel, G. (1963). LAL report 1053, University of Oway. Amsel, G., Nadai, J. P., D’Artemare, E.. David, D., Girard, E. & Moulin, J. (1971). Nucl. In.skr. .Wethods, 92, 481498. Hiildt. (;.. (ially. H. U., Seelig, J. & Zaccai, G. (1979). J. X01. Biol. 134, 673-691. Dodge, J. T., Mitchell, C. & Hanahan, D. J. (1963). Arch. Biochem. Biophys. 100, 119. Janiak, 11. .I., Small, D. M. & Shipley, G. G. (1976). Biochemistry, 15, 45754580. Lesslaucr, \\‘., Cain, ,J. E. & Blasie, .J. K. (1972). I’roc. ,Vat. dead. Sci.! U.S.A. 69, 1499-1503. Lwiw, Y. K. Br Wilkins, M. H. F. (1971). Satwe ,Vew Biol. 230, 69-72. Lorenz-\\‘irzba. H.: Schmalbrork, P., Trautvetter, H. P., Wiescher, M., Rolfs. C. & Rodney. IV. S. (1979). Nucl. Phys. ser. A. 313, 346. Xadai. .I. I’. (1967). Thesis, University of Orsav. Ranck. .I. I,.. Mateu. L., Sadler. D. M., Tardjeu. A.. Gulik-Kryzwicki, T. & Luzzati. V. (1974). J. Jol. Biol. 85, 249-677. Torlwt. .J. & \I’ilkins, M. H. F. (1976). J. Theoret. Biol. 62, 447-458. Williamson, (1. T., Boujot, J.-P. & Picard, J. (1966). Rapport CEA-R 3042. Commissariat L I’Energie Atomique, Paris. Worchester, D. L. (1976). In Biological Membranes (Chapman, D. & Wallach, D. F. H., eds), vol. 3, pp. l-46, Academic Press, London. \Vowhest,er. D. L. & Franks, N. P. (1976). J. A’ol. Riol. 100, 359-378. Zawai. (i.. Blasie. .I. K. & Srhoenborn. B. P. (1975). Proc. Sat. ilrad. Sri., l’.fJ.A4. 72. 3763x0.

Edited by H. E. Huxley