Biochimtca et Biophysica Acta, 457 (1976) 41-55

© Elsevier Scientific Pubhshlng Company, Amsterdam - Printed irt The Netherlands BBA 85157

NEUTRON

SCATTERING

FOR THE ANALYSIS OF MEMBRANES

BENNO P SCHOENBORN Biology Department, Brookhaven National Laboratory, Upton, N.Y. 11973 ( U.S A )

(Received August 1lth, 1975)

CONTENTS I. Introduction. II_ Neutron scattering from biological molecules _

41 42

III. Comparative X-ray and neutron analysis of myelin

43

IV. Phasing of membranes by lsomorphous replacement A The structure of phosphaUdylcholine bilayers B The structure of retinal photoreceptor membranes

46 46 50

V. Conclusions

53

VI Summary

54

Acknowledgments

54

References

54

I INTRODUCTION N e u t r o n scattering [1 ] is an ideal technique for the analysis o f biological m e m branes [2], since it permits the elucidation o f structural details n o t a t t a i n a b l e by X - r a y scattering. Basically, n e u t r o n a n d X - r a y scattering are rather similar, except t h a t X-rays are scattered by the electron cloud while neutrons are scattered by the a t o m i c nucleus a n d the a t o m i c scattering factors are therefore quite different While X - r a y a t o m i c scattermg factors increase with a t o m i c n u m b e r , neutron scattering factors are all o f the same o r d e r o f m a g n i t u d e b u t vary often significantly even for isotopes o f the same element (Table I). The large difference between the scattering factors o f h y d r o g e n ( ~ 0 3 8 - l 0 -12 cm) and d e u t e r i u m (0.65 10 -12 cm) are o f p a r t i c u l a r interest as described below A general description o f the p r o p e r t i e s o f n e u t r o n s in diffraction experiments IS gwen by Bacon [I]. F o r t u n a t e l y , a p a r t f r o m different scattering, f o r m a n d a b s o r p t i o n factors, the two techniques are similar, p e r m i t t i n g the use o f the well d e v e l o p e d X - r a y diffraction techniques. While p r o b m g different features o f a structure, b o t h techniques reconstruct the spatial a r r a n g e m e n t

42 TABLE I

Element

Cross section (10 24 cm 2) Neutron mass absorption coefficient (#/~, cm2/g) Total Coherent

Scattering length (10 -12 cm 1) Neutrons X-rays (sin 0 = 0)

H 2H C

81 5 7.6 55

N

11 4

O

42

--038 0.65 0 66 094 058

1.8 5.4 55 11_0 42

011 0 0001 0 0002 0048 00

028 0 28 1.69 1 97 2,25

wtthin the sample b e m g studied. In its most simple form, n e u t r o n scattering can, therefore, be used to test the validity o f a structure deduced from an X-ray investigation. N e u t r o n scattering factors are calculated from the p r o p o s e d m o d e l and c o m p a r e d with the experimental d a t a to determine if the p r o p o s e d m o d e l is consistent with the observed data. Analysis of the resultant F o u r i e r m a p will show that the n e u t r o n m a p has much larger density fluctuations t h a t can easily be Interpreted In terms o f observed c o m p o n e n t s A m o d e l is, however, n o t a prerequisite to phase n e u t r o n d a t a ; the structure can be d e t e r m i n e d ab lnitlo by using the large scattering difference between h y d r o g e n a n d d e u t e r i u m In a heavy a t o m type analysis This isotopic scattering difference can also be used to locate particular constituents by selective deuteratlon. The scattering c o n t r a s t between different c o m p o n e n t s can be adjusted (density m a t c h m g ) by simply replacing or mixing H 2 0 with 2H20 or a d d i n g probes such as alcohols, ethers, detergents or even gases.

lI NEUTRON SCATTERING FROM BIOLOGICAL MOLECULES To assess the differences between X - r a y and neutron scattering magnitudes, it is best to c o m p a r e relative scattering factors The scattering lengths for some c o m m o n elements are listed in Table I for b o t h neutrons and X-rays N o t e that the values are given as scattermg length m cm to allow direct c o m p a r i s o n o f X - r a y a n d n e u t r o n parameters. F o r m e m b r a n e - l i k e materials it is, however, more instructive to c o m p a r e the average scattering lengths for whole constituents such as proteins, water, h y d r o c a r b o n chains, lipid p o l a r groups, etc. in terms of scattering length per unit v o l u m e or per unit area, the latter being particularly useful for lamellar samples. Table II lists such group scattering factors a n d illustrates why neutrons are better p r o b e s t h a n X-rays to determine m e m b r a n e structures N e u t r o n scattering densities o f m e m b r a n e constituents vary by nearly three orders o f magnitude, c o m p a r e d to a factor o f two for the equivalent X - r a y scattering densities It also should be n o t e d that d e u t e r a t i o n o f any c o m p o n e n t drastically alters the scattering density, while relative c o n t r a s t variations can be achieved by mixing H 2 0 with 2 H 2 0 The average scattering for a

43 TABLE II AVERAGE NEUTRON SCATTERING LENGTH PER UNIT VOLUME OF SOME COMMON BIOLOGICAL CONSTITUENTS The exact volumetric scattering length depends on the actual atomic compositions and densities Scattering length (10-'* cmlA3) H20 2H20 Hydrocarbon hydrogenated Hydrocarbon deuterated Lipid polar group hydrogenated Lipid polar group deuterated Proteins hydrogenated Proteins 20 % deuterated Protems 100% deuterated RNA hydrogenated RNA 100% deuterated

06 6.3 - - 0.3 70 17 2.6 40 5_0 9.0 42 72 -

-

lipid head group is seen to be 1.7 10-14 cm/A3. This group, if immersed in a mixture of 64 % H 2 0 and 36 °//o2H20, cannot be distinguished from the aqueous phase, but it is clearly observable in pure 2H20. Such density matching techniques not only provide a unique approach to membrane structural problems but also can be used to determine the molecular volume of proteins or even the quaternary structure of protein complexes [3].

III COMPARATIVE X-RAY AND NEUTRON ANALYSIS OF MYELIN The early neutron scattering experiments on myelin (refs. 4-7 and Klrschner, D A., Caspar, D. L. D., Shoenborn, B. P. and Nunes, A. C., in preparation) were carried out to test the validity of the structure elucidated by Caspar from comparative X-ray studies on sciatic and optic nerves A general review on myelin structure can be found in ref 8 From the proposed structure (derived from X-ray results), the equivalent neutron scattering density profile was determined (Fig. 1) and then used to calculate the expected scattering transform (structure factors). This continuous transform was then compared with the experimentally observed structure factors (Figs. 2 and 3) These calculations and the neutron experiments were carried out for dxfferent H 2 0 / 2 H 2 0 concentrations to assess the densities of the membrane's constituents. The resultant density profiles calculated from the observed structure factors are shown in Fig. 4. These results are consistent with the proposed myelin structure and yielded better values for the water content in the membrane. Structural modifications of the myelin sheath (ref 7 and Klrschner, D A., Caspar, D. L. D., Schoenborn, B. P and Nunes, A C., in preparation) were characterized by analyzing

44

0.5 0.4 0.5

I I

I

0.2-

I

I

0.10 -"J-i

f

42.5 r(A) I

0

-o.J~

-

l

.

_

_

0/2=90

o

Fig. 1 Neutron scattering amphtude density profile for a simple step function approximation of myehn Ordinate is the neutron scattering density given m l0 l~ cm -2 umts, ( ) myelin m 100 K 2H20, (- - -) 65 ~ 2H20 Ringer's solution, and (-- --) 20,% 2H20 (after Klrschner et al, refs 4-7 and KIrschner, D A , Caspar, D L_ D , Schoenborn, B P and Nunes, A C., m preparation)

10 5

303

10 2

I

O*

I

i

I

5Q

i

1

28

i

i

i

10 °

i

i

i

15 °

Fig 2 Diffraction patterns for rabbit sciatLc nerve myelin membrane in 100~ ( ) and 2 0 ~ (- - -) 2H20 Ringer's solution The scattering angle (absossa) is given in degrees of 2 0 for a wavelength 2 4.1 ,~ The intensity is given on a log scale (ordinate) (after Klrschner et al_, refs 4-7 and Klrschner, D A_, Caspar, D L D , Schoenborn, B. P and Nunes, A C , m preparation)_

the effect o f d l m e t h y l s u l f o x i d e ( M e 2 S O ) , a reversible n e r v e b l o c k i n g a g e n t

Scattering

d a t a f r o m m y e l m p r e p a r a t i o n s with M e 2 S O c o n t a i n i n g R i n g e r ' s s o l u t i o n s h o w e d t h a t the m y e l i n r e p e a t d i s t a n c e c o n t r a c t e d f r o m 180 A to 120 A. F u r t h e r studies w i t h p r o t o n a t e d a n d d e u t e r a t e d M e 2 S O s h o w e d this c h a n g e in the bllayer s p a c i n g to be c a u s e d by the loss o f water, w i t h a small a m o u n t o f M e E S O localized in the b o u n d a r y

45

0.8 0.6 F(R) 0.4 0.2

o

/>'~.

-0.2-

h

2

I

iI

4

~" 6V

I i]

8

I r I

II

0.02

0

,'-------~

]

0.04

10

I

I

ii 0.06

R(~-~) Fig 3 Continuous structure factor transforms calculated from the simple step function approximation of myelin (Fig 1) ( ) myelin in 100% 2H20 , (- - -) 65 ~ 2H20, ( - - - - ) 20~ 2H20 Ringer's solution. The observed structure factor data points measured as Bragg reflections are given for (@) 100% 2H20, (©) 65% 2H20 and ( × ) 2 0 ~ 2H20 Ringer's solution (after Kirschner et al_, refs 4--7 and Klrschner, D. A , Caspar, D. L. D_, Schoenborn, B P. and Nunes, A. C., in preparation).

_•/?\ ~(,,

~

I 0

i~'/

\\,

I 20

I

/ / / ~

I 40

I

I 60

"-'

I

I 80

Fig. 4 Fourier density profiles for rabbit sciatic nerve membrane. ( ) 100 % 2H20 neutron density profile calculated from the observed structure factors. ( - - -) Equivalent X-ray density profile The large density difference observed at both ends is due to the large difference in heavy water scattering for X-rays and neutrons; the low density region in the middle spans the hydrocarbon region

region of the membrane bdayers. The observed very large second order reflection of the myelin sample in 2H20 Ringer's solution contains more than 1 ~ of the incident beam intensity, produced by less than 0.01 ~ of the spectmen's mass. This phenomenon is caused by the lipid membrane, which is composed of a double bdayer of alternating sheets of 2H20 and lipids. This remarkable diffraction power of such alternate posmve and negative scattering layers can be utilized to yield efficient wide wavelength band pass monochromators [9].

46 IV PHASING OF MEMBRANES BY ISOMORPHOUS REPLACEMENT

1VA. The structure o f phosphandyleholine bilayers The large difference between the neutron scattering of hydrogen and deuterium (Table I) provides a convenient solution to the phase problem. Since all biological membranes contain water, a simple isomorphous replacement of H 2 0 with 2H20 is sufficient to determine at least a low resolution structure The exact localization of water within the membrane will determine the resolution achievable by this simple method The necessary diffraction data are collected for the membrane soaked in H 2 0 and in 2H20 Ringer's solution The intensities are corrected for absorption with account taken of the quite different absorption corrections for H 2 0 and for 2H20, due to the large incoherent scattering of hydrogen The Lorentz corrected structure factors are then used in a difference Patterson function with (AF) 2 = ( IF] H2o-I F I 2 H 2 0 ) 2 as coefficients This difference Patterson function thus contains the information regarding distribution of water. The above definition of (AF) 2 uses the absolute values of the observed structure factors ignoring possible changes in phase of any diffraction orders A gradual H20/ZH20 exchange permits, however, direct observation of phase changes of particular reflections. Such a phase change observed for h -- 2 m a dipalmitoyl phosphatldylchohne/cholesterol sample [10,11], is shown in Fig. 7 The presence of exchangeable hydrogen on lipid head groups and in proteins poses a problem, particularly for high resolution structures, and adds to the difficulty of interpreting the Patterson function In most cases it is possible to estimate the number of exchangeable protons that can be used to unravel the difference Patterson functaon with the aid of the autocorrelatlon function [12] that extracts lnvarJant segments of the structure For determining phases for a high resolution structural analysis it is best to use a particular deuterated constituent, especially if data are obtained for both the H and the 2H velsions Determination of phases by the heavy atom technique is straightforward, but other techniques, particularly for noncentrosymmetric structures [13], have now also been developed. In the lsomorphous replacement method the calculated structure factor (fc) is obtained for the "heavy group" from the positions given by the Patterson map Interpretation The "heavy group" scattering factor in this case is the difference between the H and 2H v e r s i o n s ( b 2 H - - bu) In cases where the heavy group structure factorfc is much smaller than either of the observed structure factors, /:2 n or FH (no phase change from F2H to FH), the stgns of F2H and FH are easily assigned by observing the following inequalities : if F2H ~;" FH then the sign for both F2H and FH IS the same as that for fc; ifF2H < FH then the sign for both FH and F2H is the opposite of that for f~. In cases where fc is of the same size or larger than FH or F2H, the sign of the larger of the two observed factors is still determined by the simple inequality given above, but the sign of the smaller observed F depends, on whether fc is larger or smaller than the difference between the two observed structure factors. Since the scaling of f~ relative to the observed structure factor is difficult, changes in sign for F2H and FH are best detected by following a gradual change in the

47

di t~

-30

-~0

-I

I0

20

30

Fig 5_ Low resolution difference Patterson maps for the first four orders of dlpalmitoy! phosphatldylchohne (a) No changes in phase (sign) between the H20 and 2H20 samples (b) Orders 1, 3 and 4 without phase change, but the weak second order changed phase (after Zaccai et al. [14]).

scattering magnitude of the heavy group, which is easily brought about by mixing the H and 2H versions of the heavy group (it is particularly easy in the case of HEO/2H20 exchange). The analyses of artificial membranes [8] like dlpalmltoyl phosphatldylchohne and dlpalmltoyl phospbatldylcholine/cholesterol serve as good examples to highlight the above-described approach. The dlpalmitoyl phosphatldylchohne structure analysis by Zaccai et al. [14] is an example of the simple HzO/2H20 exchange technique. Lamellar neutron intensity data were collected from oriented dipalmltoyl phosphatidylchollne/H20 and dlpalmitoyl phosphatidylchohne/2H20 samples at low relative humidity. The solution to the low order difference Patterson function (h = 1-4) shown in Fig. 5 IS consistent with a strip of water l0 A wide separated by the repeat distance. Structure factor calculaUons for this strip of water compared with the observed data can be used to obtain phases (Table III). The resultant Fourier transforms for the H 2 0 , 2H20 and difference structure are depicted in Fig. 6. Note that the change in phase of the weak second order has no significant effect on the low order difference Patterson, which is dominated by the strong first order. At higher resolution the effect of changes in sign is, however, severe; lteratlve difference TABLE III No.

Observed dipalmltoyl phosphatidylchohne 2 F(-2o) AFob F(H20)

1

(--)

190

2 3

(--) (+)

20 77

4

(--)

85

(--) (+) (+) (--)

317 49 43 67

(--) (--)

~Fc~L* 127 69 34

--93 68 - - 39

18

18

* Structure factors calculated for a 10 A wide Gausslan band of water at center of symmetry;

AF =

F2.2o

-

-

F.2 o

48

,

(a]

[b) /

'\ _J (~)

J

-d12 30

~0

-I0

2Lo

+ d12 2L 30

FIGURE 6

Fig 6. Fourier densRy profile for dJpalmltoyl phosphatldylchohrte calculated to 15 ~ resolution. Trace (a) depicts dlpalmltoyl phosphatldylcholme in H 2 0 , trace (b) lrt 2H20, and trace(c) shows the dtfference Fourier depicting the water structure (after Zaccal et al [14])_

20

I0

F

~H2C)

Fig 7 Structure factor amplitudes for dlpalmltoyl phosphatldylchohne/cholesterol wgh a hydrogenated and a deuterated hydrocarbon tall_ The reflection orders are indicated. The data have been corrected only for absorption and sample geometry.

49 Patterson methods with the help of other structure reformation and swelling techmques were used to phase the high resolution dipalmitoyl phosphattdylcholine structure map [14]. It is best to observe changes in phase directly, by means of gradual or stepwise changes in the H 2 0 / 2 H 2 0 ratio. In the structural analysis of dipalmitoyl phosphatidylcholine/cholesterol the deuterated hydrocarbon chain of cholesterol served as the heavy group. Fig. 7 shows the observed structure factors as a function of ZH20 concentration for samples that differ only in the replacement of the hydrogen atoms of the cholesterols tail with deuterium. Both data sets were collected on a two-dimensional position-sensitive counter [15]. The reflections were corrected for changes m absorption due to increasing H concentration and for differences in path length depending on the Bragg angle. In such cases, the "heavy group" is truly lsomorphous and does not present problems due to exchangeable hydrogen positions. If the difference scattering factors of the H- or ZH-labeled heavy groups are so large that they could cause changes in the sign of the structure factor, their magnitudes can be reduced by proper mixing of the H- and 2H-labeled marker. H20/ZH20 exchange studies can also be used to observe changes in phase. Scaling of the two data sets can be a problem, however, especially if the "heavy group" scattering magmtude is large. Iterative adjustment of scale factor is possible by adjusting Fourier density levels within a part of the structure that is invariant. This approach ts particularly important in cases where data of different d spacings have to be compared [11]. Data can be directly scaled by comparing the differences at two or more H 2 0 / 2 H 2 0 concentrations. Clearly, the scale factor k is equal to the ratio of the slopes calculated from the observed structure factor changes caused by the variations in 2H20 concentration. The inspection of such 2 H 2 0 / H 2 0 concentration-dependent structure factor data is also a help m checking absorption and extinctton, as well as relative Lorentz correction of different data sets; th~s is especially ~mportant in cases where data sets collected under shghtly d~fferent condtt~ons are to be compared Ftg. 8 shows the dtfference Patterson for the dipalmltoyl phosphatidylcholine/

[

I

i

C

~v

,

0

I

10

I

20

I

30

/

I

40

I

50 Angstroms

I

60

I

70

[

80

90

F~g 8 Difference Pattersons from the low and high order dlpalmitoyl phosphatldylcholine/cholesterol data with the structure factor difference being caused by the deuterium- vs. hydrogen-containing hydrocarbon chain of cholesterol, origin at 30 A.

50 cholesterol structure with the structure factor differences being caused by the deuterated vs. hydrogenated cholesterol tall. The resultant analysis of the F o u r i e r profiles based on ten diffraction orders shows that the deuterium-labeled cholesterol tad extends to the methyl terminus of the phosphatidylchohne chain, which places the hydroxyl group of cholesterol just below the glycerol moiety [10] The chohne phosphate group is oriented again in the plane of the bilayer, as f o u n d in d~palmltoyl phosphatldylchohne [14]. The recent analysis of egg lecithin cholesterol bilayers by Worcester [16] confirms the above results. I n this case, the location of the cholesterol was determined by substituting deuterium for the hydrogen on c a r b o n (Ca) linked to the hydroxyl group. It ts remarkable that the exchange of just one hydrogen a t o m can be detected m such structures.

IVB. The Structure of Retmal Photoreceptor Membranes The analysis of retinal rod outer segments by Yeager [17-19] is so far the most

10 6

105

104 COUNTS 103

9 I

---3 10 2

10

0

I 2 4 6 8 SCAq'TERING ANGLE (o)

I 10

Fig 9. Neutron diffraction data from intact retinas. Profiles 1-3 show diffraction data collected from 10 retmas in 2H20 Ringer's solution at 5°C Data were collected on the beam pipe station [2] at the high flux beam reactor using 4/~ neutrons with a conventional scanmng counter The enhanced pattern of profile 2 was achieved by improvements m dissection and sample mounting techniques Improvements observed in trace 3 were obtained by continuouslyper fusing the retinas with oxygenated Ringer's solution containing antibiotics and a carbon source. The striking amprovement in trace 4 was due to the use of a two-dimensional pos~non sensitive detector [16]; in this case only two retinas were used.

51

105

I

I

I

I

r

I

1 I 104

3 I

2 I

~i0

4

-

_

10 0

I

I

11 2t ~-1 (x 102)

7

[

I

31

Fig 10 Background fitted dtffraction patterns of retinas. The dotted curve is a power series fit to the equatorial scattering obtained by rotating the retinas by 90 °.

ambitious use of neutron scattering for the analysis of a biological membrane system. Rod outer segments are particularly suited for diffracUon experiments [20] because the disc membranes are stacked periodically. Unfortunately, data collection from retinas is difficult because of their finite stabihty. Fig. 9 summarizes the advances in data collection techniques: the first three traces show the effects of improved sample preparation and the fourth shows the tremendous gain due to the use of a twodimensional counter system. The background underlying the Bragg reflection was obtained by rotating the retinas 90 ° to obta,n the featureless equatorial scattering The dotted line in Fig. 10 depicts the power series fit to the background [21] Integrated intensities were obtained from Gaussian peaks fitted to the background subtracted Bragg reflecUons. A Lorentz factor of h 2 was apphed as determined by slit height and tilt experiments as well as by rocking curve analysis. Phases were based on interpretation of Patterson maps calculated from diffraction data on retinas in different mixtures of H20/2H20 Ringer's solution as well as Ringer's with different osmolarittes. Fig ll shows the low order neutron scattering density profiles for different 2H20 concentrations. The low density areas centered at 110 and 190/l are interpreted as the hydrocarbon domain of the disc membrane The 50 ~ width of these features is compatible with a hpid bilayer structure. The Founers are distinctly asymmetric, with a lower density on the external disc side than in the mtradmsc space. This asym-

52 °/o

2N20

I

I

I

I

I

I

I

I

I

I

IO0 BO 6O 4O 3O

I

50

I

100

I

150

200 250 D)stance (~,)

I

300

I

350

I

400

450

Fig l 1. Low resolution Fourier density profile of rod outer segments for different 2H20 concentration. Note that the repeat distance is 300 A.

metry suggests that models which place protein symmetrically in the disc membrane or exclusively on the intradlsc face can be excluded as possible structures. These neutron Fourier syntheses can be placed on an absolute scale by assuming that the highest density (intradlsc region) in the 2H20 profile has the scattering density of pure 2 H 2 0 Ringer's solution of 6.35 l0 -14 cm/A ~. The corresponding densities in other H 2 0 / 2 H 2 0 profiles are easily determined, since this exchange is isomorphous. From this scaling procedure the scattering density in the center of the disc membrane lipid bilayers is estimated as 0.5 l0 -j4 cm/A3, which suggests that some protein resides in the membrane interior on the external disc side and extends into the cytoplasmic space. Such detailed model building calculations depend strongly on the shapes and densities of the protein molecules apart from resolution and series termination effects. Knowledge of the shape of rhodopsln, the major protein component of rod outer segments, is therefore a prerequisite for any detailed structural interpretation of the above-described Fourier transforms. The shape of opsin and rhodopsln was therefore determined from solution scattering experiments in detergent solutions [19] In these studies the 2 H 2 0 / H 2 0 ratio can be manipulated so that the solvent and detergent scatter equally (contrast matching) [21,22], leaving the protein as the differential scatterer. The resulting Gulnier plot (Fig. 12) shows the scattering observed from solutions of purified and delipidated bovine rhodopsln and opsin in Ammonyx-LO detergent. The observed radius of gyration of 21 A for rhodopsin shows this protein to be strongly elongated; a spherical rhodopsin with a partial specific volume of 0.74 cm/g would have a radius of 17.3 A. The curvature in the opsin Guinier plot indicates aggregation. Comparison of the zero angle scattering of rhodopsin with that of known proteins yielded a molecular weight of 40000, which is close to the accepted value of 38000 (g = 0.74 cma/g). These values yield a molecular volume of about 48000 A a that fit an oblate ellipsoid of about 75 A by 15 A by 15 A. In order to match the Fourier density profiles with the above general shape information, rho-

53 I

I

I

I

I

I

1

~

I--T--"

Opsln

Rhodopstn

103

I I(]2

"lO 0

I

I

I

I

5

10

5

20

202(

1

I

5 25 x l O 4 r o d l c l n s 2)

~l 10

I 1.5

I 20

I 25

Fig 12. Guinier plots for purified and dehpidated bovine rhodopsin and opsm in detergent solutions.

dopsin can be pictured as a mushroom with the stem buried m the lipid bilayer and the head extending into the cytoplasmic extradisc aqueous space. Chabre [23] recently improved the data collection considerably by magnetic orientation of isolated disc membranes. Magnetically oriented isolated rods yielded much higher diffraction intensities than intact retinas, so that Chabre was able to collect data from one sample m different H 2 0 / Z H 2 0 ratios and thus ehmmate scaling problems. Since his data processing and phasing of the seven observed diffraction orders are not yet complete, any direct comparison with Yeager's results is precluded. Studies have just begun on purple membranes from Halobactertum haloblum, another retinal-containing membrane Worcester [24] collected data from purple membranes oriented in a strong magnetic field Continuous profile data to 0.03 A- ~ were obtained as well as a few m plane reflections from the hexagonal crystalline lattice King (King, G , unpublished) carried out similar studies by using samples oriented by sedimentation In th~s case, the continuous scattering profile was observed to be 0 07 A- i as a function of 2H20 concentration. These data are consistent with a purple membrane model [25] deduced from earlier X-ray studies.

v_ CONCLUSIONS It ts clear that analysis of membrane structures by neutron scattering has just begun. The initial studies show how powerful this technique is and demonstrate that slgmficant results on membrane structure and organization can be obtained. However, in order to make full use of the capabilities of neutron analysis, better oriented samples, particularly of artificial lipid bllayers, are needed; magnetic orientation might provide the answer to this problem [23]. At present, httle attention has been gwen to the evaluation of data correction factors resulting from absorption, extraction [26], sht smearing and Lorentz factors, parameters that depend strongly

54 on beam geometry and wavelength band width, and whtch differ slgmficantly from the X-ray case, Obviously, these detads have to be considered carefully to obtain accurate results. Most present experiments have also been hindered by the relatively low flux of neutron sources. While httle improvement in reactor flux can be expected, better focusing devices wdl often zmprove the effectwe flux [27,28]. Apart from improvements m beam geometry, the most significant gain is obtained by the use of htgh efficiency two-dimensional counters [15] with high resolution The degree of success of membrane structural analysts will, however, not only depend on good instrumentatton, but is largely dependent on the skill of producmg selectively deuterated constituents [24,29,30]. From the number of papers presented at the recent Symposium on Neutron Scattermg for the Analysis of B~ologzcal Structures at Brookhaven National Laboratory, it is indeed clear that a rapid escalation of neutron studies is m the making

Vl SUMMARY The advantageous use of neutron scattering techniques for the determination of membrane structures is described. Constituents of biological membranes show much larger differences in their scattering factors for neutrons than for X-rays, permitting the assignment of chemical groups to features in the Fourier map. Deuteration of particular components further enhances this difference and can be used to phase neutron data similar to the heavy atom techmque of protein crystallography. Methods of contrast enhancement using H 2 0 and 2H20 exchange, as well as specific deuterations, are outlined with examples from studies of sciatic nerve myelin, artificial membranes and retinal rod outer segments.

ACKNOWLEDGMENTS Research was camed out at Brookhaven National Laboratory under the auspices of the U S. Energy Research and Development Administration, The author wishes to thank M Yeager and G King for help in preparing the manuscript and M. Dlenes for reading the manuscript.

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