myelin basic protein multilayers

J. Mol. Biol. (1984)

174, 385409

Lipid/Myelin A Model

Basic Protein Multilayers

for the Cytoplasmic J. S~DZIK,

Space in Central

A. E. BLAUROCK~

Nervous

System Myelin

AND M. H~CHLI

Laboratories for Cell Biology Department of Anatomy School of Medicine University of North Carolina Chapel Hill, N.C. 27514, U.S.A. (Received 2 September 1982, and in revised form 16 IVovember

1983)

A multilayered complex forms when a solution of myelin basic protein is added to single-bilayer vesicles formed by sonicating myelin lipids. Vesicles and multilayers have been studied by electron microscopy, biochemical analysis, and X-ray diffraction. Freeze-fracture electron microscopy shows well-separated vesicles before myelin basic protein is added, but afterward there are aggregated, possibly multilayered, vesicles and extensive planar multilayers. The vesicles aggregate and fuse within seconds after the protein is added, and the multilayers form within minutes. No intra-bilayer particles are seen, with or without the protein. Some myelin basic protein, but no lipid, remains in the supernatant after the protein is added and the complex sedimented for X-ray diffraction. A rather variable proportion of the protein is bound. X-ray diffraction patterns show that the vesicles are stable in the absence of myelin basic protein, even under high g-forces. After the protein is added, however, lipid/myelin basic protein multilayers predominate over single-bilayer vesicles. The protein is in every space between lipid bilayers. Thus the vesicles are torn open by strong interaction with myelin basic protein. The inter-bilayer spaces in the multilayers are comparable to the cytoplasmic spaces in central nervous system myelins. The diffraction indicates the same lipid bilayer thickness in vesicles and multilayers, to within 1 A. By comparing electron-density profiles of vesicles and multilayers, most of the myelin basic protein is located in the inter-bilayer space while up to one-third may be inserted between lipid headgroups. When cytochrome c is added in place of myelin basic protein, multilayers also form. In this case the protein is located entirely outside the unchanged bilayer. Comparison of the various profiles emphasizes the close and extensive apposition of myelin basic protein to the lipid bilayer. Numerous bonds may form between myelin basic

protein

and

between diacyl-lipid t Author

to whom

lipids.

Cholesterol

may

enhance

binding

by

opening

gaps

headgroups.

all correspondence

should

be addressed. 385

0022.-2836/84/100385-25

$03.00/O

0 1984 Academic

Press 1~.

(London)

Ltd

3%

J. SEDZIK,

A.

E.

HLAIJROCK

AND

M.

HOCHLI

1. Introduction Myelin basic protein? (M, 18,500) is the best characterized of several basic proteins found in nerve myelin (Braun & Brostoff, 1977; Matthieu, 1980; Weise et al., 1982). MBP$ accounts for 30% of the total protein in myelin isolated from bovine central nervous system (Eng et al., 1968) or for 8% of the dry mass of the myelin (Norton, 1977). There are similar percentages of MBP in the CNS myelins of other species (Norton, 1977). MBP also is present, in smaller amounts, in myelin isolated from the peripheral nervous system (Braun & Brostoff, 1977; Norton, 1977). Isolated MBP forms complexes with various lipids. The strongest affinity is for negatively charged lipids (Palmer & Dawson, 19696; Demel et al., 1973; London et al., 1973; Mateu et al., 1973; Verkleij et al., 1974; Papahadjopoulos et al., 1975; Steck et al., 1976; Boggs & Moscarello, 1978; Boggs et al., 1981a; Brady et al., 1981; Young et al., 1982; Lampe & Nelsestuen, 1982). This affinity is based on its positive charge around neutral pH (~1 > 10; Eylar & Thompson, 1969; Palmer & Dawson, 1969a). The complexes usually are layered structures, although apparently not always (Steck et al., 1976). Two X-ray diffraction studies of multilayered complexes of MBP with lipids have been reported. Mateu et al. (1973) added MBP to a mixture of acidic myelin lipids, which by themselves formed two lamellar phases. They found a single phase having two different bilayers per repeat (d = 154 A). A plausible electrondensity profile of the pair of membranes was calculated. The MBP was thought to be sandwiched between lipid bilayers. Brady et al. (1981) reported that MBP forms multilayers with egg phosphatidylglycerol. These authors assumed that MBP is located at the bilayer surface. No profile was shown. Contrary to the interpretations of X-ray data, it has been proposed that MBP penetrates a dipalmitoyl-phosphatidylglycerol bilayer and greatly alters its structure, on the basis of calorimetric (Papahadjopoulos et al., 1975; Boggs et al., 1981b) and electron spin resonance data (Boggs & Moscarello, 1978). Given the disparate structural proposals. we have undertaken to locate MBP and study its effect on the bilayer structure. In our study, complexes are formed in a novel way by adding MBP, or cytochrome c, to single-bilayer vesicles of myelin lipids. Start’ing with vesicles rather than lipid multilayers exposes at least half the total bilayer surface to the protein, thereby facilitating the interaction between the two and also avoiding the complex phase formed by Mateu et al. (1973). Freeze-fracture electron microscopy is used to study the interaction as a function of incubation time. The integrated intensities from the multilayers vary in a simple way with the d-spacing but are independent of the amount of MBP bound. These observations are best explained by a constant lipid/MBP ratio within the multilayers. A less likely alternative of dual binding sites cannot be eliminated. Given the former t Myelin basic protein also is termed: BP, LBP. EAE antigen. A,. B,, P,, BP, ISM in peripheral nervous system. 1 Abbreviations used: MBP, myelin basic protein; CNY, central nervous system; PLP, proteolipid protein.

B,.

EF

in central

nervous

system:

and

nervous

system;

PNS,

peripheral

LIPID/MYELIN

BASIC

PROTEIN

MULTILAYERS

3X7

interpretation, there would need to be one or more non-multilayer phases in order to account for the variable amount of MBP bound, but such phases have not been characterized. For the first’ time, we compare electron-density profiles of lipid bilayers with and without MBP. The two differ significantly, revealing the MBP largely in the inter-bilayer spaces. The shape of the MBP distribution is in clear contrast’ t’o the distribution obtained for cytochrome c. The distributions indicate that each MBP molecule is in close apposition to the lipid bilayer. We propose that the energy (LeNeveu et al., 1976) required to displace water of hydration (Small, 1967; Levine & Wilkins, 1971; Worcester & Franks, 1976) from the bilayer surface comes from the formation of charge bridges and hydrogen bonds between amino acid residues and lipid headgroups. Many more bonds are indicated for MBP than for cytochrome c.

2. Materials

and Methods

(a) Chemicals

All inorganic chemicals were supplied by Fisher Scientific Co. Lyophilized myelin and MBP, both isolated from bovine spinal cord, were kind gifts from Dr Steven W. Brostoff of the Medical University of South Carolina. Lipids were extracted in our laboratory. A “whole-lipid” fraction was obtained by extracting the lyophilized myelin with chloroform/methanol according to Bligh & Dyer (1959). The extract contained 5 to 7% protein. Gel electrophoresis showed this to be mainly proteolipid protein, with lesser amounts of other proteins including MBP. To check the effect of PLP on our complex. lipids were extracted according to Folch et al. (1957) and then re-extracted 6 times using chloroform/methanol/water (7:7: 1, by vol.), leaving I 1% protein. Our results were not significantly different in this case. Lipids were kept in the cold until used. (b) Preparation

of lipid

vesicles

Single-bilayer vesicles were prepared by sonicating the myelin lipids (Bronson Sonifier with microtip probe; 22W). Generally, 6 mg of lipids were dissolved in chloroform/ methanol, the solvent was evaporated using a stream of oxygen-free nitrogen, and then 2 to 3 ml of buffer (10 mi%r-sodium phosphate, 10 mM-Na,EDTA, pH3:4) were added. Immersing the lower part of the conical tube in ice-water prevented overheating, and nitrogen was blown over the open end of the tube. A cycle of sonication for 1 min followed by 1 min rest was repeated 10 times. As sonication proceeded, the solution changed from milky white to faint foggy white, usually by the 4th step of sonication. The sonicated lipids were checked by thin-layer chromatography. X-ray diffraction and freeze-fracture electron microscopy techniques. Aft,er sonication, half of the suspension of vesicles (3 mg lipid) was used to bind MBP: the other half was centrifuged and used as a control for biochemical and X-ray analyses. The starting lipids, lipids in the vesicle and lipid/MBP pellets, and lipids in the supernatants were assayed by thin-layer chromatography on silica gel (Fisher G, 250 pm) using chloroform/methanol/ammonia (65 : 25 :4, by vol.). (c)

Binding

of MBP

to

vesicles

MBP was dissolved in the same buffer as for the lipid vesicles, and the solution was added to the vesicles at room temperature. After a few seconds, particles appeared; these gradually got larger and settled out. The suspension was stirred for 1 h at 4°C to ensure complete reaction.

388

J. SEDZIK,

A. E. BLAUROCK

AND M. HC)CHLI

In one experiment, a solution of cytochrome c (Sigma, horse-heart the vesicles instead of MBP. A similar precipitate formed. (d) Detection

of bound

type VI) was added to

protein

Both samples, vesicles alone and the lipid/MBP complex, were centrifuged for 1 h at 60,000 revs/min (+4”C) using a fixed-angle rotor (Spinco Ti 65; 314,000 g max.). Portions of the starting MBP solution, and of the supernatants above the vesicle and lipid/MBP pellets, were assayed for protein (Lowry et al., 1951). Bovine serum albumin (Sigma) was the standard. The buffer itself served as the blank for the supernatants above the vesicle and lipid/MBP pellets. Bound protein was assumed as the difference between the starting amount of MBP and the amount remaining in the supernatant. (e)

Freeze-fracture

electron

microscope

preparations

The lipid vesicles without MBP were examined before centrifuging. Small portions of the vesicles were clamped between thin copper sheets (0.1 mm) and frozen in a jet of propane (Muller et al., 1980). The copper was previously etched with 40% (v/v) HNO,, washed with distilled water, dried, and coated with 0.1% Formvar in dichloroethane in order to eliminate the interaction of the vesicles with the copper surface. Specimens of lipid vesicles and of the lipid/MBP complex. which had been centrifuged and then exposed to X-rays, also were examined. In a time-study of the effects of MBP, small portions of the vesicles were mixed with MBP, and the mixtures were incubated for various lenths of time before being frozen. Through the use of very thin sandwiches (about 20 ,um), freezing rates of more than 10,000 deg.C/s were achieved (Costello et al., 1982). Fracturing and platinum/carbon replication of samples were carried out at - 100°C and lOm6 Torr (1.3 x lO-4 N/m*) in the freeze-etching apparatus (Balzers BA 360 M) equipped with electron guns and a quartz thin-film monitor. The replicas were floated on distilled water and cleaned in 50% household bleach (Clorox) and then in a 1: 1 mixture of chloroform/methanol. Electron micrographs (Kodak EM 4489) were taken with a JEOL 1OOCX transmission instrument operated at 80 kV. (f) X-ray

diffraction

procedures

Pellets of the lipid vesicles and of the lipid/MBP complex were sealed in Lindeman glass capillaries (1 mm diam.; Blake Industries) together with a small amount of buffer and exposed at 35( +O.Z)“C. Some specimens were exposed 2 or 3 times; these were kept at 35°C between exposures. The rotating-anode microfocus X-ray generator (Elliot GX6) was operated at 40 kV and 25 mA. A double-mirror, point-focusing Franks camera was used to record the X-ray diffraction pattern under vacuum using Ni-filtered radiation (2 = 1.542 A: Blaurock, 1973b; Nelander & Blaurock, 1978); the X-ray beam was -0.2 mm diameter. A stack of up to 4 films (Kodak Kodirex) was exposed. Exposure time generally was 1 to 3 days. A few very long exposures were made. Optical density was measured using a microdensitometer (JoyceeLoebl type III CS). The repeating distance (d-spacing) in a multilayer was calculated using Bragg’s Law. In the case of diffuse scatter, d does not refer to a repeating distance, but the d-spacing and the reciprocal spacing, l/d, nonetheless are convenient measures of position on the diffraction pattern. In order to locate the bilayer bands precisely, inflection points were measured between the bands (arrows in Fig. 2). The inflection points were found by constructing a y/R’ curve, where R is the distance from the center of the diffraction pattern and the constant, y, is chosen to make the curve tangent to the tracing. Our method is equivalent to correcting each tracing by RZ (Wilkins et al., 1971) and then finding the minima between the broad

LIP1

I)/MYELIN

BASIC

PROTEIN

MCLTILAY

ERS

38!1

bands. Radii to the inflection points were then converted to reciprocal spacings. After correcting by R*, the minima between bilayer bands are sensitive to a change in the positionsof the bands (Engleman, 1971;Torbet & Wilkins, 1976). An electron-density profile depicts the projected electron density versus distancethrough the membrane.Profiles were calculated according to the formula: hm.. p(z) = a 1 Fh cos (2nh(r/d))+fi.

(1)

h=k

whet-r, F,, = + (Z,,h2)1’2 is the structure factor of order h with phasesign ( + or - ) and x is the distance. The Zh terms are the integrated intensities, i.e. the areas under the narrow peaks (Fig. 2(b)). The constantsa and fl were fixed by criteria stated in Results?.

3. Results We obtained freeze-fracture, biochemical, and X-ray diffraction data from lipid vesicles and from lipid complexed with various amounts of MBP (Table 1). Multilayers invariably formed when MBP was added, and they formed before centrifuging, not simply as a result thereof. In all specimens, with and without MBP, the fatty chains were packed in the bilayer in the L, state characterized by a diffraction band peaking at d = 4.6 A. (a) Basic observations

on the lipid

vesicles

Thin-layer chromatography showed the same classesof lipids in the starting material, in the vesicle pellet, and in the supernatant (< l”/b of total lipids). Proportions of the different lipids were comparable to published values (Norton. 1977). Freeze-fracture electron microscopy of the sonicated lipids showed mainly single-bilayer vesicles. Many vesicles in Figure l(a) are about 1000 A in diameter, but vesicles up to 3000 b can be seen. Some vesicles are elongated. Some are in contact over large areas, possibly bound together by residual MBP. Multilayers were seen, and also vesicles within other vesicles. The X-ray diffraction patterns from pelleted vesicles showed concentric, broad bands similar to those reported by Wilkins et al. (1971) from sonicated egg lecithin. The diametric densitometer tracings in Figure 2(a) show, on each side of t)he beam-stop shadow, one strong band (I) and two weaker bands (II and III). Band III is stronger than band II, as expected (Wilkins et al., 1971) in view of the large amount of cholesterol in the myelin lipids (Norton, 1977). Weak, sharp rings superimposed on the predominant diffuse intensity showed that multilayers sometimes were present. Based on the integrated intensity in the sharp peaks, in ratio to the total intensity in the broad bands (Fig. 2(a)), generally from 0 to 3% of t’he lipids were in the form of multilayers; in one case, 17%. The repeating distance in the lipid multilayers ranged from 125 to 160 A. The t In the absence of precise knowledge of specimen concentration. thickness and degree of orientation, and integrated flux in the incident X-ray beam, the scaling factor. CC and the average rlrctron density, j?, in eqn (1) are experimentally indeterminate. As a result, some means must be found for comparing diffracted intensity in different patterns.

390

J. SEDZIK,

A. E. BLAUROCK

AND

M. HC)CHLI

FIG. 1. Freeze-fracture electron microscopy of lipid vesicles and lipid/MBP complex. (a) Sonicated lipids (not centrifuged). (b) Complex 6 min after adding MBP (not centrifuged). Both rounded, possibly multilayered, vesicles (upper left) and large flat multilayers (lower right) can be seen. Magnification, 62,500 x ; the bar represents 1000 A.

LIPID/MYELIN

BASIC

PROTEIN

39 1

MULTILAYERS

(a)

Shadow Of stop

beam I

0.08

,

0.06

1

I

0.04

0.02

I

0.0

Reuprocal

0.02

spacing I

0.04

I

O-06

I

0.08

(H-‘) I

(b)

I

0.08

I

006

I

0.04

I

0.02 Reoprocol

0.0 spacmg

,

002

0.04

0.06

O-08

(He’)

FIG. 2. Diametric densitometer tracings of X-ray diffraction films. The optical density I is plotted W-sus reciprocal spacing (l/d). (a) Diffraction from single-bilayer vesicles: 201 h exposure. s = 92.7 mm. Two tracings are shown. The inner, lower tracing is of the 3rd film in a stack and shows bilayer band I on both sides of the beam-stop shadow. Two multilayer reflections were visible on the original negative; an estimated baseline for the peaks corresponding to one of them (h = 2, at 0.014 .%-I) is sketched here (- - -). The outer tracing is of the first film and shows bands II and III. The arrows indicate inflection points; these become minima after I(R) is multiplied by R*. Integrated intensities of orders 1 and 2 from a lipid/MBP complex are superimposed ( x ; expt 8). (b) Diffraction from a lipid/MBP complex; 144 h exposure, s = 92.7 mm. The inner tracing is of the 4th film; the outer tracing is of the first film in a stack. The peaks are indexed as orders 1, 2, 3 and 5 of a repeating distance of 75.4 A (expt 8). A background has been drawn beneath the peaks ( - - - - ), Order 4 was not visible on the original negative. FWHM, full width at half the maximum height. 14

392

J.

repeating distance (Palmer & Schmitt,

SEDZIK,

A.

presumably 1941).

E.

BLAUROCK

was

(b) Basic observations

AND

M.

HOCHLI

large because the ionic strength

on the lipid/MBP

was

low

complexes

Much of the added MBP was bound to the lipids although the proportion varied (Fig. 3 and Table 1). The best-fitting line in Figure 3 shows a linear trend-of MBP bound versus MBP added. No saturating amount of MBP was found. Thin-layer chromatography showed the same classes of lipids in the lipid/MBP pellets as in the starting material, and in similar proportions. No lipids were detected in the supernatant. Two types of structure were seen by freeze-fracture electron microscopy after adding MBP to the vesicles. The majority of the fracture area consisted of aggregated vesicles (Fig. l(b), upper left). These are considerably larger than in Figure 1(a), showing that MBP caused the original vesicles to fuse. Flattening and close apposition within the aggregates indicate strong interaction over large areas of the vesicles. The interaction evidently is mediated by the MBP. Individual vesicles may be multilayered. The second type of structure was regularly observed within the heavily aggregated regions. This structure was remarkably flat and obviously multilayered (Fig. l(b), lower right). In places, the length of the shadow at steps in the fracture plane is regular, indicating even spacing between bilayers. More than 20 steps were counted. The planar fractures extended several micrometres. The fracture faces in Figure 1 look smooth and finely textured, both in the presence and in the absence of added MBP; no “particles” are seen. Assuming that the fractures occurred at the centers of bilayers, we find no indication that MBP is a trans-bilayer protein. Similar observations were made on freeze-fracture specimens after they had been centrifuged and exposed to X-rays.

+

MBP

FIG. 3. Binding of MBP to the myelin lipids. sonicated lipids. The broken line is a least-squares fair correlation between MBP added and bound

(y

added

hgl

Various amounts of MBP were fit to the + symbols (Bevington, = 044).

added to 3 mg of 1969); there is a

LIPID/MYELIN

Binding

BASIC

PROTEIN

TABLE 1 or cytochrmne c to single-&layer

of MBP

Protei@ Added

Bound(mg)

oal

0.06

0.3 0.4

0.3

d-spacing

(A)’

Number layers,

of N

n.d. n.d.

77.2

0.5

0.16 0.5

0.9

0.3

79.5,

82.7,

84.5

n.d.

27, 30. 32

W!J

0.6

76.6,

73.3,

74.7

n.d.

24,

20. 22

0.9

0.9

76.8,

77.8,

77.4

21

24.

25, 24

I .3

0.8

1.4 2.0

0.7 1.8

75.4 78.3, 82.0,

8 n.d. n.d.

22 25, 29,

24 29

2.1 2-l 2-l 2.2 2.2

0.9 1.2 n.d. I.6

24 23, 24

n.d.

0.5

77.8

nd. nd. nd. nd. 8

IV Ii' IX I9 20g

2.4 2.7 2.9 2.9 n.d.

I.1 I.8 2.4

nd. 11

1.5’ n.d.

75.5 75.8, 77.2, 74.6, 76.5

21b

2.4

0.53

85.9

77.1 76.0, 77.4

’ ’ s ’ ’ j

85.6,

76

75.9 75.0 74.4

33, 23

n.d. 3.42f1.64 5.74+0.63 5.61f0.53 5.29f0.99 3.99f0.67 4.69kO.57

(d = 79.5) (d = 82.7) (d = 84.5)

5.29+1.07 4.35kO.37 +96+0.29 5.32kO.86 4.74+ 190 4.55k0.53 4.7OkO.31 4.12kO.73 4.7OkO.69

n.d.

25

4.43io.37

5.00+

n.d.

23 23, 23 24, 22 22, 21 24

nd.

33

n.d.

6 9

c in expt

28. 24 25 21 21

IllI,’

n.d.

-----------------------------

a Per 3 mg lipid. b MBP in expts 1 to 20 and cytochrome ’ d-spacings accurate to kO.4 A. dd-A3

5 12

vesicles

Inter-bilayer space (A)d

81.4, 77.6 73.7 74.1

77.0 82.1

393

MULTILAYERS

5.62kO.25 5.79f0.55 6.58kO.88 4.83f0.41

(d = 76.6) (d = 76.8) (d = 77.8)

(d = 78.3) (d = 82.1)

(d = 85.6)

1.09 (d = 75.9) (d = 75.0) (d = 74.6)

21.

A.

Excluding diffuse background (Fig. 2(b)). Per 6 mg lipid in expt 19. Lipids re-extracted as in Materials and Methods. Not centrifuged; hence binding not determined (n.d.). Lipids plus 3 pg gangliosides. Lipids plus 30 pg gangliosides.

Freeze-fracture electron microscopy showed that aggregates formed within 20 seconds of adding MBP, and large planar multilayers were visible after six minutes (Fig. 1(b)). Since sharp X-ray rings were recorded from non-centrifuged precipitate (Expt 13 in Table I), our various observations consistently show that multilayers formed before centrifuging the complex. As noted, adding MBP to the lipid vesicles changed the X-ray diffraction pattern from broad bands to concentric sharp rings. The rings indicate large numbers of multilayer regions with various orientations (Blaurock, 1982). In a

394

J. SEDZIK,

A. E. BLAUROCK

AND

M.

HC)CHLI

diametric densitometer tracing, each diffraction ring gives rise to two narrow peaks (Fig. 2(b)). The reciprocal spacings of the peaks were precisely in the ratios of 1:2:3..., indicating a single type of multilayer in each specimen. The d-spacing varied from specimen to specimen, and from exposure to exposure, over a range of 73 to 86 A (Fig. 4 and Table 1). In addition to sharp rings, the lipid/MBP complexes gave diffuse X-ray intensity. In Figure 2(b), the diffuse intensity is weak around peaks 1 and 2, relative to the peaks themselves, but it becomes relatively stronger with increasing reciprocal spacing. The diffuse intensity and a greater FWHM (full width at half the maximum height) of the fifth-order peaks together are evidence of a variable repeating distance (stacking disorder) within each multilayer region (Blaurock, 1982). In most cases rings up to h = 5 were observed (Fig. 2(b)), while in a few cases two sharp rings were observed (h = 1 and 2) and then only broad bands at larger reciprocalspacings. The comparison indicates that the degree of stacking disorder varied from specimen to specimen. The diffuse scatter in Figure 2(b) includes bands II and III similar to those in Figure 2(a). This observation indicates that the lipid bilayers in this complex (Expt 8) all had much the same structure (Blaurock, 1982). We conclude that the stacking disorder was the result of variable spaces between bilayers. This conclusion was confirmed by a model calculation described below. The X-ray diffraction pattern had the same general form when a specimen was exposed a second and a third time. The d-spacing sometimes changed by a few angstrom units, however (Table 1). Often the d-spacing decreased, with a limiting value of dmin = 73 A. In two cases the d-spacing increased markedly. A complex also formed when cytochrome c was substituted for MBP. Again there were sharp rings superimposed on diffuse scatter. At least six rings could be seen, which were all orders of a spacing of d = 85.9 A (Table 1)

MBP

FIQ. depend

bound

(mg)

4. Repeating distance in the multilayer complex in any simple way on the amount of MBP bound.

zxrs~g

MBP

bound.

The

d-spacing

does not

LIPID/MYELIN

BASIC

PROTEIN

MULTILAYERS

39.5

The average number of layers in the multilayer regions, N, can be estimated from the width of the first-order peak (FWHM in Fig. 2(b): see Nelander & Blaurock, 1978). The estimates of N in Table 1 vary from 5 to 21. In plots of the data, N did not appear to correlate. with the amount of MBP added, or bound. N is regarded as a lower limit on the actual number of layers, since stacking disorder may broaden the first-order peak, reducing N (Blaurock, 1982). In two cases, gangliosides were isolated from lyophilized myelin (Svennerholm & Fredman, 1980) and added to the lipids before sonicating them. In complexes including gangliosides, d-spacings and values of N were comparable to those in t)he absence of gangliosides (Table 1). (c) Structural analysis The five-part analysis of the lipid/MBP complexes is based on d-spacings, MBP contents, electron microscope observations, and diffracted X-ray intensity, both Hragg reflections and diffuse scatter. In subsection (c)(i) we confirm that the diffuse scatter in Figure 2(b) arises from the multilayers, and we then show that the lipid bilayers in the vesicles and in the multilayers have similar profiles. In subsection (c)(ii) we show that MBP is a regular part of the multilayers. In subsection (c)(iii), theoretical electron-density profiles are developed. These indicate that the phasing is independent of the choice of model, within very broad limits. In subsection (c)(iv) we consider the consequences of the fact that the models predict changes in the diffraction as a function of MBP content, which are not observed. We are therefore led to consider two different models of binding. Finally, in subsection (c)(v) profiles of the vesicle bilayer and of the multilayers are calculated. When the profiles are scaled according to either of two binding models, they indicate similar distributions of MBP. (i) Constant Mayer

thickness

We first considered the different d-spacings. Plots of these versus MBP added (not shown) or MBP bound (Fig. 4) did not show a significant correlation. The t,endency of the d-spacing to change, in repeated exposures of a given specimen, underscores the fact that the d-spacing is independent of the MBP bound. Next, we tested our interpretation that the diffuse scatter in Figure 2(b) is due to stacking disorder in the multilayers, and does not arise from some nonmultilayer phase. The test was to calculate a theoretical diffraction pattern for comparison with Figure 2(b). To do this, one needs to assumea model profile and a function describing the distribution of widths of the inter-bilayer spaces. In t,heory, any distribution can be approximated by a Gaussian function (Guinier, 1963). In practice, different groups have found that the Gaussian distribution predicts diffuse scatter in good agreement with observations, while some other plausible distributions are unsatisfactory (Hosemann & Bagchi, 1962; Schwartz et (~1.1975; Blaurock & Nelander, 1976). The most probable width of the inter-bilayer spaces in the specimen of Figure 2(b) was 22 A (Expt 8 in Table 1). Given a plausible profile (model (3) in Fig. 6), we then varied the width of the Gaussian distribution. When this width

390

J. SEDZIK,

A. E. BLAUROCK

AND

M. HOCHLI

was 9 A FWHM, the predicted diffraction (Sedzik & Blaurock, unpublished results) was in good agreement with Figure 2(b) in respect of the width of peak 5 and the ratio of the height of peak 5 to the height of the underlying band III. In addition, the peculiar shape of band II in Figure 2(b) was accounted for, in the predicted pattern, by a small peak 3 superimposed at one edge of the band. We conclude that the diffuse scatter beyond 0.05 A-’ in Figure 2(b) is due largely to stacking disorder; i.e. peak 5 and band III both arise from the same multilayers. Part but not all of the diffuse scatter from the origin out to 0.05 A-’ is accounted for in the same way. We apply this conclusion to show that the lipid bilayer in the multilayers had nearly the same structure as in the original vesicles. Thus the relative heights of bands II and III in Figure 2(a) and (b) are comparable, and the inflection points defining the bands (arrows) are at similar reciprocal spacings. The inflection points (Table 2) shift very slightly, suggesting a thinning of the lipid bilayer by 0.7 A when MBP binds (P < 1% by Student’s t-test). Measurements on other patterns confirmed that the binding did not greatly affect the lipid bilayer. The dimensions allow just one bilayer per repeat in the multilayer. After multiplying the curve in Figure 2(a) by R2, the bands were centered approximately at n/43 A, n = 1, 2 and 3, indicating layers of headgroups centered 43 A apart in the vesicle bilayer (Wilkins et al., 1971). This dimension was confirmed in a calculated profile (Fig. 7). Allowing 5 A from the center to the outer surface of each headgroup layer, the estimated overall thickness of the vesicle bilayer is 53 A; of the lipid bilayers in the multilayer, 52 A. Given the d-spacings in Table 1, there can be one 52 to 53 A thick bilayer per repeat. In addition, there is room for a 20 to 33 A space between bilayers (“Inter-bilayer space” in Table 1). (ii) A new membrane Adding MBP to the single-bilayer vesicles created a new membrane. If the multilayers consisted simply of hydrated lipid bilayers identical to the vesicle

Reciprocal

TABLE 2 spacings of the injlection points between bands I to III in the small-angle patterns Minimum

9, pecimen Single-bilayer vesicles Lipid/MBP complex Lipid/cytochrome e complex

I-II 0.03827 0.03883

(A-‘) +040035 f 0@0037’ n.d.b

between II-III

(A-‘)

0.04964 +040077 0.05051+ 0@O070a 0.04970 & 0@0077’

Inflection points were measured as described in Materials and Methods on diametric densitometer tracings made at intervals of 30” azimuthal angle. Each value in the Table is the mean of 13 measurements f the standard error of the mean. ’ The larger reciprocal spacings of the inflection points in the lipid/MBP pattern indicate a thinning of the lipid bilayer by 0.7 A. b Inflection too broad to locate precisely. ’ The inflection point is at the same position as in the vesicle pattern, to within the error of measurement, indicating the same bilayer thickness.

LIPID/MYELIN

BASIC

PROTEIN

39;

MULTILAYERS

hilayer, then the integrated intensities of the sharp rings (1,) would “sample” the intensity from the vesicles. In fact, there is no way to scale the I, terms onto the vesicle curve, as is illustrated by the crosses in Figure 2(a). That this is true of the complexes generally, is illustrated by Figure 5. Figure 5 is a plot of the ratio I,/I, for various complexes ver.sus the d-spacing (filled squares). In Figure 5 we have also plotted the ratio obtained by sampling t,he intensity from the vesicles at h/d, h = 1 and 2 (crosses). For all complexes save one, Ii/I, is significantly different from the ratio for the vesicles. However, two possible corrections need to be considered. First, the thinner lipid bilayer in the complex (Table 2) is predicted to have a smaller ratio of Z,/I, (Sedzik & Blaurock, unpublished results). Hence, the effect of adding MBP is understated in Figure 5. Second, given any reasonable model of the disorder, both intensities are predicted to decrease, but I, will decrease more than /1 (Blaurock, 1982). Assuming the 9 A variability in the case of experiment 8. the ratio will have increased by 5%. This is less than the standard deviation in Figure 5. i.e. the effect of the stacking disorder is insignificant. This is generally the case, and we conclude that adding MBP increases I,/I, significantly. It follows that the MBP is a regular part of the new structure. The MBP is located in the new membrane by calculating electron-density profiles from the experimental data. After suitably scaling two profiles, one with MBP and the other without, the electron-dense protein can be located by subtracting one profile from the other (Fig. 7). The necessary phase-signs (eqn (1)) are chosen as follows.

Complex ,

2Vewles 0

73

75

77

79

81

83

85

d-spacing(8) FIG. 5. Experimental intensity ratio, 1,/I, verse d-spacing. (m) For the complexes; error bars indicate the standard deviation of 8 measurements. (+) For the single-bilayer vesicles; in this case the standard deviation generally is too small to indicate. Curves similar to those drawn here are predicted generallv from the lipid bilayer profile and models (3) and (4) in Fig. 6.

398

J. SEDZIK,

A. E. BLAUROCK

AND

M.

HOCHLI

(iii) Choosing phases 1971; Blaurock, 1973a; Blaurock, 1982) and Theoretical studies (Engelman, experience with natural and reconstituted membranes (Wilkins et al., 1971; Blaurock, 1973c) show that membrane-associated protein perturbs the lipid bilayer diffraction. Despite some striking effects on diffracted intensity, the phasing remains basically that for the lipid bilayer (Blaurock, 1982). Widely divergent models of the lipid/MBP complex (Fig. 6) confirm the previous results. The theoretical models in Figure 6 all assume the same profile for the lipid bilayer. This profile was derived from relevant data in the literature (Rand & Luzzati, 1968; Blaurock & Nelander, 1979). The diffraction calculated from the model bilayer profile was similar to experimental diffraction from t,he vesicles and from myelin-lipid multilayers (Franks et al., 1982). Figure 6 also depicts four possible models for associating protein (hatched areas) with the membrane and shows their effects on Z,/Z,. The integral protein and a related model (( 1) and (2)) predict a decrease in Z,/Zz when protein is added (A and A, respectively). This behavior is flatly contrary to observation (Fig. 5), and these models are therefore eliminated. The peripheral protein and a related model ((3) and (4)) correctly predict an increase in Z,/Z, when protein is added (+ and x , respectively). Thus the modeling study points to MBP being a peripheral protein, consistent with electron microscope observations. Equally important, all models predict the same phases at all protein contents considered?. It follows that the crucial information is the intensities themselves. Profiles were calculated accordingly (Fig. 7; see below). (iv) A discovery Figure 5 does not fully bear out our modelling, leading us to an important discovery. A simple curve can be fit to the various intensity ratios in Figure 5, i.e. most of the filled squares are within one standard deviation of the curve shown. When intensity ratios predicted from either model (3) or (4) were plotted versus d-spacing, a similar curve could be drawn through all points corresponding to a given MBP content. However, we found no correlation between the residuals of the experimental points and amounts of MBP ranging from 0.15 to 2.4 mg per 3 mg of lipid. This observation contradicts the prediction of our modelling, that curves of Z,/Z, versus d-spacing will be quite different for different amounts of MBP (Fig. 6). A s a result, none of the four models fully accounted for the data in Figure 5. In order to deal with this discrepancy, we propose two models of binding in stages. While they have distinct characters, the two models nonetheless lead to similar conclusions as to the precise location of MBP and its interaction with the bilayer. First two-stage model of binding. In the first stage of this model, MBP would bind to vesicles until a critical surface density (not necessarily saturating) was reached. At this point, the second stage would begin. In this stage, two vesicles t The phases are the same for all points in Fig. 6. For if the phase of either I, or I, changed, the intensity would be zero at some point, and the ratio 1,/Z, would be either zero or infinite. Neither event occurs in Fig. 6.

LIPID/MYELIN

BASIC

MBP

PROTEIN

bound

Fro. 6. Predicted intensity ratio 1,/I, ‘ucrsw MBP model profiles including protein (hatched areas): (A) (model (1)); (A) predicted from trans-bilayer protein (2)); (+) predicted from entirely superficial (extrinsic) protein partly superficial and partly inserted into distance of 750 A was assumed for the calculations.

(mg/mg

MULTILAYERS

Iapld)

bound. Ratios were predicted from 4 theoretical predicted from tram-bilayer (intrinsic) protein also extending 10 A outside the bilayer (model protein (model (3)); and ( x ) predicted from the headgroup layer (model (4)). A repeating Predicted phase signs: - , - in all cases.

would collide, stick together, and then break open so as to form parallel bilayers with the MBP sandwiched between them. The newly exposed inside surface of the broken vesicles would bind MBP, and other vesicles would stick and break open, thereby generating the multilayer. The multilayer would contain a definite amount of MBP, independent of the amount added. Second two-stage model of binding. In the second model, MBP would saturate primary sites on the bilayer during the first stage, and then additional MBP would bind at secondary sites of lower affinity during the second stage. Given either model of binding, the data in Figure 5 indicate that the intensity ratio would need to increase rapidly in the first binding stage and then remain constant during the second stage. The first model assuresthis behavior: once the critical surface density was exceeded, bilayers would come together and further binding in the inter-bilayer spaces would be blocked. As a result, the intensity ratio would not depend on the amount of MBP added. This model offers a plausible explanation for the X-ray data, all the more so since visible aggregates generally formed before all the MBP had been added to the vesicles. The second model of binding requires that adding MBP to the primary sites would change the ratio Ii/Z,, but adding to the secondary sites would not, i.e. I,/Z, for all complexes containing MBP in the secondary sites would need to be t.he same. This puts a special constraint on the distribution of MBP at the secondary sites, making the second model less likely. Nonetheless, we have pursued both models, leading us to similar although not’ identical conclusions about the structure of the complex.

J. SEDZIK,

400

A. E. BLAUROCK

AND

M. HOCHLI

(v) Calculated profiles Profiles of the multilayers were calculated using the same phase signs as for the vesicle diffraction. This is the phasing expected on the basis of a perturbation of the lipid bilayer pattern (Blaurock, 1973a), and also the phasing predicted by all of the models at all MBP contents (Fig. 6). Figure 7(a) shows calculated profiles of the vesicle bilayer and three lipid/MBP complexes. To obtain I, and I, for the vesicles, the curve in Figure 2(a) was sampled at h/75*0 A, h = 1 and 2. For the complexes, the Z,, terms are the areas under peaks 1 and 2 (Fig. 2(b)). Figure 7(b) shows difference profiles obtained by subtracting the bilayer profile in (a) from the upper three profiles. The profiles in Figure 7(a) have been scaled to one another under the assumptions (1) that the lipid bilayer has nearly the same thickness with and without MBP, and (2) that MBP is not an integral protein. Accordingly, the three profiles of complexes in Figure 7(a) were scaled to the vesicle profile by superimposing all four profiles over the core. The profiles of the lipid/MBP complexes in Figure 7(a) all lie above the profile of the vesicle bilayer in the inter-bilayer space (I-B space) and over the lipidheadgroup layers (HG). This is the effect expected assuming that MBP (0.45 e/A3; Blaurock & Nelander, 1979) displaces water (O-334 e/A3) in the inter-bilayer space and, possibly, from between lipid headgroups. The peak-to-peak distance in the lipid-bilayer profile is 43 A compared to 45 A in the profiles of complexes

Bllayer

I - 100.0

I - 50.0

I 0.0

I 50.0

I 100-O

FIG. 7. (a) Electron-density profiles of the vesicle bilayer and of lipid/MBP complexes. The electron density p(r), in arbitrary units, is plotted versu8 distance through the bilayer. Three repeats are shown. The lowermost profile (- ) shows the vesicle bilayer, and the upper three profiles ( -) show complexes having d 1 7.5 A (expts 3, 8 and 18 in Table 1). The profiles were calculated as in the text and then scaled as for the $ral two-stage binding model (see Results). A profile for bovine brain-stem lipids (Franks et al., 1982) calculated by sampling their Fourier transform at h/75 A, h = 1 and 2, looks like the lowermost profile here except that the peak-to-peak distance is 42 A. HG, headgroups layer; Core, lipid fatty chains; I-B space, inter-bilayer space. (b) Difference profiles showing the excess density attributed to MBP (hatched area). These 3 profiles were obtained by subtracting the lower profile in (a) from the upper 3 profiles. By comparison to Fig. 9, the hatched area here indicates 0.53 mg MBP per 3 mg lipid.

LIPID/MYELIN

BASIC

PROTEIN

MULTILAYERS

401

containing from 0.15 to 2.4 mg MBP. As noted above, the lipid bilayer in the complex is, if anything, slightly thinner than the original vesicle bilayer. Our interpretation of Figure 7(a) is that the increase to 45 A is an apparent effect of the added MBP, and that the 2 A increase does not represent a true thickening of the lipid bilayer. The difference profiles in Figure 7(b) are 22 to 24 A wide (FWHM). They have the width and shape predicted, at the present resolution, for a uniform distribution of MBP just wide enough to fill the inter-bilayer space. Thus the observation, that about one-third of the hatched area is in the headgroup layers, is equivocal. We can say only that MBP may possibly penetrate between lipid headgroups. Higher-resolution profiles will better indicate the degree of penetration. Consistent with the first model of MBP binding, the profiles in Figure 7 indicate much the same amount of MBP, independent of the amount added or bound (Table 1). The amount of MBP has been estimated by using the cytochrome c complex as a calibration: given the hatched area in Figure 9 and the amount of cytochrome c bound (Expt 21), the hatched area in Figure 7 represents 0.53 mg of MBP per 3 mg of lipid. A re-scaling of the profiles is required to conform to the second binding model (Fig. 8). The profiles need to meet scaling assumptions (1) and (2) above, but they need also to meet a further condition. Starting with two of the profiles of complexes in Figure 7 (0.8 and 2.4 mg MBP bound), the profile corresponding to the largest amount of MBP was scaled up (greater a in eqn (1)) in order to make the areas in the corresponding difference profiles (Fig. S(b)) have the same ratio as the amounts of MBP bound, 3 : 1. The two difference profiles now have different shapes, indicating some difference in MBP binding (Fig. S(b)). The higher difference profile also is the broader one (41 1%FWHM). It is too wide to be consistent with a uniform distribution of MBP

Dlstonce

FIG. 8. (a) Profiles

(8,

of the vesicle bilayer and 2 lipid/MBP the text and then scaled as for the seeolld two-stage binding by the same process as in Fig. 7. The areas are in the ratio

complexes. Profiles were calculated as in malel. (b) Two difference profiles obtained of the amounts of MBP bound, 3 : 1.

40%

J. SEDZIK,

A. E. BLAUROCK

AND

M. HUCHLI

between bilayers; instead, if valid, it unequivocally indicates MBP in the lipid headgroup layers. The profiles in Figure 9 were computed similarly for the repeating unit in the lipid/cytochrome c multilayers and for the vesicle bilayer. The profiles superimpose nicely over the entire bilayer. The good fit indicates much the same bilayer thickness with cytochrome c as without. Constant bilayer thickness also is indicated by the data in Table 2. The peak-to-peak distance in the bilayer is smaller by 1 L! than in Figure 7(a), owing to the different resolution in Figure 9. The hatched area in .Figure 9(b) shows the excess- density attributed to cytochrome c. This area is entirely in the inter-bilayer space, indicating that cytochrome c does not penetrate the bilayers, with the possible exception of a few low-density (Blaurock, 1972), hence undetectable, amino acid side-chains. In sum, the difference between profiles of the lipid/MBP multilayer and the vesicle bilayer shows MBP in close association with the lipid bilayer. The contrast with the lipid/cytochrome c profile emphasizes the extensive contact of MBP with the bilayer. Neither protein has much effect on the structure of the myelin-lipid bilayer.

4. Discussion Our freeze-fracture electron microscopy and X-ray diffraction observations demonstrate a strong affinity of the myelin lipids for MBP. The single-bilayer vesicles are stable; there are few multilayers even after centrifuging at high g-forces and incubating at 35°C for days. When MBP is added, however, vesicles aggregate and fuse, and extensive multilayers form quickly and without centrifuging. Our results, in conjunction with published work, provide strong support for the hypothesis that MBP is required to form and maintain the cytoplasmic junction

Bllayer I-B space

(b)

I

I -IOQO

I -500

I 0.0 Distance

I 50.0

I 100.0

(X)

FIQ. 9. Profiles of the vesicle bilayer and the lipid/cytochrome c multilayer. Three repeats are shown. (a) Profiles of the vesicle bilayer (-) and the repeating unit in the multilayer (expt 21; d = 85.9 A). The vesicle profile was calculated as in the text. The 2 profiles have been scaled by superimposing the bilayers. The peak-to-peak distcmce, 42 8, is smaller by 1 A as a result of the 14 A resolution here (d/(2 h,,)) compared to 19 A in Fig. 7. Note the wider inter-bilayer space than in Fig. 7. (b) Difference profile showing the excess density attributed to cytochrome c (hatched area).

LIPID/MYELIN

BASIC

PROTEIN

103

MULTILAYERS

in CNS myelin. Privat et al. (1979) and Matthieu et al. (1980) show that, in genetic mutant mice lacking MBP, CNS myelin forms with the normal spiral structure: the eytoplasmic space is, however, much wider than in normal myelin. We now show that MBP/lipid ratios similar to that in normal CNS myelin (10% by weight) cause multilayers to form, and the multilayers are as compact as normal CNS myelin. Presumably, MBP causes the cytoplasmic membrane junction to form in CNS myelin once the membranes have been brought close enough by some means during growth; the micrographs presented by Privat et al. (1979) suggest how close they need to come before MBP can complete the junction, MBP bound to a monolayer or bilayer is protected to some extent from the action of trypsin (London et al., 1973; Wood et al., 1974); from covalent labeling (Wood et al., 1977); and from antibody binding (Boggs et al., 1981a). MBP reduces the temperature and enthalpy of the thermotropic transition of dipalmitoyl-phosphatidylglycerol (Papahadjopoulos et al., 1975; Boggs $ Mosoarello, 1978; Boggs et al., 1981b). Our X-ray results place the MBP molecule in a position to associate with many lipid headgroups, which may explain these effects. (a) Experimental

model of the cytoplasmic

space

Several labeling studies have located MBP on the cytoplasmic side of the CNS my&n bilayer. Thus lactoperoxidase-catalyzed iodination (Poduslo & Braun. 1975) and salicylaldehyde and pyridoxal phosphate labeling of protein (Golds & Braun, 1976) indicate this location. Histochemical (Adams et al., 1971) and immuno-labeling electron microscopy studies (Herndon et al., 1973; Omlin et aE., 1982) also suggest the cytoplasmic location. We therefore compare the interbilayer spaces in our complexes to the cytoplasmic spaces in CNS myelins. First, a range of 20 to 33 A was obtained for the inter-bilayer spaces (Table 1). The limiting width, 20 A, is comparable to the widths, 15 to 20 A, obtained for the cytoplasmi; spaces in CNS myelins from various species (Blaurock, 1982). Second, the inter-bilayer spaces vary within each multilayer region, as do the cytoplasmic spaces in native myelin (Nelander & Blaurock, 1978). Third, the shapes of the respective profiles are similar (Blaurock, unpublished results). Lastly, the MBP/lipid ratio indicated by Figure 7, 0.18 mg of MBP per mg of lipid, is comparable to the ratio in native myelins: 0.2 mg of MBP per mg of lipid, including the two half bilayers adjacent to the cytoplasmic space and excluding the two half bilayers adjacent to the extracellular space. Thus the inter-bilayer space in our complex resembles the cytoplasmic space in CNS myeiin. Based on the comparable inter-bilayer spaces, we suggest that the forces forming and maintaining them may be similar in native myelin and in our multilayers, and that MRP may assume similar conformation(s) in the two structures. Since the inter-bilayer space is similar to the cytoplasmic space, the multilayer appears to be a good model for investigating processes in native myelin. (b) Formation Experiments demonstrating are cited in the Introduction.

of the complex

the affinity of MBP for negatively In our experiments, the positively

charged lipids charged MBP

464

J. SEDZIK,

A. E. BLAUROCK

AND

M.

HOCHLI

molecules will have diffused to the negatively charged vesicles (Norton, 1977) via electrostatic attraction. As discussed below, the actual binding may involve various interactions. It was suggested to us that the lipid vesicles might aggregate, due to MBP bound on the outer surfaces (Young et al., 1982; Lampe & Nelsestuen, 1982), and then collapse as a result of centrifuging. Both electron microscope and X-ray experiments demonstrate, however, that multilayers with a normal d-spacing formed without centrifuging (Table 1 and Fig. 1). The d-spacings are too small for two lipid bilayers in the repeating unit, and we have concluded that MBP reached every space between bilayers. Finding MBP in every inter-bilayer space (Fig. 7) implies, remarkably, that the vesicles broke open as the multilayers formed. Vesicles have fused into larger vesicles, and extensive planar sheets have formed (Fig. 1). Pressing together the halves of the electron microscope sample holder, after incubating and before freezing, may have forced the multilayers to become planar (Wu et al., 1977). It has been proposed that MBP molecules on the surfaces of proximate bilayers may form dimers to bind the bilayers together (Smith, 1977; Golds & Braun, 1978) or, alternatively, that each MBP molecule may span from one bilayer to the next, across the inter-bilayer space (Boggs et al., 198lc). Although the latter model is the simpler, our structural analysis does not distinguish between these two possibi1ities.t The lipid/MBP complex is surprisingly variable in respect of the fraction of MBP bound and the d-spacing (Figs 3 and 4, Table 1). Regarding the variable binding, we note that P, exhibits simple saturating behavior when used in place of MBP (Sedzik & Blaurock, unpublished results). Hence the variable binding appears to be characteristic of MBP under our experimental conditions. Potentially important factors are the rates of adding MBP and stirring as it is added; but if, as we have suggested, vesicles stick together and convert to multilayers when a critical surface density of MBP is reached, then these rates need not be critical. The balance of the bound MBP, above 0.53 mg (Fig. 7), may possibly form a second phase in the centrifuge pellet; this MBP might be aggregated and rendered sedimentable by limited amounts of lipid. The balance of the MBP might in some caseshave bound to the surfaces of large particles of complex, but such binding cannot reasonably account for the largest amounts bound (Table 1). Binding to the surfaces of laboratory vesselsprobably does not account for the balance, since a full monolayer of MBP 20 A thick on these vessels would amount to 1 to 10 pg, i.e.

< 1o/o of the balance

in several

cases. Further

studies

may

decide

between

various possibilities, e.g. a protein-rich phase would be expected to separate from the multilayers under density-gradient centrifugation. The variable d-spacing is based on a variable inter-bilayer space (Table 1). + The area attributed to MBP in Fig. 7 can be interpreted in various ways. It. can be divided in the middle of the inter-bilayer space and assigned half to each bilayer, it can be interpreted in terms of MBP molecules interdigitating within the space, or interpreted in some other way. Similarly, when profile (2), (3) or (4) in Fig. 6 is repeated in a mode1 multilayer, and the resulting multilayer profile will be the same as for interdigitation of protein molecules projecting 20 A from the bilayer.

LIPID/MYELIN

BASIC

PROTEIN

MULTILAY

ERS

105

Assuming the MBP molecules associate with the lipid bilayer in different orientations, or with different conformations, then the usual tendency of d to decrease with time might mean that the association fluctuates due to thermal motions and tends toward a most tightly bound structure having minimal t)hickness. In two casesthe d-spacing increased with time (Table 1); we are as yet unable to interpret this behavior. (c) Distribution

of MBP

in the reconstituted

membranes

In order to scale the profiles, we assumed that the lipid bilayer did not change when MBP was added, and that MBP was not a trans-bilayer protein. The grounds for the latter assumption are the electron microscope observations (Fig. l), and X-ray diffraction data coupled with the modeling study (Figs 2, 5 and 6). We also note the extractibility and hydrophilic nature of MBP: MBP extracts like a peripheral protein (Lowden et aE., 1966; Eng et al., 1968); it is more hydrophilic, overall, than most proteins (Blaurock & Nelander, 1979); and the more hydrophilic residues (Wolfenden et al., 1981) are distributed rather uniformly in the sequence (Braun & Brostoff, 1977). The profiles in Figure 7 therefore were matched over the core of the bilayer. The sealing of the profiles of the three complexes in Figure 7(a), relative to the profile of the vesicle bilayer, is the minimal scaling consistent with our assumptions. A scaling-up of the three profiles is possible by increasing c1(eqn (1)) and keeping a match at the center of the bilayer. After re-scaling, most of the difference area remains in the inter-bilayer space (Fig. 8). The difference profiles in Figure 8 have different shapes, however, indicating different binding sites as post,uiated for the second two-stage binding model. The ultimate limitation to scaling-up is that the highest electron densities implied must be physically reasonable (Kirschner, 1974; Blaurock & Nelander, 1979). MBP is distributed in our artificial membrane somewhat similarly to basic proteins in native PNS myelin. Kirschner & Ganser (1980) have calculated electron-density profiles for PNS myelin without (shiverer mouse) and with the basic proteins I’, and P,. (In contrast to the CNS, normally compact PNS myelin is found in the absence of these basic proteins.) A small difference in the profiles indicates the absence of I’, and P, from the cytoplasmic space and either t’he absenceof these proteins from, or else a rearrangement of headgroups in, the lipid headgroup layer (Kirschner & Ganser, 1980). (d) Interaction

of MBP

with the bilayer

A strong interaction is indicated, not only by the collapse of the vesicles, but also by the very position of MBP. Whatever its precise distribution, it is apparent that many MBP residues are in a position to interact with the myelin lipids. The MBP will have displaced water that is normally at the biiayer surface (Small. 1967; Levine & Wilkins, 1971; Worcester & Franks, 1976), and this is water strongly bound to the bilayer (LeNeveu et al., 1976). In order to explain the displacement of water, the interaction energy of MBP with the lipids must be

406

J. SEDZIK,

A. E. BLAUROCK

AND

M. HOCHLI

greater than the interaction energy of the water. We therefore suggest that MBP forms numerous specific bonds with the lipid headgroups. Charge bridges and, particularly, hydrogen bonding between MBP and lipid headgroups are quite possible. As depicted in Figure 10, charge bridges would be expected to form between charged MBP residues (positive: Arg and Lys; negative: Asp and Glu) and oppositely charged groups on the lipids (negative: phosphate, carboxyl and sulfate; positive: ammonium and trimethyl ammonium). Hydrogen bonds can mediate between oppositely charged groups (Cotton et al., 1973) and also link uncharged groups. Hydrogen bonding might account for an apparent non-ionic contribution to the binding, noted by Palmer & Dawson (1969b), as well as a change in the optical rotatory dispersion/circular dichroism spectrum. (e) Lipidlcytochrome

c complex

The bilayer thickness does not change when cytochrome c binds (Fig. 9 and Table 2). Cytochrome c is a globular molecule 25 A x 25 A x 37 A (Dickerson et al., 1967), and it appears to retain its shape when it binds (Gulik-Krzywicki et al., 1969). Thus the inter-bilayer space in the complex, 33 A (Table l), is wide enough to accommodate just one layer of cytochrome c. Cytochrome c is distributed differently from MBP (compare Fig. 9 with Fig. 7 or 8). (f) Structural

interpretations

of calorimetric data

As noted, adding MBP or cytochrome c reduces the transition temperatures and enthalpies observed for dipalmitoyl-phosphatidylglycerol bilayers (Papahadjopoulos et al., 1975; Boggs &z Moscarello, 1978; Boggs et al., 1981b). These authors suggest that both proteins deform the bilayer structure, causing it to become much thinner. Large parts of the proteins might insert between lipid fatty chains (Papahadjopoulos et al., 1975; Boggs et al., 19816). Such thinning of the bilayer is, however, not supported by the present work, nor by previous work with phosphatidylinositol (Gulik-Krzywicki et al., 1969) or asolectin complexed with cytochrome c (Blaurock, 1973b). The calorimetric data might be interpreted differently. Papahadjopoulos et al. (1975) emphasize the correlation between a protein expanding lipid monolayers and its lowering the transition temperature. Accordingly, the MBP molecule may exert a force tending to separate lipid headgroups, causing the fatty chains to go from gel-state to liquid-like packing at a lower temperature. Proteins or other agents may raise the transition temperature (Verkleij et al., 1974) by holding lipid headgroups in the gel-state array. Cholesterol may be important in lipid/MBP complexes (Fig. 10): di-C,,phosphatidylglycerol vesicles formed at least two different multilayer phaseswith MBP, whereas a single multilayered phase formed when MBP was added to cholesterol/di-C,,-phosphatidylglycerol (Sedzik & Blaurock, unpublished results). We note that the low ionic strength of our buffer favors a strong interaction between myelin lipids and both proteins (Gupte et al., 1983). Tn conclusion, our experiments demonstrate remarkably strong interactions

LIPII~/MYELIN

BASIC

I--__ -I&=20-338-I-

PROTEIN

Repeat,nq \

Fro. 10. Catalog of’ possible interactions Hydrogen bonds link oppositely charged

un,, (d=73-868)

Bllayer

between

*0;

MULTILAYERS I

(8=538)

common

+-18=20-33h

MBP

amino

acids and lipid

headgroups.

&s well &9 uncharged groups. The interaction between an Arg and a phosphate group is after Cotton et al. (1973). Note gaps between diecyl-lipid headgroups where cholesterol intervenes. H, hydrogen; C and 0, carbon; 0 and 0, oxygen; N and 0, nitrogen; P. phosphorus; CHOL, cholesterol.

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