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The two faces of the purple membrane
J. Mol. Biol.
(1978) 123, 235-2413
The Two Faces of the Purple Membrane Structural Differences Revealed by Metal Decoration DOROTHEA-CH. NEUGERAVER~ Institut
fiir
Uiochemie der Gniversittit Wiirzburg, Qerwucny
N’iirzburg
AND H. P.
!ikNGSIIEIM:j:
I~~UX-Pla,rzck-Illstitut fiir Biophysikalisehe Chemie (Karl-Friedrich-Bonhoeffer Institut) dbt. Xolekulurer Systemaufbau Gttingen, (ierrnany (Received
6 October 1977)
TIN%t,wo faces of the purple mrmbranc arc lularrl~)i~llously tlist~inguishablo from c,ach other in tile electron microscope aft,cr metal decnration. (Jrystalline order is rcxvpaled on one of tllc t,wo surfaces of the membrane. Membrane cracking and fragmentation upon drying is strongest on llydrophilic supports and bacomcss less severe with increasing hydrophobicity of ttlc srlpport. This behaviour is almost
Ilnaffect,ed
by the presence
of lmv nmlccnlar
\veight,
ac~t~eo~~s “sustains”, int,erasselnblics in mew.
stlch as ghcosc or sucrose, and dernoltst.rates t.tlth irnportanc~~ of surface art ioIls for tile preserrat,iotl
of t hirl rnacrornolt~crIl;~r
1. Introduction I’ery little information is available on t,hr nature and propcrt,ics of the t.wo surfaces of the purple membrane (Henderson, 1977). Thih membrane, isolated from Halobacterium haZobiwm, exists as flat, sheets in suspension (Blaurock & Stoeckenius. 1971). Ba&riorhodopsin, which is t,he only protein found in this membrane, forms a twodimensional crystal with a hexagonal la,ttice of plane group ~3. The three-dimensional structure of the protein at a resolution of 7 A has been determined by low-dose electron microscopy (Unwin & Henderson, 1975: Henderson & Unwin, 1975). Freeze-et.ch electron microscopy (Blaurock 8r. St.oeckenius, 1971) and S-ray diffraction (Blaurock, 1975; Blaurock & King, 1977) have provided some evidence that protein and lipids may be distributed asymmc~trically across the width of thcl membrane. This information pert’ains mainty to t hr> strucbure of t,he membrane interior. X-ray results suggest that the membrane surfaces arc smooth within about, 7 A (Henderson, 1975). It is not known. however. whether the crystalline order of the purple membrane extends t,o it.s surfaces (Henderson, 1975). Whether any asymmetry is also expressed in different physical and chemical properties of the two surfaces has not been shown. t Prosent~ address: Max-Plan&-Institut fiir Bio[~hysikalischr~ (‘hcmie (Karl-FriedrichBonhoeffer Institut), D-3400 GBt~tingen, Germany. $ To whom all corruspondenco should be addrcssocl. 235 0022-2836/78/1232-3546 $02.00/O
C 1978 Academic Press Inc. (London) Ltd.
236
D.-CH.
NEUGEBAUER
AND
H.
P. ZINGSHEIM
When isolated purple membranes are dried onto a solid support (e.g. mica) and shadowed with heavy metal, they display cracks and fissures coinciding with the hexagonal protein lattice (Blaurock & Stoeckenius, 1971). The observation that only about half of the membranes display these cracks (which themselves are thought to be a drying artifact), led to the assumption that the cracking behaviour is evidence for the structural asymmetry of the purple membrane (Kushwaha et al., 1976: Oesterhelt, 1976). Accordingly, cracking and fragmentation would depend on the face, which comes to rest on the solid support, as well as on the nature of the support itself. The verification of this hypothesis requires a labelling met,hod by which the two faces can be unambiguously distinguished. It is also known that the detrimental effects of drying can be avoided for certain molecular crystals by the addition of hydrophilic “sustains” such as glucose or sucrose. This was of decisive importance for the molecular structure determination of bacteriorhodopsin by low-dose electron microscopy (Unwin & Henderson, 1975). In this paper we report on t’he finding that the two faces of t,he purple membrane can be labelled and unambiguously distinguished by metal decoration?. A rigorous test of the asymmetry hypothesis for cracking was possible, therefore. In addition, we report
on the effect
of various
of the two faces of the purple fragmentation
and
cracking
support,
membrane. is largely
surfaces
and of sustains
The experiments
determined
by surface
on the morphology
revealed
that membrane
interactions
and,
thus,
by the nature of the solid support.
2. Materials and Methods (it)
Purple
membranes
Purple membranes of H. halobium, strain l&M,, wore prepared as described by Oesterhelt $ Stoeckenius ( 1974). After sucrose gradient-centrifugation they were dislysed overnight against 0.02% (w/v) NaN,, washed onto and st’ored in 0.0276 azide at 4°C: for up to 5 months at concentrations of 3 to 6 mg/ml. (b) Specimen
preparation
Freshly cleaved mica, hydrophilic carbon films and hydrophobic carbon films were used. Hydrophilic carbon films were obtained by carbon arc evaporation at 10m6 Torr onto freshly cleaved mica in a vacuum unit equipped with a turbomolecular pump and a liquid nitrogen trap. The films were floated off onto the surface of double-distilled water and picked up from underneath with copper grids. They were used the same day. After several days at room atmosphere these carbon films became hydrophobic, as indicatecl by the failure of distilled water to wet the carbon surface. A portion (50 ~1) of a fairly concentrated membrane suspension (1.5 to 3 mg/ml) was put onto a PTFE surface. After 15 to 30 min the carbon-coated grids (using the formerly dry carbon surface), or small pieces of mica, were brought into contact with the surface of the droplets for 1 to 2 s. Subsequently, they were washed 3 times with the same buffer t Metal decoration is a simple, but very sensitive method for detecting differences in surface properties (Bassett, 1958). When met,al is deposited onto a solid surface by vacuum evaporation, the formation of the thin metal film proceeds viu thermal accommodation, nucleation, and crystal growth (Bassett et al., 1969). When sufficiently small amounts of metal are deposited, the thin film is discontinuous, consisting of small (10 to 50 A) individual crystals. The probability of nucleation need not be t,he same all over the surface; instead, there may be sites where preferential nucleation occurs (decoration). This effect depends on the nature of the surface and, obviously, also on the surface mobility of the thermally accommodated metal atoms. Low melting point metals such as Ag, Au and Pt show pronounced decoration effects. The technique has been used so far mainly to study inorganic crystal surfaces.
THE
TWO
FACES
OF
THE
PURPLE
MEMBRANE
237
solution in which the membranes were suspended, followed by 3 washes on droplets of double-distilled water (for 5 min each). The specimens were then air-dried by suction from a Pasteur pipette connected to an aspirator. In the “sustaining” experiments all solutions contained 1 o/o (w/v) of glucose or sucrose.
and
(c) Decoration
shadowing
Decoration and shadowing were carried out in the same vacuum unit as described above. Tile amount of metal deposited was measured by a quartz cryst’al thickness monitor. Silver was evaporated by resistance heating from a small molybdenum boat; tantalum/ tungsten was evaporated by an electron beam evaporator (Zingsheim et al., 1970). Silver ws,s deposited at normal incidence, whereas for tantalllm,‘tungsten shadowing an angle of 45” wa,s chosen. In order to reduce short-term secondary morphological changes (Bachmann & Hilbrand, 1966) of the thin metal layers, a final layer of carbon, approx. 10 A thick, was deposited at normal incidence onto the grids. When mica was the support, this layer was the only carbon layer present and was made sufficiently thick to be floated off and picked up with specimen grids. When several materials had to be deposited consecutively, thra vacuum was not broken in between. (d) Electron microscopy Tllr specimens were examined with a Siemens Elmiskop I at 80 kV using magnifications of 20,000 and 40,000 x . The magnifications were calibrated with the aid of a replica from nn optical grating. Aggregation of the silver particles on decorated specimens occurred within a few days. Therefore micrographs had to be taken within 48 11. Micrographs of decorated and shadowed specimens were generally taken 0.5 to 0.7 pm under focus in order to somewhat enhance tire contrast of the smell silver particles (approx. 20 .S, diarn.). Kodak electron microscopic film was used and developed for 4 min in Gevaert (: 170 C developer. All micrographs are presented as poait,ives with tile shadows appearing whit?. TIlta arrolvs indicate the shadowing direction. (e) Optical diflraction Optical diffraction was performed with and a camera length of 1 m. The original patterns recorded on 35 mm film (llford developer.
a diffractometer employing a 1 mW He/N? laser micrographs were diffracted and the diffraction FP 4) drrelopecl for 4 min in Geraert G 170 C’
3. Results (a) The general morphology
of the two mem,brane faces
The purple membrane, when dried onto solid supports and general morphology illustrated in Figure 1 (see also Blaurock Both surfaces of the membrane are essentially smooth. The larger cracks and pits or dimples are typical for dried purple diffraction of such micrographs occasionally reveals periodicities 62 A lattice constant of the hexagonally arranged protein, but any of these cases to decide whether this was caused by genuine merely by minute cracks, which were always present. (b) Metal
shadowed, shows the & Stoeckenius, 1971). numerous smaller or membranes. Optical compatible with the it was not possible in surface variations 01
decoration
Figure 2(a) demonstrates that the purple membrane displays pronounced decoration effects when silver is deposited at an incidence perpendicular to the plane of the specimen, thus eliminating any shadowing effects. As it is nearly impossible to observe the overall morphology of the specimen by decoration alone, we have adopted the practice of shadowing additionally with a 6 a layer of tantalum/tungsten at 45” incidence after decoration (Fig. 2(b)).
238
D.-CH.
NEUGEBAUEK
AND
H.
t’.
ZINGSHETM
air-dried onto solid supports and shadowrtl lvith Ta/W. They arc an illustration of t,hv nmbigltitirs arising in any att,empt’ to USC thr cmcking patt,wn as a marker for thrk two (lifI&wnt, facts of th,s membrane. (a) A membranr~ depwitcd onto a hydrophobic carboll film. Thr: rrwmbrarw has folded over, thus displaying both faces. C’racks can be visualized on both facw. Sot,<> that OII both faccx the surface between t,he cracks appears wsrwtially smooth. The strong background granlllarity is mainly caused by a mow than optimal thickness of shariowing mat,tvial on this sprcirnrn. Magnification 100,000 x . (b) Membranes deposited onto freshly clcavd m&t. Various forms ant1 degrees of cracking can be observed (from top to bottom) : climplw, small c~wks, large cracks tt~l(l wide gaps (fragmentation). All forms can occur wit,hin one and thr same nwmh~~w (bottom right), along with completely smooth areas. Magnificatilvr 40,000 x
The silver particles are arranged in a two-dimensional hexagonal lattice with a lattice constant of 62 ,A. The small fissurrs or cracks visible in Ngurtb 2(I)), and in many other micrographs. are always pa,ra,llel to the lattice lines rc~caled by metal decoration. Figure 3(a) shows an optical diffraction pattern from an area of a micrograph containing approximately 2000 unit cells. It shows a clear directionalit,v because each individual silver particle is shadowed by tantalum/tungsten. Shadowing leads to a substantial increase in the contrast of the silver grains. Bat’isfactory optical diffraction patterns showing tho lattices orient,ation can easily 1~ ol)tained from micrograph areas as small as 4 i’ 1 unit cells (Fig. 3(b)).
(b) FIG. 2. Metal decoration can reveal t.he hexagonal lattir? of thp purple membrane. The mom(a) Decoration after vacuum branes were air-dried on carbon films. Magnification 160.000;\. deposition (at normal incidence) of a thin layer of silver (average thtrkness 10 a). In this case, thts 6% A periodicity of the hexagonal purple membrane lattice is rrvea.lcd. (h) Decoration (as for (a), but, only 6 A of silver), followed by shadowing et 4.5” incid?ncf> with 6 x of Ta/W. Although they Aver islands are much smaller now, t,hey are still clearly risiblr. The additional shadowing layer reveals the surface relief of the membrane. Small cracks and dimples an- clearly visualized. Sot(, tha,t by decoration alone (a), it. would be extremely difficlllt to cl&>ct th(lsts tltlf?ct.s, whcrcas 1”~’ shadowing (Fig. 1) cannot reveal the hexagonal lattice.
On membranes that can unambiguously be identitied as having folded over during preparation (thus displaying both faces of one and the same membrane, see Fig. l(a)) only one. and always one. of t,htt faces reveals t,h(b hexiLgona1 lattice after m&al decoration (see Fig. 4). A total of 30 such membranes ww tbxamined and no exception was found. Therrfore, metal decoration is an unambiguous marker for one pnrticular face of the purple membrane. The quality of the decoratiou pattern, as judged tly the ease with which the difference betwet>n the two faces of a folded mernbran~ can bc detected visually, depends criticall) on thtn amount of metal depositrd. +th measured in terms of the
(a)
(b)
FIG. 3. Optical diffraction patterns f’rorn micrographs of drcwratctl and shadowctl pnrplc mcmbranea. (a) %rom an area comprising about 2000 unit ~11s. Loss of the thirtl set of srcon~l-or~lr~ diffraction spots is due to the directionality of shadowing, which cnhtuw~v thv rmtrast of thr tlccoration particles. (b) From a 4 Y 4 unit ~11 arca.
avcragc met,:11 film different amounts avcragc silver layer average diameter decorated in order
t’hickness. WC compared folded meml)ra,nt:s, \\.hich had recciwd of silwr. The wsults arc presented in Pigurcb 5. ‘I’hc optimum thickness for visual inspect~ion is about8 5 .A. This corresponds to an of the silver grains of 20 A. Since not cwry unit cell riced hc to produce nn optical diffractSion pattern, it is possible t#o use crven
242
I).-CH.
NEC’GKBdUE:K.
ANI)
H.
t’. %Ih’GRHE:IM
between membrane fragments, generally increases when the supporting surface becomes hydrophilic. On folded membranes, cracks can occur on both faces, although only one face shows regular decoration. From these results it is clear that the asymmetry hypothesis for cracking (Kushwaha et aZ., 1976; Ocsterhelt, 1976) requires some qualifications. Table 1 shows the correlation of regular decoration with the cracking behaviour of isolated purple membranes. This correlation can be used as a test of the above hypothesis because the already described results from decoration of folded membranes show that decoration is an unambiguous indicator for membrane orientation. TABLE
1
Correlatiorl of regular decoration with the cracking behaviour mem,bmnes dried onto hydrophilic carbon Ji1m.s Hexagonal lattice Yf!S
revealed
of
by decoration No
Y’es
lS(j,, (33)
33yJo (ti0)
SO
L”3Y, (52)
“00/b (36)
Cracks
The values are thta percentages absolute numbers countrd.
of thr total
numbs
of membranes
and, in parentheses,
the
As a result’, the cracking behaviour of the purple membrane is indeed correlated with the asymmetry of its surface propert,ies. However, the probability of correctl) a particular surface of a particular membra,ne by its cracking behaviour identifying is only 0.6, meaning that almost every other identification will be wrong. Nevertheless, the increased tendency for cracking. when the surface normally revealing the hexagonal lattice by decoration faces t’he support film. is statistically significant (>95:,, significance, tested via kinomial distribut’ions (Bevingt’on, 1969): meaning that upon repetibion of our experiment t,he sa.me correlation would be obtained in more than 95’yA of all cases). (d) The effect of hydrophilic
sustairzs on the morphology
of the purple
m,embrane
Dried layers of glucose or sucrose on a carbon film can be clearly revealed by decoration, even if they are so thin that they cannot be visualized by conventional electron microscopic methods (see Fig. 6(d) and (e)). The density of the silver particles on the sugar layer is six to ten times smaller than t,hat on plain carbon films. This effect forms the basis for detecting thin layers of sugar covering the membrane faces. Even in the presence of such a layer, cracks can still be seen (Fig. 6(a)). This holds only for the face that normally shows a random distribut’ion of silver grains. It is true even when the sugar layer is sufficiently thick to mask the membrane edges completely (Fig. 6(c)). The already described dependency of membrane cracking on the nature of the solid support is maintained for this face in the presence of the sugars. For the other membrane face, normally showing the hexagonal array of silver grains, the situation is different. This face is hardly ever covered with a sugar layer,
I).-CH.
NEUGEH.L\UElC
9x11)
H.
I’.
ZINGSHE
R
Membrane
illustrating the obscrvcxl drying FIG. 7. Schemat,ic cross-sect,ion of the purple rnembmno, bchaviour, its correlation with thp two faws as distinguished by metal decoration. and the effect of aqueous sustain. xxx Indicate thr regularly spmxxt skrs of prcfcrrntisl nuclwtion, which pivv rise to the hexagonal array of mct,al drcoration particlw.
effect of its structural asymmetry schematically in Figure 7.
on the cracking
hehaviour.
This
is illustrat,ed
4. Discussion Metal decoration is a method of considerable value, because it can reveal periodicities even on extremely smooth surfaces, where other methods fail. Moreover, it allows for an ana,lysis of the crystallinit,y and lattice orientation of extremely small areas of purple membrane (see Neugebauer et al., 1978). Our work represents the first systematic application of this method to a biological object. Tt is important to appreciate t.hat decora,tion does not provide morphological information in the same sense as sha.dowing or negat~ivr staining (Abermann d nt.. 1972: Zingsheim. 1972). The mecha,nism of decoration is not well-understood; not even on simple inorganic surfaces. We cannot drcidc M,hether the nucleation sites art: situated on certain portions of the I)act’eriorllodopsirl t’rimcrs or on some of the membrane lipids. We should also like t,o stress t)hat t,he decorat,ion rclsult,s can neither confirm nor contradict any other findings concerning t.he surfact smoothness of’ the purple membrane (Henderson, 1975: Hlaurock & King. 1977). distribut~ion of metal However, the structural order cxprclssed as a non-random particles on one fact is obviously a consequence of the crystalline arrangement of 1)acteriorhodopsin. because the lattice constant of t,he purple membrane and that of metal decoration are identical. Moreovc~.. regular metal decoration occurs on neithrr face of t.he apomrmbrane. which is purplcl mem bran<> after t,hc removal of t,he rct,inal (Oest,erhclt et al., 1974). Thcb apomembrane lacks t,hc crystalline arrangement of protein. as shown directly b,v S-ray diffraction (R. Henderson. personal communication) and indicated by the complrtc lack of cracks whc~n dried onto a Ilydrophilic carbon film. The absence of a regular array of metal decoration particles on one face of the purple membrane is more difficult to explain. It could bc due to either a lack of order on the surface or a lack of specific nucleation sites. Observahions on a number of other surfaces and molecular arrays provide no evidence that specific surface groups are required, but indicate that a regular spatial arrangement is decisive (unpublished results). Thus, the following hypothesis is plausible and compatible with our results.
THE
TWO
FrZC’ES
OF THE
PCRI’LE
MEMHRANI?
245
although other alternatives cannot be ruled out: It is known that about 30% of the total lipids of purple membrane possess glycolipid headgroups (Plachy et al., 1974; Kushwaha et al.. 1975). If these lipids were asymmetrically distributed between the two membrane halves similar to other membranes (Bretscher, 1973; Rothman & Lenard, 1977), one surface could predominantly carry the glycolipid headgroups, which are not rigid.Thereislittle reason to assume that their long sugar t’ails are found in a regular array after drying. A random arrangement of t’hese surface groups could well obscure any regularity of the molecular arrangement of the membrane. The observf>d preference of added sugar for this membrane surface could then be explained as a rrsult of the formation of hydrogen bonds with the glycolipid headgroups. Membrane cracking occurs whenever the interaction (adhesion) of the membrane with t,he solid support is stronger than its cohesion. Direct int,erfacial interaction is reduced on hydrophobic supports or by thin interstitial layers (e.g. of sugar). As we have shown, thcb original intuitive asymmetry hypothesis for cracking (Kushwaha rt al.. 1976; Oesterhelt, 1976) was correct, although no reliable indicator for membrant~ orientation was available at that t’ime and no attention \vas paid t,o the effect of tht, support surface. Our experiments have revealed an interesting facet of the much more general problrm of t’he preservation of macromolecular assemblies i/~ VUCUO.While the protjertivtl csffect of aqueous sustains on hydrated crystals can be explained by assuming that the sugars act as substitutes for water, their effect on the drying of the purple membrane cannot be understood entirely in the same terms. The purple membrane ,,,rr .SYis not a very hydrated structure and high resolution structural information remains almost unaffected by drying (Blaurock & Stoeckenius, 1971; Blaurock, 1975: Henderson, 1975). meaning that the sustains should act mainly on the membrane surfaces. This we have experimentally observed. Our results illustrate quite clearly thaf the disruption of supramolecular structures by surface interactions can 1~ dominating, even when aqueous sustains are applied. Obviously: such effects are most rrlernnt for the very thin molt~cular crystals used in low-dose electron microscopic investigations. Tlris work 11ns benefited greatly by the generolls support which the Max-Planck (:esellscllaft has provided to one of us (D.-Ch. N.). We arr particularly indebted to I’rofessor H. Kuhn for initiating this collaboration and to Professor D. Oesterhelt for many tlelpful suggestions during the preparation of the manuscript. Tllis Lvork is part of a Ph.D. thesis submitted at the University of Wiirzburg by one allthor (D.-Cll. N.), who gratefully acknowledges a Ph.D. grant from t)he Studionstiftung dcs DcLlltscllen Volkes. REFERENCES .4b(~rn1ann,
R., Salpeter, M. M. 62 Bachmann, L. (1972). ln Principles and Techniprces iv ,~&osco~y (Hayat., M.A., ed.), vol. 2, pp. 195-217, Van Nostrand Reinhold Co., New York. l~achmarur. L. 62 Hilbrand, H. (1966). In Basic Problems in Thin Film Physics (Niederrnayer, R. & Mayer, H., eds), pp. 87-92, Vandenhoeck & Ruprc&t,, Giittingen. Bassett, G. A. (1958). Phil. Mag., ser. 8, 3, 1042-1045. Bassett, G. A., Menter, J. W. & Pashley, D. W. (1959). In Structure and Properties of Thin Films (Neugebauer, C. A., Newkirk, J. B. S: Vvrmileyea. D. A.. eds), pp. 11-4.5. .Jolm Wiley & Sons, Inc., New York. Hevington. R. (1969). Data Reduction and Ermr Analysis for the Physical Sciences. McGraw-Hill Book Co., New York. Hlaurock, A. E. (1975). J. Mol. l&l. 93, 139--158. !I* Electron
246
I).-C’H.
NEUGEHAUEK
ANI)
H.
1’. ZINGSHETJl
Blaurock, A. E. & Kirq, (:. 1. ,lYi’i). Science, 196, 1101 1104. Blaurock, A. E:. & Stoeckcnius, 11’. (197 1). iYatrrre ;Yew Rid. 233, I52 IRS. Bretschrr, M. S. (1973). Science, 181, 622 629. H~ndcrsor~, It. (1075). .I. .\lol. Hiol. 93. 123 138. Hentlrrso~~, K. (1977). .4fL,nu. Rev. Ihphys. Bioe7bg. 2, 87 IO!). Henderson, K. & Unwill, P. N. T. (1975). h’atwe (London). 257, 28 32. Kusll~vaha, S. C., Kates. M. K- Martin, IV. (:. (1975). Can. ./. Hiochem. 53, 284 292. Kusl~ud~a, 8. C’., Katw. M. & Stoeckf~ttirls, 14.. (1970). Hiochim. Biophys. Acta,, 426, 703 710. Ncugebaucrr, D-CJI., Zingslrrim, H. I’. & Oest~erllelt~, D. (l!J78). .7. ilId. h’iol. 123. 1277137. Oosterhelt. D. (1976). ,4ngew. Chemie, Intern. Edit. 15, 17 24. Oesterhelt~, D. & Stoeckenius, W:. (1974). ,VIethods h’ruymol. 31, 667-678. Oestorhelt, D., Schulmxmn, L. & Grubrr. H. (1974). FEBS Letters, 44, 657 261. Plachy, 11:. Z., Larlyi, J. K. dz Kates, M. (1974). Biochemistry, 13, 4906 4913. Rothman, .J. E. 6: Lenard, .J. (1977). Science, 195, 743-753. Unwin, P. N. T. & Henderson, R. (1975). ,J. Mol. Biol. 94, 425 -440. Zingsheim, H. P. (1972). Biochim. Biophys. A&. 265, 339 366. H. P.. Ahcrrnarrtr, K. & Haclrrnatrtl, I,. (1970). ./. I’hys. IC: Nci. f~str. 3, 39-42. Zingslwim.
(1978) 123, 235-2413
The Two Faces of the Purple Membrane Structural Differences Revealed by Metal Decoration DOROTHEA-CH. NEUGERAVER~ Institut
fiir
Uiochemie der Gniversittit Wiirzburg, Qerwucny
N’iirzburg
AND H. P.
!ikNGSIIEIM:j:
I~~UX-Pla,rzck-Illstitut fiir Biophysikalisehe Chemie (Karl-Friedrich-Bonhoeffer Institut) dbt. Xolekulurer Systemaufbau Gttingen, (ierrnany (Received
6 October 1977)
TIN%t,wo faces of the purple mrmbranc arc lularrl~)i~llously tlist~inguishablo from c,ach other in tile electron microscope aft,cr metal decnration. (Jrystalline order is rcxvpaled on one of tllc t,wo surfaces of the membrane. Membrane cracking and fragmentation upon drying is strongest on llydrophilic supports and bacomcss less severe with increasing hydrophobicity of ttlc srlpport. This behaviour is almost
Ilnaffect,ed
by the presence
of lmv nmlccnlar
\veight,
ac~t~eo~~s “sustains”, int,erasselnblics in mew.
stlch as ghcosc or sucrose, and dernoltst.rates t.tlth irnportanc~~ of surface art ioIls for tile preserrat,iotl
of t hirl rnacrornolt~crIl;~r
1. Introduction I’ery little information is available on t,hr nature and propcrt,ics of the t.wo surfaces of the purple membrane (Henderson, 1977). Thih membrane, isolated from Halobacterium haZobiwm, exists as flat, sheets in suspension (Blaurock & Stoeckenius. 1971). Ba&riorhodopsin, which is t,he only protein found in this membrane, forms a twodimensional crystal with a hexagonal la,ttice of plane group ~3. The three-dimensional structure of the protein at a resolution of 7 A has been determined by low-dose electron microscopy (Unwin & Henderson, 1975: Henderson & Unwin, 1975). Freeze-et.ch electron microscopy (Blaurock 8r. St.oeckenius, 1971) and S-ray diffraction (Blaurock, 1975; Blaurock & King, 1977) have provided some evidence that protein and lipids may be distributed asymmc~trically across the width of thcl membrane. This information pert’ains mainty to t hr> strucbure of t,he membrane interior. X-ray results suggest that the membrane surfaces arc smooth within about, 7 A (Henderson, 1975). It is not known. however. whether the crystalline order of the purple membrane extends t,o it.s surfaces (Henderson, 1975). Whether any asymmetry is also expressed in different physical and chemical properties of the two surfaces has not been shown. t Prosent~ address: Max-Plan&-Institut fiir Bio[~hysikalischr~ (‘hcmie (Karl-FriedrichBonhoeffer Institut), D-3400 GBt~tingen, Germany. $ To whom all corruspondenco should be addrcssocl. 235 0022-2836/78/1232-3546 $02.00/O
C 1978 Academic Press Inc. (London) Ltd.
236
D.-CH.
NEUGEBAUER
AND
H.
P. ZINGSHEIM
When isolated purple membranes are dried onto a solid support (e.g. mica) and shadowed with heavy metal, they display cracks and fissures coinciding with the hexagonal protein lattice (Blaurock & Stoeckenius, 1971). The observation that only about half of the membranes display these cracks (which themselves are thought to be a drying artifact), led to the assumption that the cracking behaviour is evidence for the structural asymmetry of the purple membrane (Kushwaha et al., 1976: Oesterhelt, 1976). Accordingly, cracking and fragmentation would depend on the face, which comes to rest on the solid support, as well as on the nature of the support itself. The verification of this hypothesis requires a labelling met,hod by which the two faces can be unambiguously distinguished. It is also known that the detrimental effects of drying can be avoided for certain molecular crystals by the addition of hydrophilic “sustains” such as glucose or sucrose. This was of decisive importance for the molecular structure determination of bacteriorhodopsin by low-dose electron microscopy (Unwin & Henderson, 1975). In this paper we report on t’he finding that the two faces of t,he purple membrane can be labelled and unambiguously distinguished by metal decoration?. A rigorous test of the asymmetry hypothesis for cracking was possible, therefore. In addition, we report
on the effect
of various
of the two faces of the purple fragmentation
and
cracking
support,
membrane. is largely
surfaces
and of sustains
The experiments
determined
by surface
on the morphology
revealed
that membrane
interactions
and,
thus,
by the nature of the solid support.
2. Materials and Methods (it)
Purple
membranes
Purple membranes of H. halobium, strain l&M,, wore prepared as described by Oesterhelt $ Stoeckenius ( 1974). After sucrose gradient-centrifugation they were dislysed overnight against 0.02% (w/v) NaN,, washed onto and st’ored in 0.0276 azide at 4°C: for up to 5 months at concentrations of 3 to 6 mg/ml. (b) Specimen
preparation
Freshly cleaved mica, hydrophilic carbon films and hydrophobic carbon films were used. Hydrophilic carbon films were obtained by carbon arc evaporation at 10m6 Torr onto freshly cleaved mica in a vacuum unit equipped with a turbomolecular pump and a liquid nitrogen trap. The films were floated off onto the surface of double-distilled water and picked up from underneath with copper grids. They were used the same day. After several days at room atmosphere these carbon films became hydrophobic, as indicatecl by the failure of distilled water to wet the carbon surface. A portion (50 ~1) of a fairly concentrated membrane suspension (1.5 to 3 mg/ml) was put onto a PTFE surface. After 15 to 30 min the carbon-coated grids (using the formerly dry carbon surface), or small pieces of mica, were brought into contact with the surface of the droplets for 1 to 2 s. Subsequently, they were washed 3 times with the same buffer t Metal decoration is a simple, but very sensitive method for detecting differences in surface properties (Bassett, 1958). When met,al is deposited onto a solid surface by vacuum evaporation, the formation of the thin metal film proceeds viu thermal accommodation, nucleation, and crystal growth (Bassett et al., 1969). When sufficiently small amounts of metal are deposited, the thin film is discontinuous, consisting of small (10 to 50 A) individual crystals. The probability of nucleation need not be t,he same all over the surface; instead, there may be sites where preferential nucleation occurs (decoration). This effect depends on the nature of the surface and, obviously, also on the surface mobility of the thermally accommodated metal atoms. Low melting point metals such as Ag, Au and Pt show pronounced decoration effects. The technique has been used so far mainly to study inorganic crystal surfaces.
THE
TWO
FACES
OF
THE
PURPLE
MEMBRANE
237
solution in which the membranes were suspended, followed by 3 washes on droplets of double-distilled water (for 5 min each). The specimens were then air-dried by suction from a Pasteur pipette connected to an aspirator. In the “sustaining” experiments all solutions contained 1 o/o (w/v) of glucose or sucrose.
and
(c) Decoration
shadowing
Decoration and shadowing were carried out in the same vacuum unit as described above. Tile amount of metal deposited was measured by a quartz cryst’al thickness monitor. Silver was evaporated by resistance heating from a small molybdenum boat; tantalum/ tungsten was evaporated by an electron beam evaporator (Zingsheim et al., 1970). Silver ws,s deposited at normal incidence, whereas for tantalllm,‘tungsten shadowing an angle of 45” wa,s chosen. In order to reduce short-term secondary morphological changes (Bachmann & Hilbrand, 1966) of the thin metal layers, a final layer of carbon, approx. 10 A thick, was deposited at normal incidence onto the grids. When mica was the support, this layer was the only carbon layer present and was made sufficiently thick to be floated off and picked up with specimen grids. When several materials had to be deposited consecutively, thra vacuum was not broken in between. (d) Electron microscopy Tllr specimens were examined with a Siemens Elmiskop I at 80 kV using magnifications of 20,000 and 40,000 x . The magnifications were calibrated with the aid of a replica from nn optical grating. Aggregation of the silver particles on decorated specimens occurred within a few days. Therefore micrographs had to be taken within 48 11. Micrographs of decorated and shadowed specimens were generally taken 0.5 to 0.7 pm under focus in order to somewhat enhance tire contrast of the smell silver particles (approx. 20 .S, diarn.). Kodak electron microscopic film was used and developed for 4 min in Gevaert (: 170 C developer. All micrographs are presented as poait,ives with tile shadows appearing whit?. TIlta arrolvs indicate the shadowing direction. (e) Optical diflraction Optical diffraction was performed with and a camera length of 1 m. The original patterns recorded on 35 mm film (llford developer.
a diffractometer employing a 1 mW He/N? laser micrographs were diffracted and the diffraction FP 4) drrelopecl for 4 min in Geraert G 170 C’
3. Results (a) The general morphology
of the two mem,brane faces
The purple membrane, when dried onto solid supports and general morphology illustrated in Figure 1 (see also Blaurock Both surfaces of the membrane are essentially smooth. The larger cracks and pits or dimples are typical for dried purple diffraction of such micrographs occasionally reveals periodicities 62 A lattice constant of the hexagonally arranged protein, but any of these cases to decide whether this was caused by genuine merely by minute cracks, which were always present. (b) Metal
shadowed, shows the & Stoeckenius, 1971). numerous smaller or membranes. Optical compatible with the it was not possible in surface variations 01
decoration
Figure 2(a) demonstrates that the purple membrane displays pronounced decoration effects when silver is deposited at an incidence perpendicular to the plane of the specimen, thus eliminating any shadowing effects. As it is nearly impossible to observe the overall morphology of the specimen by decoration alone, we have adopted the practice of shadowing additionally with a 6 a layer of tantalum/tungsten at 45” incidence after decoration (Fig. 2(b)).
238
D.-CH.
NEUGEBAUEK
AND
H.
t’.
ZINGSHETM
air-dried onto solid supports and shadowrtl lvith Ta/W. They arc an illustration of t,hv nmbigltitirs arising in any att,empt’ to USC thr cmcking patt,wn as a marker for thrk two (lifI&wnt, facts of th,s membrane. (a) A membranr~ depwitcd onto a hydrophobic carboll film. Thr: rrwmbrarw has folded over, thus displaying both faces. C’racks can be visualized on both facw. Sot,<> that OII both faccx the surface between t,he cracks appears wsrwtially smooth. The strong background granlllarity is mainly caused by a mow than optimal thickness of shariowing mat,tvial on this sprcirnrn. Magnification 100,000 x . (b) Membranes deposited onto freshly clcavd m&t. Various forms ant1 degrees of cracking can be observed (from top to bottom) : climplw, small c~wks, large cracks tt~l(l wide gaps (fragmentation). All forms can occur wit,hin one and thr same nwmh~~w (bottom right), along with completely smooth areas. Magnificatilvr 40,000 x
The silver particles are arranged in a two-dimensional hexagonal lattice with a lattice constant of 62 ,A. The small fissurrs or cracks visible in Ngurtb 2(I)), and in many other micrographs. are always pa,ra,llel to the lattice lines rc~caled by metal decoration. Figure 3(a) shows an optical diffraction pattern from an area of a micrograph containing approximately 2000 unit cells. It shows a clear directionalit,v because each individual silver particle is shadowed by tantalum/tungsten. Shadowing leads to a substantial increase in the contrast of the silver grains. Bat’isfactory optical diffraction patterns showing tho lattices orient,ation can easily 1~ ol)tained from micrograph areas as small as 4 i’ 1 unit cells (Fig. 3(b)).
(b) FIG. 2. Metal decoration can reveal t.he hexagonal lattir? of thp purple membrane. The mom(a) Decoration after vacuum branes were air-dried on carbon films. Magnification 160.000;\. deposition (at normal incidence) of a thin layer of silver (average thtrkness 10 a). In this case, thts 6% A periodicity of the hexagonal purple membrane lattice is rrvea.lcd. (h) Decoration (as for (a), but, only 6 A of silver), followed by shadowing et 4.5” incid?ncf> with 6 x of Ta/W. Although they Aver islands are much smaller now, t,hey are still clearly risiblr. The additional shadowing layer reveals the surface relief of the membrane. Small cracks and dimples an- clearly visualized. Sot(, tha,t by decoration alone (a), it. would be extremely difficlllt to cl&>ct th(lsts tltlf?ct.s, whcrcas 1”~’ shadowing (Fig. 1) cannot reveal the hexagonal lattice.
On membranes that can unambiguously be identitied as having folded over during preparation (thus displaying both faces of one and the same membrane, see Fig. l(a)) only one. and always one. of t,htt faces reveals t,h(b hexiLgona1 lattice after m&al decoration (see Fig. 4). A total of 30 such membranes ww tbxamined and no exception was found. Therrfore, metal decoration is an unambiguous marker for one pnrticular face of the purple membrane. The quality of the decoratiou pattern, as judged tly the ease with which the difference betwet>n the two faces of a folded mernbran~ can bc detected visually, depends criticall) on thtn amount of metal depositrd. +th measured in terms of the
(a)
(b)
FIG. 3. Optical diffraction patterns f’rorn micrographs of drcwratctl and shadowctl pnrplc mcmbranea. (a) %rom an area comprising about 2000 unit ~11s. Loss of the thirtl set of srcon~l-or~lr~ diffraction spots is due to the directionality of shadowing, which cnhtuw~v thv rmtrast of thr tlccoration particles. (b) From a 4 Y 4 unit ~11 arca.
avcragc met,:11 film different amounts avcragc silver layer average diameter decorated in order
t’hickness. WC compared folded meml)ra,nt:s, \\.hich had recciwd of silwr. The wsults arc presented in Pigurcb 5. ‘I’hc optimum thickness for visual inspect~ion is about8 5 .A. This corresponds to an of the silver grains of 20 A. Since not cwry unit cell riced hc to produce nn optical diffractSion pattern, it is possible t#o use crven
242
I).-CH.
NEC’GKBdUE:K.
ANI)
H.
t’. %Ih’GRHE:IM
between membrane fragments, generally increases when the supporting surface becomes hydrophilic. On folded membranes, cracks can occur on both faces, although only one face shows regular decoration. From these results it is clear that the asymmetry hypothesis for cracking (Kushwaha et aZ., 1976; Ocsterhelt, 1976) requires some qualifications. Table 1 shows the correlation of regular decoration with the cracking behaviour of isolated purple membranes. This correlation can be used as a test of the above hypothesis because the already described results from decoration of folded membranes show that decoration is an unambiguous indicator for membrane orientation. TABLE
1
Correlatiorl of regular decoration with the cracking behaviour mem,bmnes dried onto hydrophilic carbon Ji1m.s Hexagonal lattice Yf!S
revealed
of
by decoration No
Y’es
lS(j,, (33)
33yJo (ti0)
SO
L”3Y, (52)
“00/b (36)
Cracks
The values are thta percentages absolute numbers countrd.
of thr total
numbs
of membranes
and, in parentheses,
the
As a result’, the cracking behaviour of the purple membrane is indeed correlated with the asymmetry of its surface propert,ies. However, the probability of correctl) a particular surface of a particular membra,ne by its cracking behaviour identifying is only 0.6, meaning that almost every other identification will be wrong. Nevertheless, the increased tendency for cracking. when the surface normally revealing the hexagonal lattice by decoration faces t’he support film. is statistically significant (>95:,, significance, tested via kinomial distribut’ions (Bevingt’on, 1969): meaning that upon repetibion of our experiment t,he sa.me correlation would be obtained in more than 95’yA of all cases). (d) The effect of hydrophilic
sustairzs on the morphology
of the purple
m,embrane
Dried layers of glucose or sucrose on a carbon film can be clearly revealed by decoration, even if they are so thin that they cannot be visualized by conventional electron microscopic methods (see Fig. 6(d) and (e)). The density of the silver particles on the sugar layer is six to ten times smaller than t,hat on plain carbon films. This effect forms the basis for detecting thin layers of sugar covering the membrane faces. Even in the presence of such a layer, cracks can still be seen (Fig. 6(a)). This holds only for the face that normally shows a random distribut’ion of silver grains. It is true even when the sugar layer is sufficiently thick to mask the membrane edges completely (Fig. 6(c)). The already described dependency of membrane cracking on the nature of the solid support is maintained for this face in the presence of the sugars. For the other membrane face, normally showing the hexagonal array of silver grains, the situation is different. This face is hardly ever covered with a sugar layer,
I).-CH.
NEUGEH.L\UElC
9x11)
H.
I’.
ZINGSHE
R
Membrane
illustrating the obscrvcxl drying FIG. 7. Schemat,ic cross-sect,ion of the purple rnembmno, bchaviour, its correlation with thp two faws as distinguished by metal decoration. and the effect of aqueous sustain. xxx Indicate thr regularly spmxxt skrs of prcfcrrntisl nuclwtion, which pivv rise to the hexagonal array of mct,al drcoration particlw.
effect of its structural asymmetry schematically in Figure 7.
on the cracking
hehaviour.
This
is illustrat,ed
4. Discussion Metal decoration is a method of considerable value, because it can reveal periodicities even on extremely smooth surfaces, where other methods fail. Moreover, it allows for an ana,lysis of the crystallinit,y and lattice orientation of extremely small areas of purple membrane (see Neugebauer et al., 1978). Our work represents the first systematic application of this method to a biological object. Tt is important to appreciate t.hat decora,tion does not provide morphological information in the same sense as sha.dowing or negat~ivr staining (Abermann d nt.. 1972: Zingsheim. 1972). The mecha,nism of decoration is not well-understood; not even on simple inorganic surfaces. We cannot drcidc M,hether the nucleation sites art: situated on certain portions of the I)act’eriorllodopsirl t’rimcrs or on some of the membrane lipids. We should also like t,o stress t)hat t,he decorat,ion rclsult,s can neither confirm nor contradict any other findings concerning t.he surfact smoothness of’ the purple membrane (Henderson, 1975: Hlaurock & King. 1977). distribut~ion of metal However, the structural order cxprclssed as a non-random particles on one fact is obviously a consequence of the crystalline arrangement of 1)acteriorhodopsin. because the lattice constant of t,he purple membrane and that of metal decoration are identical. Moreovc~.. regular metal decoration occurs on neithrr face of t.he apomrmbrane. which is purplcl mem bran<> after t,hc removal of t,he rct,inal (Oest,erhclt et al., 1974). Thcb apomembrane lacks t,hc crystalline arrangement of protein. as shown directly b,v S-ray diffraction (R. Henderson. personal communication) and indicated by the complrtc lack of cracks whc~n dried onto a Ilydrophilic carbon film. The absence of a regular array of metal decoration particles on one face of the purple membrane is more difficult to explain. It could bc due to either a lack of order on the surface or a lack of specific nucleation sites. Observahions on a number of other surfaces and molecular arrays provide no evidence that specific surface groups are required, but indicate that a regular spatial arrangement is decisive (unpublished results). Thus, the following hypothesis is plausible and compatible with our results.
THE
TWO
FrZC’ES
OF THE
PCRI’LE
MEMHRANI?
245
although other alternatives cannot be ruled out: It is known that about 30% of the total lipids of purple membrane possess glycolipid headgroups (Plachy et al., 1974; Kushwaha et al.. 1975). If these lipids were asymmetrically distributed between the two membrane halves similar to other membranes (Bretscher, 1973; Rothman & Lenard, 1977), one surface could predominantly carry the glycolipid headgroups, which are not rigid.Thereislittle reason to assume that their long sugar t’ails are found in a regular array after drying. A random arrangement of t’hese surface groups could well obscure any regularity of the molecular arrangement of the membrane. The observf>d preference of added sugar for this membrane surface could then be explained as a rrsult of the formation of hydrogen bonds with the glycolipid headgroups. Membrane cracking occurs whenever the interaction (adhesion) of the membrane with t,he solid support is stronger than its cohesion. Direct int,erfacial interaction is reduced on hydrophobic supports or by thin interstitial layers (e.g. of sugar). As we have shown, thcb original intuitive asymmetry hypothesis for cracking (Kushwaha rt al.. 1976; Oesterhelt, 1976) was correct, although no reliable indicator for membrant~ orientation was available at that t’ime and no attention \vas paid t,o the effect of tht, support surface. Our experiments have revealed an interesting facet of the much more general problrm of t’he preservation of macromolecular assemblies i/~ VUCUO.While the protjertivtl csffect of aqueous sustains on hydrated crystals can be explained by assuming that the sugars act as substitutes for water, their effect on the drying of the purple membrane cannot be understood entirely in the same terms. The purple membrane ,,,rr .SYis not a very hydrated structure and high resolution structural information remains almost unaffected by drying (Blaurock & Stoeckenius, 1971; Blaurock, 1975: Henderson, 1975). meaning that the sustains should act mainly on the membrane surfaces. This we have experimentally observed. Our results illustrate quite clearly thaf the disruption of supramolecular structures by surface interactions can 1~ dominating, even when aqueous sustains are applied. Obviously: such effects are most rrlernnt for the very thin molt~cular crystals used in low-dose electron microscopic investigations. Tlris work 11ns benefited greatly by the generolls support which the Max-Planck (:esellscllaft has provided to one of us (D.-Ch. N.). We arr particularly indebted to I’rofessor H. Kuhn for initiating this collaboration and to Professor D. Oesterhelt for many tlelpful suggestions during the preparation of the manuscript. Tllis Lvork is part of a Ph.D. thesis submitted at the University of Wiirzburg by one allthor (D.-Cll. N.), who gratefully acknowledges a Ph.D. grant from t)he Studionstiftung dcs DcLlltscllen Volkes. REFERENCES .4b(~rn1ann,
R., Salpeter, M. M. 62 Bachmann, L. (1972). ln Principles and Techniprces iv ,~&osco~y (Hayat., M.A., ed.), vol. 2, pp. 195-217, Van Nostrand Reinhold Co., New York. l~achmarur. L. 62 Hilbrand, H. (1966). In Basic Problems in Thin Film Physics (Niederrnayer, R. & Mayer, H., eds), pp. 87-92, Vandenhoeck & Ruprc&t,, Giittingen. Bassett, G. A. (1958). Phil. Mag., ser. 8, 3, 1042-1045. Bassett, G. A., Menter, J. W. & Pashley, D. W. (1959). In Structure and Properties of Thin Films (Neugebauer, C. A., Newkirk, J. B. S: Vvrmileyea. D. A.. eds), pp. 11-4.5. .Jolm Wiley & Sons, Inc., New York. Hevington. R. (1969). Data Reduction and Ermr Analysis for the Physical Sciences. McGraw-Hill Book Co., New York. Hlaurock, A. E. (1975). J. Mol. l&l. 93, 139--158. !I* Electron
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H.
1’. ZINGSHETJl
Blaurock, A. E. & Kirq, (:. 1. ,lYi’i). Science, 196, 1101 1104. Blaurock, A. E:. & Stoeckcnius, 11’. (197 1). iYatrrre ;Yew Rid. 233, I52 IRS. Bretschrr, M. S. (1973). Science, 181, 622 629. H~ndcrsor~, It. (1075). .I. .\lol. Hiol. 93. 123 138. Hentlrrso~~, K. (1977). .4fL,nu. Rev. Ihphys. Bioe7bg. 2, 87 IO!). Henderson, K. & Unwill, P. N. T. (1975). h’atwe (London). 257, 28 32. Kusll~vaha, S. C., Kates. M. K- Martin, IV. (:. (1975). Can. ./. Hiochem. 53, 284 292. Kusl~ud~a, 8. C’., Katw. M. & Stoeckf~ttirls, 14.. (1970). Hiochim. Biophys. Acta,, 426, 703 710. Ncugebaucrr, D-CJI., Zingslrrim, H. I’. & Oest~erllelt~, D. (l!J78). .7. ilId. h’iol. 123. 1277137. Oosterhelt. D. (1976). ,4ngew. Chemie, Intern. Edit. 15, 17 24. Oesterhelt~, D. & Stoeckenius, W:. (1974). ,VIethods h’ruymol. 31, 667-678. Oestorhelt, D., Schulmxmn, L. & Grubrr. H. (1974). FEBS Letters, 44, 657 261. Plachy, 11:. Z., Larlyi, J. K. dz Kates, M. (1974). Biochemistry, 13, 4906 4913. Rothman, .J. E. 6: Lenard, .J. (1977). Science, 195, 743-753. Unwin, P. N. T. & Henderson, R. (1975). ,J. Mol. Biol. 94, 425 -440. Zingsheim, H. P. (1972). Biochim. Biophys. A&. 265, 339 366. H. P.. Ahcrrnarrtr, K. & Haclrrnatrtl, I,. (1970). ./. I’hys. IC: Nci. f~str. 3, 39-42. Zingslwim.