Refolding of bacteriorhodopsin in lipid bilayers

J. Mol.

Riol.

(1987) 198, 655-676

Refolding of Bacteriorhodopsin in Lipid Bilayers A Thermodynamically

Controlled Two-stage Process

Jean-Luc Popott, Sue-Ellen Gerchmanl and Donald M. Engelmanh department of Molecular

~jocherni~tr~~ and Biophysics Yale University, 260 Whitney Averwe New Haven, CT 06511. C.S.A.

(Received 27 October 1986, and in revised form 17 June 1987) Possible steps in the folding of bacteriorhodopsin are revealed by studying the refolding and interaction of two fragments of the molecule reconstituted in lipid vesicles. (1) Two denatured bacteriorhodopsin fragments have been purified starting from chymotryptically cleaved bacteriorhodopsin. Cleaved bacteriorhodopsin has been renatured from a mixture of the fragments in Halobacterium lipids/retinal/dodecyl sulfate solution following removal of dodecyl sulfate by precipitation with potassium. The renatured molecules have the same absorption spectrum and extinction coefficient as native cleaved bacteriorhodopsin. They are integrated into small lipid vesicles as a mixture of monomers and aggregates. Extended lattices form during the partial dehydration process used to orient samples for X-ray and neutron crystallography. (2) Correct refolding of cleaved bacterioopsin occurs upon renaturation in the absence of retinal. Regeneration of the chromophore and reformation of the purple membrane lattice retinal. are observed following subsequent addition of all-trans (3) The t,wo chymotryptic fragments have been reinserted separately into lipid vesicles and refolded in the absence of retinal. Circular dichroism spectra of the polypeptide backbone transitions indicate that they have regained a highly a-helical structure. Thtb kinetics of chromophore regeneration following reassociation have been studied by absorption spectroscopy. Upon vesicle fusion, the refolded fragments first reassociate, then bind retinal and finally regenerate cleaved bacteriorhodopsin. The complex formed in t,he absence of retinal is kinetically indistinguishable from cleaved bacterioopsin. The refolded fragments in lipid vesicles are stable for months, both a,s separate entities and after reassociation. These observations provide further evidence that the native folded structure of bact,eriorhodopsin lies at’ a free energy minimum. They are interpreted in terms of a twostage folding mechanism for membrane proteins in which stable transmembrane helices are first formed. They subsequently pack without major rearrangement to produce the tertiary structure.

1. Introduction

in crystallization methods has led to the highresolition three-dimensional map of a complex containing three integral membrane proteins (Deisenhofer et al.. 1985). As was the case three decades ago for soluble proteins, the question arises as to the extent and manner in which the sequence debermines of amino acids their spatial organization, and of whether our understanding of the rules governing this process is suficient to permit folding models to be derived from sequence data. As has been proposed for soluble proteins (see e.g. Kim & Baldwin, 1982; Creighton, 1985; and references therein), it seems possible that some information relevant to these questions can be

of intrinsic membrane Our understanding proteins has long been hampered by the scarcity of structural data. This situation has changed rapidly during the past few years. With the advent of techniques for cloning DNA, nucleic acid sequences coding for many proven or presumed integral membrane proteins have been established. Progress 7 l’ermanent address: lnstitut de Biologie Physico1:himiqur & Colli?ge de France, F-75005 Paris, France. f Present address: Brookhaven Xational Laboratory. Ilpton. NY 11973, 1J.S.A. fi Author to whom reprint requests should be sent. 655 ~2.-2836/87/%40655-SB

$03.00/O

0 1987 Academic l’rws

Limited

656

J.-L.

l’opot

&rived from a study of t’hc pat’hway(a) of’ protein rr~llat uration from unfolded to folded stat’tls. The anisotropicb lipid bilayer environmrnt of integral membrane proteins makes the development of such systems more complex. On the other hand. the &rong constraints it provides on folding represent an asset in attem@ing structural predictions. 12a~t,eriorhodopsin (HRT), the light-driven proton pump from liiclobacterium halobium plasma membrane (for reviews. see St,oeckenius & Bopomolni, 1982; Ijencher, 1983). is the only mcmhrant~ protein to have hcen successfully renatured from a completely unfolded state (Huang rt al., 1981). About three-quarters of the mass of this small retinal protein (AI, 26,653) is folded into sevcLn transmembrane electron-dense rods. most likely a-helires (Henderson & Cnwin, 1975; Leifer & Henderson, 1983: Tsygannik & Haldwin. 1986). The sequence of BR (Ovchinnikov et al.. 1979: Khorana P#(L/.. 1979) ineludes seven conspicuous stretches of mainly hydrophobic amino acid residues. and it has been suggested t’hat they fold into as many t ransmemhrane a-helices (Ovchinnikov ef nl.. 1979: Engelrnan et c&t.. 1980). Calculations of free energy (Ii:ngelman ut (lb., 1982, 1986) predict that each of these sequence segments. considered in isolation, would tend to partition into a lipid bilaycr as a Stahk x-helix. Estimates of t,heir probable boundaries in the sequence are in good agreement with biochemical observations (see Engelman rt al., 19X6, and references therein). Such predictions rest on thr> assumption that the transmembrane domain of RR, and similar proteins is formed by the assembly of helices t’hat would he individually stable as isolated transmembrane entities. Kacteriorhodopsin is a particularly att.ractivr system in which to t’est this idea. Starting from the completely unfolded polypeptidr in formic acid. refolding can be achieved irr ul;tro to the point at which the chromophore environment and the ability to putnp protons are regenrratjed. This suggests that. as for most soluble pro&ins, the native state lies at, a free energy minimum (Huang rt ~1.. 1981). Furthermore. renaturation in lipid/bile salt mixed micelles has been demonstrated starting from two denatured chymotr;vpt,i(b fragment,s, thought to contain, respectively. fire and two transmembrane helices in t’hr folded protein (Huang et ctl., 1981). i’ircular dichroism (r.d.) measurements in the far ult~raviolet, region and t’he kinetics of chromophorr regeneration indicated that helix formation

within

each fragment

was followetl

hy

t Abbreviations used: BR, bact’eriorhodoptiin: BC). bactrrioopsin: cKR and cHC). respectively, KO and BR c+a\rd brtwrrn residues 71 and 72: c.d.. circular dic4~roisrri; C-1. fragment 72-248 of HO: (‘-2, fragment I 7 I of 130: c.m.c.. critical micellar concentration:

DRllY~, dim~ristoylphosphatidylcholine; DPIY‘, c~i~)almito~lphoYphatidylcholine: HI,. Hnlobactrriurrr hnlobiu,m hpids: l’(‘, phosphatidyloholine: PIN. pot.assium dodeqvl sulfate: PM, purple membrane: TI,(?. thin-layrr chromatography: TLCK. iV*-p-tosvl-I,-

Iysine chloromethylketone; u.v.. ultraviolet iight.

et al. t,heir association and the rebinding of retinal to yield renatured (but cleaved) bacteriorhodopsin. F’ollowing addition of lipids and removal of’ detergent. vesicles formed and light-driven proton pumping was demonstrated (Liao et al., 1983). We have extended these pioneering stjudies hy developing conditions for the renaturaiion of int’actt or ch,vrnotryptically cleaved RR (cHR) under which: (1) refolding and reassembly occur in a bilayer environment and; (2) detailed st rnctural st,udies of the refolded material are possible. The new protocol permits the following. (1) Renaturation of BR or cHR at’ such low lipidto-protein ratios that reformation of a twodimensional latt.ice can be induced. The rrnaturetl rnateriaJ has been c+haracaterized by biochemi4. spectroscopic and c~r-yst,allographic~ methods. and the conditions t,hat lead t,o lattice formation have been determined. A detailed crystallographic analysis has been published (Popot ef aE., 1986). In view of the potential applications of the protocol to the study of BR and. possibly, its extension to other membrane proteins, the influence of some experiment,al parameters 011 renaturat,ion awl crystallization has been charact~rrized. \Vtb show that t’he presence of retinal is not needed for correct refolding of cleaved bacterioopsin (cH0) to o(‘cur. (2) Isolation of stable “folding irttermc,tiiut,~,s” inserted in lipid bilayers (individual fragmrnts anal cBO). The fragment,s have been separately reinserted int’o lipid vesicles and subsec~ut~ntl~ brought together t)g vrsi& fusion. 1Ve havy st.utlicqj their secondary struct’ure by c.d. and used absorption spectroscopy to examine t,he kinrticss of chromophore regeneration upon vesicle fusion. These observations suggest a t.wo-stage mcy:hanisrn for memhranc protein folding.

2. Materials and Methods (i) I’w$

ntembrrr~n.(~ halobium.

fialobacteriurn

strain

89 (originally

a gift

f’rom W. Stoeckenius), was grown as described (Hngelman & Zacca’i, 1980). (‘ulturrs were grown to st,ationary phase at 37 Y’ with controlled illumination and aeration. Purple membranes (PM) were isolated from washed cells by the

rnrthod of Orstrrhelt, & Stoeckenius (1’371).The isolated tnrrnbrsnes were st,ored frozen at - 20°C‘ in 45?J, (w/v) su(‘rose. Concentrations were drtclrmintxd from the absorbance of light-adapted I’M near 5fi5 nm and hi

amino acid analysis. Cultures labeled with either [3HJleuc+nr 01’1“(!]valine wpre grown on the defined medium described by (‘respi (1988). The radioactive amino acids were added to media containmg lO(& or 1000,,, respectively. of the st,andard concentrations of either r,-leucine or I.-saline. About 04105 or O4300.5~~,of the label was incorporated into PM, respectively. resulting in preparations wit,h spccitic activities of about, 100 (li/rnol ((‘H]f’M) or 0.2 (li/mol ([‘V\PM).

Acrylamide, bisacrylamide, glycinr and SDS (elrctrophoresis purity) were from Kio-Rad (Richmond. (‘A).

Refolding of Bacteriorhodopsin L-amino acids (standard kit.), cc-chymotrypsin (TLCKtreated), all-truns retinal. sodium azide and Trizma base (reagent grade) from Sigma, (St Louis, MO). chloroform and methanol (Omnisolv) from MCB (Darmstadt. FR(:). ethyl alcohol (200 proof. anhydrous) from WarnerGraham Co. (Cockrysville. MD), acetone (Photrex) and formic acid (90%) from Baker (Phillipsburg. N,J). sucrose and urea (ultra-pure) from Schwartz/Mann (Spring Valley. SY) and formic acid (88?/,) from Mallinckrodt (Paris. KL). All other chemicals were from Eaker (analytical grade). ;1!! solutions were prepared in water purified on a Milli-Q system (Millipore Co.. Bedford. MA).

SIN buffer: s’$, SIN. O@Bf,O;, XaX,. iit) nrM-sodium phosphate (pH 6.0) unless otherwise indicated. K buffer: IfiO InM-Kc!, OG%“& x’sKi,. 30 mM-potassium !)hos!)hatr (pH 6.0). K/5 buffer: 30 mm-KU. 0.005% NaS,. 6 mMpotassium phosphate (pH 6.0).

The gel s.yst.em used was similar to that descaribed b> Dumont rl al. (1986). A lfi”i; t.o 19% gradient ge! was made fresh weekly and prepared using solutions containing !9q/, acrylamide and O%3o/h bisacrylamide or in 0.375 Mlfj 91, acrylamide and 0+0/b bisacrylamide Tris.HCl (pH 8.8). 6 M-urea and 0.1 y& SDS. Sucrose (1.5%) was added to the !90/,, solution to stabilize the gradient. The gel was ovrrlwid with a stacking gel containing 5?/, acrylamide. 0.17 % bisacrylamidr. 0.1 ?$ SDS and 0.1.53 M-Tris. HU (pH 6.8). The tank buffer contained 25 rnM-Tris HC!l (pH 8*3), 0.2 ,M-glycinr and ().I(>& SDS. Electrophoresis was carried out. at !6”(’ fol about I 1 h (1Fi mh/grl). Aft,rr staining with (‘oomassir brillant. blue and destaining. gels were scanned on an LKH densitometer using t,he tungsten lamp. Drying without crarking was achieved by soaking the gels in NT{, methanol (reagent grade) and placing them between 2 sheets of BioRad csrllophane membrane barking on a pre-heated RioRad slab gel dryer. Vacuum was applied for 1 h with heating and maintained unt,il cbooling was c~ornplrtc

Dried fracbtions (about 1 nmol BR or fragments) c*ontaining 10 nmol of norleucinr as an internal standard were overlaid with 200 ~1 of 6 M-HCI rontaining 0.2% phenol. The gla.ss t,ubes were sealed under vacuum at (~25 mTorr: I Torr z 133.3 Pa) and maintained 110 “(I for 20 h. The hydrolyzates were dried by c:entrifugat,ion untier vacuum and 60 p! of 0.2 M-sodium citrate (pH 2.2). added t.o each in preparation for amino acid analysis. which was carried out on a Durrum D-500 analyzer. SDS up to 500 /(g/fraction did not interfere with the analysis. (ireater amounts resulted in uninterpretable rlution pat.terns. (d) Halobacterium

halobium lipids

(i) t’rupamtion lipids were prepared following Kates et (1982). modified as follows. A 12-l culture of H. halobium strain S9 was harvested at the end of the logarithmic, phase and the cells resuspended in 4 M-NaCI to a final volume of 500 ml; 1.2 1 of methanol and 600 ml of chloroform were successively added to the suspension and the mixture stirred at room temperature under Halobacteriwn

(II.

in

Lipid

Rilayer7~

657

nitrogen atmosphere for 4 to 14 h. Aft.er I() min centrifugation in glass bot,tles in the *JAI4 rotor of a Becskman ,721 centrifuge (FiOOOrevs/min. 3800 g), the supernatant was filtered through Whatman no. 1 filter overnight in rhloroformjmethanol !)aper y-washed (I : 1. v/l-). The pellets were resuspended in 500 ml of water ant! m-extracted for 1 h as described. The filtrates were c*ombined and I.2 1 of chloroform and I.2 1 of water were added to them. The mixture was shaken and allowed to separate overnight in separatory funnels. The lower chloroform phase was withdrawn and brought to dryness in a rotary evaporat,or at 30°C’. The residue was dissol\.rd in 20 ml of chlorofortn and renbrifuged for 20 min al. OY’ in the ,JA20 rotor of a Berkman 521 centrifuge (10.000 revs/min, 12,OoOg). The white pellet was discarded. The clear orange supernatant was mixed with PO0 ml of ice-cold acetone and rentrifuged in the same r’ot,or for B min at 0°C (3000 revs/min. 1000 g). The suprrnatant was discbarded. The pellets were dried under nitrogen. resuspended in 20 ml of chloroform. and the preci!)itation wit.h acet.one was repeated. The final extracst in 20 ml of cahloroform. containing 30 to 40 g lipids IXY l&r. wax stored either at 4°C’ or at -2O”(‘.

Total phosphorus was determined by the method of Fiske-Sub!)aRow (Biittcher rt nl., 1961) after mineralization by perchloric acid (20 min or until colorless at t 8OY’). Extracts were qualitatively analyzed by 2-dimensional TLC’ (Kates et (LE., 1982). Plates were stained either with iodine or with the phosphate 01 sr-naphthol st,ains in order to identify phospholipids and gly~~olipids. The composition of the 5 different lipid extracts used in this study was very similar to that descaribed by Katrs ut al. (1982). The proportion of the ma,joi (phosphatidyl#~cerophosphate. phoslipids phat.idyiglycrrol a.nd glycohp!d sulfate) appeared identical from one preparation to the next. rvrn t,hough marked differences in roloration suggested a variable contamination with rarotenoids and/or bacterioruberins. Phospholipids romprised 68( +2)9; of t.he dry weight. (assuming an average of 66Og of phospholipidimol phosphorus), caonsistmt with earlier analyes (Kates et tel.. 1982). For the purpose of reronstitut.mn tbxperimrnt,s. lipid amounts were based on dry wright. (t’) I’rPpnmtion

of chymotryptic

RR frugrwnts

(‘hymotrypticb fragments were prepared basically as by Khorana & co-workers (Gerber et al.. 1977. 1979; Huang el a.1.. 1981). with a number of modifications made necessary either by the different renaturation procedure to be used or by the large amounts of mat,erial needed for neutron diffraction measurements;. Because we have found the success of our renaturation J)ro&ures t.o be dependrnt on the exact manner in whirh the fragments are !)urified. our Jbrotocot is given here in full. Apomemhranes were prepared essent,ially as destbribed by Qerhrr Pt ~2. (1977); losses and aggregation were minimized by substit,uting dialysis for centrifugation steps. Purple membranes were supplemented &her with L3H]PM or [‘4C]PM and transferred by dialysis into 4 MNaCl (final protein concentration. 5 to 7 g/l). The suspension was mixed with an equal volume of 2 MNH,OH . H(‘1 (pH 7.0), and irradiated at room t,emperature with a NO W xenon lamp through a heatabsorbing filter and an orange Melles Griot 0(X15 Schott glass filter (515 nm rut-off). Depending on the age of the lamp. complet,e bleaching of a 100 mg sample rtquired

descrlbrd

from less t,han 24 to more than 48 h. Apomrmbranrs were dialyzed t,wice for 10 t,o 15 h against 50 to 200 vol. water at ‘LT. (‘hymot,ryptic cleavage closely followed the protocol of (ierber et rrl. ( 1979). Apomembrane suspensions (2 to 3 g/l) were supplemented with Tris . HCI and Ca(I, (final c*oncentrations 50 mw and 5 mM, respectively, pH = 8) and TLCK-treated chymotrypsin (1 g/60 g ISR), and incubated for 3.5 h at 37°C. Proteolysis was terminated by cooling to 4°C: and centrifuging 2 h at 44,000 revs/min (220,OOOg) in a Beckman 45Ti rotor. The prllet8s were resuspended in ice-cold distilled water. divided into 50 to 100 mg samples in 30-m] (Iorex centrifugat,ion tubes. quick-frozen, lyophilized and stored at - 20°C’. (The remove single centrifhgatiori step does not entirely chymotrypsin; resuspension of such samples in an aqueous medium followed by dilution into SDS/rlrct~rophoresis sample buffer results in extensive degradation of UK. This degradation is not observed, however. after solubilization in formic acid.) Separation of the fragments was adapted from the f)roredurt: of Huang ul al. (1981). When large amounts of material had to be prepared. particular attention was paid to achieving sufficient separation of intact RR, from its (‘-1 fragment in a limited time while maintaining relatively low voncent.ratione of the fragments in organic solution. T,yophilized clea.ved apomembrane (up to 300 mg) was brought to room temperature and extracted with 0.06 to 0.1 ml of formic acid (88 or 90’:“) per mg RR for 5 min, under constant stirring: after dilution to a 3 : 7 (v/v) formic acid/ethanol ratio. the extract was crntrifuged for 10 min at 10.000 revs/min (8500 g) in a Heckman JA2O rotor. If necessar?;. the clear yellow superriatant was concentrated to _
success of subsequent reconstitution experimrnts that this step be conducted at a low concentration of protein. The C-1 and C-2 pools were diluted to between 0.5 and 1 g/l with a 40/‘, (w/v) solution of SDS in formic acid/ ethanol (3 :7, v/v: final SD8 concentration 1 to 30, ((:-I) or 0 to 29;, (C-2)). The pH was raised to -5 \)y addition of 28.4’?,, BH,OH, taking care to avoid excessive heat build-up, and the samples were dialyzed against, 15 to 30 vol. SDS buffer (pH W), using Spectra/I’or 3 dialysis tubing (X, rut-off - 3500: 11.5 mm diam.). The protein I)rec*ipit.atrd almost, immediately. During the tirst 2 to 3 h the pH of’ the dialysis buffer was cxonstantly monitored and adjusted to 8 with c*oncent,rated NaOH as required. /Vhen t.hr pH stabilized. the buffer wa.s changed and t,hr dialysis continued for another 8 to IO h at room temperature. by which tZimr the content of the bags was c+ar. The buffer was changed again, the dialysis continued for 6 to 8 h. and the buffer replaced with pH 7 bu& (same composition). After 12 to 24 h of dialysis. the pH of’ the buffer was brought t)o pH 6.0 wit,h (aoncrntrat.ed HCY and the dialysis continued overnight. The samples were c~ollt&ed and analyzed by gel rlr&ophoresis. Protein concentration, typically 0.5 to I g/I. was determined from radioactivity counting and checked by measuring absorbance at, -180 nm (2 mm l)athlrngth vuvet,tes). using the rxtinct,ion c:orfficients given hy Lao of al. (1983). The overall yield of the preparation depended rssrntiall>, on reclovery from the column and etfectivrnrss of separation. In large-scale experiments (starting with 200 t,o 300 mg of cleaved apomembrane). yields ranged from 30 to 600,, for C-l and from 45 t,o ‘i.‘,?,, for (‘-2.

lVe describe here the standard reconstitution procedure used for lattic*r reformation. The effects of several modificat.ions t,o this procedure are described in the next srttion. The protocol can be applied to unclravrd RR wit,h higher yields of rhromophore regeneration. Chymotryptic fragments in SDS buffer were mixed in a I : 1 molar ratio and supplemented with Hakhctw’cun lipids (st)ock solution, I.27 0,) (w/v) in SDS buffer) and sodium tauroc+holat,ta (stock solution. 1 t)o KO(, (w/v) in SDS buffer) in a protein/lipid/taurocholat’e ratio of I : I : I (t)y weight,). 811~trccnsretinal (10 mM in ethanol) was added in a I.1 to I.5 molar ratio t,o the fragments: the final c.oncLrntration of ethanol never exceeded O..ic)i; (v/v), The final c.oncentration of protein was typically 0.5 g/l. Test, rrconstitutions were generally conducted on 5 or 10 nmol samples (0.13 or 0.27 mg; 200 t,o 500 ~1). The procedurr could be scaled up to 150 mg without modification. We have not invest.igated t,he dependence of renaturation on SDS purity but this parameter should not be overlooked. Tatradt~~yl and hrxadecyl sulfates are nearly absent in SIN from Bioltad. but can represent up to 35a,, of .‘SDS” from other sources. and have been found to impair renaturation of nucleasrs on polyacrylamidr gels (Lacks rt trl.. 1979). Dotircyl sulfate was precipitated as its potassium salt (PIN) hy addition of 4 M-KU with vigorous stirring. The amount of KC!1 t,o be added was calculated so that. following 1’1)s precipitation. t,he concentration of frer potassium ions would be 150 tTlM, assuming stoichiometric c*onlplrx formation of K + wit’h dodecyl sulfate: e.g. a 500.~1 sample would be treated with 42 ~1 of 4 N-KU. A white precipitate appeared instantaneously. The samples were incubated in t,hr dark at room temperature for

Refolding of Bacteriorhodopsin about half an hour, with occasional stirring. The PDS precipitate was then removed by 2 successive centrifugations (-5 min each) at 2000 revs/min in either a Speed-Vat (35Og), a Beckman J21-C (rotor JA20, 340g) or a Sorvall RC-5B centrifuge (GSA rotor, 65Og), depending on sample size. The supernatant was t.ransferred into Spectra/Par 2 dialysis tubing (6.4 mm or I I .5 mm dry diameter) and dialyzed for at least 2 days at room temperature in the dark against at least 100 vol. K buffer, changed at least twice. Traces of powdery PDS precipitate, when initially present, had usually disappeared following 24 h of dialysis. The protein yield of the renaturation step depends on losses during centrifugation. For sma.11 (O-1 to 0.2 mg) samples, final recovery is tjypicalIy 50 to 600/b. For larger samples ( 2 1 mg) it is usually in excess of 80 to 90%. In preparations where Cl and C-2 were differently radiolabeled, no significant discrepancy between the recoveries of the 2 fragment,s was noted. When taurocholate is omitted (see next section), losses are much more significant unless the lipid-to-protein ratio is raised. (‘entrifugation t,imrs must then be determined by trial and error. We have not directly measured the amounts of dodecyl sulfate and taurocholate that remain associated with Halobacterium lipid vesicles following PDS precipitation. From the solubility data given by Goto & Sakamoto (1971). the solubility product of PDS can be estimated to be -1.5x IOw5 M at 2O”C, 3.2 x low5 M' at 27°C and 1.0 x IO-’ M at, 37°C. At dO”C, in the presence of 150 mM-K’, doderyl sulfate solubility should be about IV4 M (i.e. 0.00346. a little over 0.0005 the initial SDS concentration and about 0.1 of the c.m.c.). The partition coefficient of dodecyl sulfate in HaEobacterium lipids is not. known. If we assume its standard chemical potential t,o he similar in these highly charged lipids and in SDS micelles, a crude calculation yields a (very approximate) ratio of I mol of dodecyl sulfate per 10 mol of lipids at equilibrium. As regards taurocholate, if we assume its part)ition coefficient in Halobacterium lipids to be roughly to t.hat of cholate in Electrophorus comparable membranes (Brotherus et al.. 1979); we obtain an estimate of 1 mol per 50 mol of lipids under the usual conditions (-0.050,, (w/v) final concentration). Elimination of residual detergents following 2 days dialysis should be very extensive. The half-time for diflusion of free (14C]choIate from 6.4 mm Spectralpor 2 tubing under similar conditions (Popot et aE., 1981) is about 45 min (J.-L. Popot, unpublished results). Elimination of dodec,vI sulfate is consistent with the disappearance of contaminat.ing PDS rrystallites. (ii) Effect of various mtdijeations to the standard protocol Doubling the amount’ of taurocholate or suppressing it altogether had only minor effects on the extent of chromophore regeneration and the quality of the lattice. Taurocholate was always well below its critical micellar concentration (which is -3 mM under our experimental conditions; see Small. 1971). Although in its absence renat,uration proceeded faster, the final extent was similar. The renatured PM sedimented rapidly. however. and became difficult to separate from the PDS precipitate. For small samples (10.3 mg), protein recovery dropped from typically 50 to 60% to - loo/, or whose residual less. dodecyl sulfate, Besides concentration after addition of KC1 is strictly determined by the final concent,ration of K+. taurocholate is the only component of the mixture to be significantly soluble as a

in Lipid

Bilayers

659

monomer. AS its concentration during the renaturation process is of secondary importance, the protocol is not very sensitive to the absolute concentrations of protein. lipids and taurocholate in the SDS solution, and diluting the mixture with SDS buffer has little or no effect. Roth the initial concentration of SDS and that of the KC1 solution used to precipitate it are important parameters. Higher concentrations of either SDS or KC1 improved renaturation. presumably by speeding up the removal of dodecyl sulfate. The effect of raising the initial concentration of SDS may seen paradoxical; it can perhaps be rationalized by considering that the rate at which dodecyl sulfat’e diffuses out of reforming bilayers ma.7 be higher when there is an abundance of crystals to which PdS molecules can adsorb. Raising the final concentration of K+ from 0.15 M to 0.5 M had little or no effect, even t.hough it should decrease proportionately the equilibrium molar ratio of residual dodecyl sulfate to lipids in the vesicles: perhaps a positive effect is counterbalanced by the destabilizing influence of high on Halobacterium lipid bilayers (see ionic strength Discussion). Lowering the final concentration of K+ or incubating for several hours before removal of PDS and dialysis was det,rimentaI. The effects of varying the lipid-to-protein ratio on renaturation and crvstallization are different. Between 0.5 and 2.0 (w/w), little effect, is consistently seen on chromophore regeneration. Outside this range, the yield of regeneration decreases slowly. Neither regeneration nor lattice were obtained at 0.3: 1 (w/w). the ratio in native PM. Good quality lattices have been observed at ratios of 0.5 and 2 (w/w). As the ratio rises. the contribution of lipid phases to the low-angle region of the X-ray pattern increases; beyond a 2 : 1 (w/w) ratio, the reflections due to the f; lattice become blurred. At 7: 1 or 10: 1 (w/w) ratios. the I>3 reflections are nearly undetectable, even though the ext,ent of regeneration (over 30:;) would ensure lattice formation at, lower lipid-to-protein ratios. Observing sharp diffraction lines, therefore, depends both on the &icienry of chromophore regeneration and on the lipid-to-protein ratios. the most favorable range of which seems to lie between -0.75: 1 and 1.0: 1 (w/w). This ratio of (I : 1. range, together with a Iipid/taurochoIate w/w). defines our st,andard conditions for la.ttice reformation (see Fig. 3). High levels of chromophore regeneration ran be obtained using other lipids. We have experimrnbd with DMPC, DPPC. asolectin and egg PC, at diRerent temperat,ures and with different protein/Iipid/taurocholate ratios. Whether with Ha,lobacterium lipids or with DMPC, chilling the sample simultaneously with PDS precipitation or raising the temperature to 37°C’ were detrimental. Both effects are readily explained; we shoa below that even when both fragments have been properly refolded. their interactions to regenerate cBR is very slow at 4°C: at 37°C. on the other hand, dodecvl sulfate is not efficiently removed. the solubility of PDk being greater t.han the c*.m.cs.(see section (f) (1). above). Chromophore regeneration was best (averaging 70%) at large DMPC to protein ratios (10 : 1. w/w), in the 18 to 28°C temperature range (i.e. both below and above the critial temperature for DMPC. t, z 23°C) and in the absence of taurocholatr. Our attempts at reforming the P3 lattice using lipids more convenient than HaZobacteriwm lipids. however. have met with limited success. Even when regeneration in cxc~~ of 30% was obtained at low lipid-to-protein ratios (- I : 1. w/w). either no BR lattice was formed (asolectin. egg I’(‘) or a very disordered one (DMPC and Dl’I’(‘: see Fig. 2(d)).

It is perhaps not surprising that only poorly ordrred were observed in samples rrconstitutjrd wit,h I)MP(‘. At high protein to DMI’C ratios, c~hromophorr regeneration was poorer t’han in Halohacteriun~ lipids (-407,,); our hrst samples. therefijro. contjainrd renatured cHR in a protein/I)Ml’(’ weight ratio of’ - I : 3. U’hile the X-ray patterns (recorded at room trmprraturr) cbharartrristica exhibited t’he 1.2 A reflection of hexagonally ordered fatty chains (Engelman. 1970). it is likely that lipid phase t,ransition was not completr mtl t,hat most of cBR was not aggrrgatrd (c&f. Fig. 3A of Uencher cutcrl.. 1983). Tt should be noted. however. t.hat preliminary experiments with DI’PC (t, z 41 ‘C’) had 110 more sur~ess. lattices

Fragments were reintegrated into separate HL vesicles following the standard reconstitutJion protocol described above, with the following modifications: (1) fragments in SDS buffer were kept separate; (2) retinal and taurocholate were omitted; (3) the HL lipid-to-protein ratio was 10: 1 (w/w), unless otherwise noted: (4) centrifugation of PDS without excessive loss of reconstituted material was more difficult; several I to 4 min centrifugations at 2000 revs/mill (340g) in thr JA20 rotor of the Beckman 521-C centrifuge t.gpicallg resulted in 60 to XOO/orecovery. After dialysis. the fragments werfi stored at room temper&ure in K buffer. Thrir c*oncrntration. determined by radioactivity counting, was typically around 0.5 g protein/l. Protein-fret, HT. vesicles. when needed. were prepared by the same method. Vesicle fusion was achieved by freeze-thawing (f’ick. 1981). Suspensions of vesicles cont)aining the 2 fragments were mixed in a l-ml glass tube in the desired proportion. generally 1 : I (mol/mol), supplemented with retinal 1.5 : 1, mol/mol), and the mixture frozen in solid (IO,/ethanol. The tube was mounted in an icr/watclr bath and allowed to t.hau- at 0°C. The tip of the 3 mm diameter probe of a set on position ‘i was Kranson model W14OD sonifirr brought in contact with the sample. The temperature was monit,orrd using a thermist~or probe (f3ailry Tnstruments, model ISAT-8) and not allowed to rise above 5°C’ during sonication. When the sample was calear. it was transferred to a plast’ic s1)e‘~t’ro1)hotometrr cuvette (10 mm lightpath. Kartell, pre-cooled at 0°C‘) and rapidly sonicaatrd until the sample reached a predet’ermined temperature. generally 20°C‘. This process took about 30 to 60 s. The beginning of the 2nd sonicat,ion was taken as time zero. t,. in kineticas experiments. Occ*axionally. freeze -thaw and sonica.tion \I ere accomplished direc%ly in the spe&rophotometer cuvrtte, with identical results. The cuvette was rapidly transferred to a Beckman modrl 26 doublebeam spe:“t’.oI’h”torn”tel. equipped with a thermostatically ront,rolled sample holder atrd thr regeneration of the chromophorr followed at 560 nm. Final spectra were recaorded 2 to 3 days after fusiotl, when regeneration was complete. The extent of rrg:rneration \vas c*al(~ulated from the magnitude of the absorbance peak at 554 nm and the fragment concentration determined from radioactivit,y counting. assuming c554 = 44,500 (see Result,s). Sonication is not required for chromophore rt’genrrat,ion. The development of the purple valor can be observcbd on samples left to thaw at room temperature. Such samples, however. are rxtrrmel,v turbid and sonication is required before a reliable absorption spectrum ran br recorded. Retinal can be omit,ted from t.hr mixturfs of fragments and added a&r fusion and souication.

(h) Sucrose gradient amlysis Xative PM and reconstituted samples were layered on 20 7; to SOoi, (w/w) sucrose gradients in K buffer (Beckman SW60 rotor) and centrifuged at 58.000 revs/min (450,00Og), 4°C for 17 to 20 h. Gradients were collected as 4 to 6 drop fractions, using a plunging capillary and a peristaltic pump. Alternat,ively, the sucrose concentration corresponding to each purple band can he determined to better than @39b by measuring the refractive index of a number of small (5 to 10 ~1) samples t,aken from immediately below, in the center of, and immediat.ely above t.he sample. Fractionation on tauroehofate-containing gradients was performed in an identical manner. Both the samples and the gradient werrl supplemented with 0.57, (w/v) taurocholate. The absorption spectrum of the purified reconstituted sample was determined direct)ly on t,he pooled fra&ions. The density of Ha~lobacterium lipids was estimated by swelling a dried sample in water and centrifuging it as described above on a 0 to 75% sucrose gradient in water. A single band was observed at 17.3 (+0.2)0/, sucrose (p = I+)72 g/l). The density of BR (p = I.235 g/l) was calculat,ed from our measuretf density for PM in K buffer (p = I.190 g/l), assuming a 3 : 1 (w/w) protein-to-lipid ratio. It is identical with the buoyant density of delipidated

BR

(p = 1.24 g/l)

given

by

(lasadio

&

Stoeckenius (1980). These values of p4 c, which differ somewhat, from the p 25,,Cvalues given by Maryue pf al. (1984). have heen used to estimate the lipid-to-protein ratio of reconstituted samples.

(i) &xctroscopy (i) A t~sorption spectra Samples w-err suspended in either K buffer or K/5 buffer. Light scattering was generally reduced by a brief sonication (3 x 10 s, 3 mm probe). Either quartz or plastic 1 cm pathlength cuvettes were used, depending on whether the ultraviolet region of the spectrum was to be examined. Spectra were recorded on the double-beam spectrophotometer with either butfer or a bleached or retinal-free sample in the reference cell. as indicated. The calibration of the monochromator was checked using a holmium oxide filter (Beckman no. 96157). Samples were either dark-adapted for at least 12 h at room temperature, or light-adapted by 7 min exposure t,o a 65 W sodium lamp (George W. Gat’es bi Co., model STAA-5C) equipped with an OG515 Schott glass filter.

Extimtion coeffcients were determined as described by Rrhorek & Heyn (1979). A 1 tnM or 1.5 mM-retinal solution in absolute et,hanol was prepared daily and it,s exact concentration calculated from the absorbance at 382 nm of a l/l00 dilution in absolut,e ethanol. taking E382 = 42.800 (see Rehorek & Heyn, 1979). TLC analysis of the solution following the procedure of Rehorek & Hegn (1979) on Merck silica gel 6OF-254 plates revealed only 1 spot when the solution was fresh. The ultraviolet absorbance spectrum of the solution was identical to published spectra (Robeson et al., 1955; Tosukhowong & OISOJI. 1978). Unless they were kept under argon, retinal solutions stored at - 20°C for extended periods (1 month) underwent, oxidation. as shown by t.he appearance of suppletnentary spots upon TLC analysis and a blue shift of the spectrum. Extinction coefficients were determined for retinal bound to: (1) bleached PM; (2) bleached and

RQoldkg of Bacteriorhodopsin chymotryptically cleaved PM; (3) a bleached reconstituted sample; and (4) a sample reconstituted in the absent-e of retinal. The samples. at about 0.4 g/l in K buffer, were distributed as 0.5 to 1.5-m] samples in 1 cm. pathlength cuvettes. Retina1 samples (1 to I.5 ~1) were added with glass micropipettes (Drumond Microcaps). The accuracy of the delivery was tested by measuring the absorbance of retinal dilutions in ethanol; the volumes of retinal solution actually delivrred were 96% of nominal values. The appropriate correction was made when ~*alculatinp extinction coefficients. The absorbance at, 382 nm of samples prepared by dilution of retinal into a suspension of HaZoOacterium lipid vesicles in K buffer was 19%, lower than that of identical dilutions into ethanol, It does not seem likely. that this difference results from delivery of less retmal (e.g. because of retinal precipitation on the inside wall of the capillary), as the same effect was observed using a 5-~1 Hamilton syringe. and the delivery of retinal into ethanol is not significantly lower when Microcaps are not rinsed. The difference could be due t,o either a lower 8382 of retinal in lipid bilayers ~~er.s~~et.hanol, although no difference in the absorption spectrum is apparent. or to instrumental error in determining the true absorbance at 382 nm of lightscat,tering samples. Spet*tra were rec*orded after regtmeration was complete. i.e. after at least overnight incubation at room temperature. Samples were light.- or dark-adapted as described above. The reference cuvette contained an identical sample but with no retinal added. Control experiments showed that identical results are obtained whether regeneration is conducted in either quartz or plastic cuvettes. and plastir was generallv used. Extinction coefficients were calculated by assuming that all t-he retinal added binds to BO (or cB0) in a 1 : 1 (mol/mol) ratio (Rrhorek & Heyn. 1979). The Beckman model 26 spectrophot,ometrr used was not equipped with an integrating sphere. Tn order to estimate whether lightscattering artifaclts could introduce a systematic* error in the determination of AZhO, the spectra of a series of samples regenerated from bleached PM were recorded in K buffer and in a 60 : 40 (v/v) mixture of glycerol and K buffer. The extinction coefficients determined for darkadapted I’M in both conditions were identical: that determined for light-adapted PM was slightly lower (by - 5’!;,) in the presencr of glycrrol. (iii) (‘irc~rlnr flickroisnr sprrtrn Visiblr spectra were recorded in t.he 400 to 800 nm region on either a Vary 60 spertropolarimeter equipped with a 6001 c..d. attachment, (3 cm pathlength cuvette) or on an Aviv 601)s spectropolarimeter (1 mm pat.hlengt,h cnvrttr). ‘l’hta variable position detector of the latter instrument was set tiirect.lji adjacent to the sample cell to rrducor light-scattering effects. At least 3 spectra at, 25°C were rollected for each specimen examined. Ultraviolet’ spectra were recorded on the Aviv 6ODS spectropolarimet~rr. The wavelength range scanned was 300 to 195 nm. Measurements were made at %7”C using a 0.1 mm pathlength cell. The spectra reported are the average of 4 scans measured every 0.2 nm and smoothed over 17 points using the Savitsky-Qolay algorithm. Data were analyzed in thr wavelength range of 195 to 240 nm by a linear least-squares fitting procedure using a referrnre of 15 water-soluble proteins (Chang et al., 1978). with thr helix length st>t at 26 residues. using the yeptide concentrations obtained from amino acid analysis (see Mao & Wallace. 1984: Wallace & Teeters. 1987). The sum of’ thr src~ondary struc.turr fractions was very close to

in Lijoid Bilayers

unit,y

and

no

normalization

wa.s

required.

The

correspondences between the measured and calculated spectra for these samples were excellent, resulting normalized root-mean-square deviation parameters 0.03 to oa4. (j) X-ray

diflraction

in of

experimente

Samples (0.25 to 0.50 mg of BR) were suspended in 4 ml of K/5 buffer and centrifuged for about 30 min at about 30,000 revs/ruin (21,OOOg) in an SW60 Beckman rotor at 20°C. The pellets were homogenized with the tip of an automatic pipet, layered on thin polystyrene sheet (0.0012 Clear UPS. Kama Corp.), and equilibrated with sa.turated ?v’aCl at room temperature (76); relative humidity). The partially dehydrated sample was mounted perpendicular to the beam of a. Baird and Tatlock Srarle toroid camera. and exposed to copper K, X-rays from an Elliott, GX-6 rotating anode source. The camera was purged for at least 10 min with helium humidified by bubbling through a saturated solution of SaCI. Diffraction patt,erns were recorded for I to 2 h WI Kodak DEF-5 X-ray film at, a specimen-to-film distanc*r of 75 mm. Samples in suspension were pelleted as described above and the soft pellet homogenized and transferred by lowspeed crntrifugation (Adams DJmac tabletop caentrifuge) into 0.7 mm diameter (10 pm wall thickness) X-ray capillaries (Charles Supper Co.. Natick. M.4). Thr capillaries were sealed and the samples mount.ed iI, thr beam of eit.her the toroid or a Franks camera (set, Popot rt al.. 1986). Exposure times werr 4 to 6 hours and I to 5 days. respectively. Visual estimat,es of the quality of the patterns on art scale from 0 (no pattern) to 6 (comparable to an t~xt*ellt~nt PM pattern) were based primarily on t,hr pr~st~~*e and sharpness of thr wrak reflections “in the 7 to X LA region. These evaluations 011 art% somewhat dPpPn’lPnt extrantaous factors such as earnera focusing anti film density. Ratings of diffraction patterns from difkrtnt portions of the same reconstituted HampI? generalI! agreed to within kO.5. Pa.tterns rated 5 a11t1 xbovc* exhibited sharp reflections out t,o 7 A4 and diffi~rt~tl from PM patterns mainly in the prrsence of strong lipid refiections in the 50 ,S region and a highfar continuous background (see Popot et al., 1986). The patt,tlrns shower in Fig. 2(a), (b), ((1) and (d) werp rated 6. 5.5. 0 and I. resppcati vely

3. Results (a) &constitution (i) Spectroscopic

in the presence of rrtinml

properties

of reconstituted

samples (a)

il h.sorbnnce

spertsum

Purified chymotryptic exhibit

indicating

no absorbance

fragments in 8J)S solution at

560,

382

or

362 nm,

the absence of int,act BR chromophore, retinal or retinal oxime (Fig. 1, curve 1). Precipitation of potassium dodecyl sulfate (PDS) from a mixture of the fragments in SDS supplemented with retinal, Halobacterium lipids and taurochofak resulted in chromophore regeneration, as judged by the development of purple color. PDS precipitate was removed by centrifugation, and t,auroc:holatr and residual dodecyl sulfate

K62

J.-L.

--

Popot et al.

i

Exposure diminution 1,,,

(+2

100

400 600 Wavelength(nm)

8

Figure 1. Absorption spectra of equimolar mixtures of bacteriorhodopsyl fragments in SDS solution and after reconstitut,ion with and without retinal. Curve 1, purified (‘-1 and C-2 fragments in SDS buffer mixed in equimolar ratio. C’urve 2, eyuimolar mixture of fragments in SDS buffer supplemented with HaloOacterium lipids, retinal and taurocholate and reconstituted by I’DS precipitation and dialysis according to the standard protocol described under Materials and Methods; K buffer, sample no. 396. Curve 3. same as curve 2 but retinal was omitted from the raronstitution mixture: K buffer, sample no. 395. The 3 wrves have been normalized to the same AZsO value.

eliminated by dialysis against K buffer. The absorbance spectrum of the resulting samples indicated the presence of some free retinal and of regenerated RR, chromophore (Fig. 1, curve 2); no regeneration was observed if either one of the fragments or retinal was omitted (curve 3). Absorbance spectra were recorded on a doublebeam spectrophotometer, taking as a reference an identical sample reconstituted in the absence of retinal. Peak absorbance was observed at 1 max = 554 (+2) nm with dark-adapted samples at room temperature in K buffer (pH 6.0: see Table 1).

Table I Visible absorption maxima and extinction coeficients of various native and reconstituted preparations

PM (‘kaved PM Sample no. 395 Sample no. 3%

DA LA DA LA DA DA

553f2 564+ 2 55652 564k2 554+2 554&d

49,400 57.200 45,100 49,700 45,000 43,400

Extinction coefficients at I,,, were determined as deswihed in Materials and Methods. Known amounts of all-trans retinal were added t,o the following samples: PM, bleached PM; cleaved PM, bleached and ohymotrypticallg cleaved PM; no. 395, OIW reconstituted from fragments in the absence of retinal; no. 396; CUR rttconstituted from fragments in the prexenre of retinal and subsequently bleached. DA. dark-adapted; LA, light-adapt,ed.

a slight shifts of

to 3 nm). some subsequent

involved experiments working at different temperatures or pH, we have examined the effect of these variables on t,he absorption spectra of reconstituted samples (not shown). Raising the temperatjure from 2°C to 45°C had little eflect on I,,,. which shifted to the blue by only 3 nm. From 45°C to 75”C, I,,, further shifted to t,he blue by 17 nm. Peak absorbance decreased 40’& over the same range of k)Y ahout) temperatures. I;p to 75°C. changes in imaX and A,,, were reversible. Tn cont,rast to native PM (Mowery rt al.. 1979; Muccio & Cassim, 1979), both ,I,,, and in the 4 to 7 pH range. ~-La, varied substant,ially 1‘mm remained essentially constant from pH 7.0 to pH 5.5 and then shifted t.o the red, reaching 600 nrn at. pH 4.0. &,, varied in a complex manner over the same pH range: at pH 7.0 and pH 5.0, A,, was about I1 7;) and 7”;, lower than at pH 6.0. respectively. As

200

to actinic light resulted in of absorbance with minimal

(b)

Extinction

coefficient,9

Extinction coeficients were determined by the procedure of Rehorek &L Heyn (1979): which relies on measuring absorbance increases at i,,, induced by adding known amounts of retinal to bleached PM. Reconstituted samples were bleached in the presence of hydroxylamine as described in Materials and Methods; samples of all-trans retinal were added and both the kinetics of chromophore regeneration (see below) and its final extent were det,ermined. For comparison, identical experiments were conducted on apornembrane and chymotrypt,ically cleaved apomembrane. Finally, we have taken advantage of the ability to renature cH0 from fragment,s by PIIS precipitation in the absence of retinal (see below) and we have Gtrated such a preparation with retinal. The results of these experiments are surnmarized in Table I. They indicate that the extinction coeffirient of t-econstituted samples is similar to t’hat of darkadapt,ed cleaved PM and about loo/,, lower t.han that of dark-adapted PM. The extinction coefficient we measure for light>adapted PM is about loo/, lower than t,hat reported by Rehorek & Heyn (1979). We have not identified the source of this discrepancy. The purity of the retina,1 solutions used and the amounts delivered have been checked as described under Materials and Met,hods. Measurements in SO?; glycerol suggest that light,-scattering artefacts do not account for the difference. Throughout this paper, estimates of chromophore regeneration in reconstituted samples are based on t.he measured average extinction coefficient. of 44,500 M - ’ cm - ‘. (c) Visible

circular

dichroiem

Exciton interactions between neighboring KR molecules are responsible for the biphasic shape of the visible cd. spectrum of native PM (Heyn rt al..

Refolding

of Racteriorhodopsin

1975; Becher & Ebrey, 1976; Brith-Lindner & Rosenheck, 1977); monomeric BR exhibits only a positive band (Dencher & Heyn, 1978; Cherry et al., 1978). In preliminary measurements, we have compared the c.d. spectrum in the 400 to 750 nm range of fully crystalline BR (native PM), monomeric BR (solubilized in Triton X-100) and a renatured sample. The spectrum of the renatured sample exhibited an intermediate shape, featuring a marked asymmetry with a reduced negative peak, suggestive of a mixture of monomeric and oligomerit BR (not shown). (ii) Composition of reconstituted samples The amino acid compositions of samples renatured from an equimolar mixture of the C-l and C-2 fragments were indistinguishable from that of native PM (not shown). The polypeptide composition of the purified fragments and that of reconstituted samples, as determined by SDS/ polyacrylamide gel electrophoresis, have been described (Popot et al., 1986). Minor contaminants were uncleaved BR (15% in ali experiments described here) and occasionally an approximately 35.000 M, species, most’ likely a dimer of the C-l fragment. The carboxy-terminal-less C-l fragment, when present at all, represented less than 3% of (Y-1. The C-l to C-2 ratio in renatured samples was examined by gel electrophoresis and by measurement of radioactivity on two samples renatured from [3H]C-2 and (‘4C]C-1 fragments. The first determinat’ion yielded a ratio of Ia03 +O*lO (mol/mol: taking cleaved apomembrane as a standard), the second yielded a ratio of 0.95 +0*02 (moljmol). It appears that neither of the fragment,s is preferentially removed from the mixture during PDS precipitation. Electron microscopy of t,wo samples negatively stained with uranyl acetate revealed vesicles 400 to 800 a (1 A = 0.1 nm) in diameter (not shown). In preliminary experiments, limited digestion with proteinase K of one reconstituted sample (no. 396) according to the procedure of Dumont et al. (1985) yielded the band pattern expected from cBR Inserted with a native t,opology and predominantly inside-out orientat8ion (M. E. Dumont, personal communication). The homogeneity of the samples was examined by sucrose gradient equilibrium centrifugation of three different samples (not shown). Two of the samples yielded a single purple band centered at 1.14 to 1.15g/l. The third one yielded a major band at 1.15 and a minor one at 1.12 g/l. The density of the major bands is that expected from a 1 : 1 (w/w) mixture of Halobactvrium lipids and BR (1.15 g/l; see Materials and Methods). Phosphate and amino acid analyses indeed yielded a lipid-to-protein ratio of 0.94 : I t,o 1.I5 : 1 (w/w; 2 samples). This composition suggests that most of the protein and lipids initially present. in the SDS solution reassociated (some free retinal, however, binds to the PDS precipitate). The similarity of the protein and chromophore profiles in sucrose gradients further

in Lipid

Rilayers

663

indicated that properly and improperly renatured RR fragments do not segregate in vesicle populations with different densities. Consistent with these conclusions, the peak fractions contained free retinal (a marker for lipids), total protein and regenerated chromophore in ratios similar to those in the samples applied to the gradients, After dialvsis to remove sucrose and orientation hi pa&al drying, these fractions generated the same X-ray diffract.ion patterns as the original samples (see below). Incubation of native PM with bile salts does not solubilize BR (Hwang & Stoeckenius, 1977). Rather, the hexagonal lattice shrinks as most lipids are extracted (Glaeser et al., 1985). Tn order to examine whether lipids and non-renatured material could be separated from renatured cBR by this approach, reconstituted samples were incubated in K buffer containing 0.5% taurocholate and centrifuged on sucrose gradients in the presence of A single purple band 0.5 9/;, taurocholate. equilibrated at a density (1.20 g/l) identical wit,11 t,hat of native PM in the same medium (not shown). Absorbance spectra indicated that, with respect, to the original reconstituted sample, the dense fraction had lost most of its free retinal and was enriched in regenerat,ed chromophore. (Assuming E,,, to bfa reunchanged in the presence of detergent! generation of the protein was lOl’$& in the purified fraction LWSUS 79% in the origina) sample.) Removal of the taurocholate and sucrose 1)~ dialysis or by dilution and centrifugation, however. inactivated part of the regenerated chromophore unless Halohcterium lipids were added t.o the purified fraction prior to detergent rrmoval. Presumably because of this requirement, X-raJ diffraction patterns generated by such purified fractions were poorer than those obtained from the original samples. (iii) Reformation

of the P, lattice

(a) C’onditions for lattice observation

Samples reconstituted according to the standard protocaol described in Materials and Methods were oriented by partial drying and placed in an X-ray beam. The diffraction patterns obta,ined (Fig. 2(h)) included reflections with the same positions and relative intensities as patterns generated by native PM (Henderson. 1975; Blaurock, 1975; Fig. 2(a)). Quantitative analysis of X-ray and neutron intensit,ies to 7 a resolution indicated that the dimensions and content of the unit cell are indistinguishable from those of native PM (Popot et al., 1986). The continuous background, however, was conspicuously higher in reconstituted samples, and reflections due t’o the lamellar or hexagonal arrangement of the excess lipids in these samples were present (note differences near beam stop in Fig. 2(a) and (b): and see Popot et al.. 1986). Tn order to ascertain whether the hexagonal 13R lattice was present hefore the samples were oriented by partial drying. diffraction patterns were samples kept in recorded from reconstituted

Figure 2. S-ray diffraction pat)trrns of native I’M and rrconstitut~ed samples. Samples were either oriented by partial dehydration ((a). (b) and (d)) or (c-) kept, as a thick suspension in K/5 buffer in a sealed capillary. (a) Native PM; (b) and (r) two samples (110s27” and 1396.respec*t~ivrly) reconst~ituted according t,o the standard protocol described in Materials am1 .\llethods. The rreiprocal spacing of the reflections marked with white bars in (a) and (b) are l/4.45, l/46 and l/H-!) .S, ‘. from left to right. The first 2 are difticnlt to print in (b) beearrse of the higher background. (d) A sample (no. Sl5) reeonstitutrd wit,h I)MFY’ instead of Hulobnct~rium lipids: the rrcipronal spacing of the reflertion marked with it white bar is lj4.2 4 ‘. The sample was rreonstit~utrtl start,ing from cleaved apomembrane solubilized in formic actid and thrrefort~ contained son~e Hnlohn~trric~nr lijkis (E”.otein/l)MP(1:HnEohwt:tPTi?~Inl.lipids I : 1 : 0.3. by wt). (!hrornqthore regeneration was 4Oc?b.assuming t,hr same rxt~inct~ion coefficient. as samples reconstit,ut,ed with Ht&mc.ter+urt~lipids.

suspension in K buffer in sealed glass capillaries. lrnlike suspensions of PM (Rlaurock & Stoeekenius. 1971), such samples did not’ generate any detectable diffraction patt)ern (Fig. 2(c)). (b)

C)ptim,al conditions for chromophore wgeneration and latticr ,formation

Several hundred reconnt,itution experiments were performed in the course of defining a satisfactory protocol. A few conclusions can be drawn regarding reproducibility and critical fact,ors. At, lipid to protein ratios between 0.7 and 1.2 (w/w), good quality lattices were not) observed unless at least 30 to 4oyy,, of the chromophore was regenerated (Fig. 3). Under the st,andard conditions described in Materials and Methods, regenerat,ion was never less t,han 4OoiO (average 53 (_+8)(&) and sharp diffraction patterns were consistently obtained; the variahilky in pattern quality is largely due t’o uncontrolled factors during drying of the reconstituted samples before X-ray measurements, to variat,ion in the sharpness of camera focusing and t,o subjective bias in visually evaluating films of Vilriable density. The extent of chromophorr

regeneration varied both within a series of reconstitution experiments with the same membrane fragments and from one preparation to the next. Det’ails of fragment preparation that appear t,o be important to the success of subsequent, re const’itution experiments are described in Materials and Methods. Titrations of fragments in SD8 one by another indicated that a larger percent,age of the f:-2 t.han the C-1 fragment could be renatured (typically 70:/o r,ersus Sric~~cr)).As a result, the optimal molar ratio in the reconstitution mixture was generally Cl /C-Z z 1 :@8 rather than 1 : 1. Purified fragments in SDS buffer stored for two months at room temperature exhibited less than a IO’%, drop in activity (not shown). We have examined the effects of variations in the reconstit,ution protIocol on chromophore regeneration, lattice formation and overall yield (see Materials and Methods). Most of t)hese observations were made using cleaved apomembrane in formic acid/et,hanol as the starting material, i.e. a preparation that, contains liralobacterium. lipids in a 1 : 3 lipid to protein weight ratio as well as retinal oxime. Any experiment that we repeated starting

Refolding

of Hacteriorhodopsin

6-

in Lipid

l 272

+

5-

x

*e . *am/

l

+t-x

F 4 G L 8 3.= 7 2-

Y x xt

x * I-

x

+

267

l

Oe-396

++

x

x

x

+

X0

x+ x

x

x

665

+ lk-x+’+ +++

xx

x.0.

l&layers

x +xxx

0

395 0

T:

+..a+...)(

.,.,..

,.

*..x

1

I 0

1

I

20

1

I

40 Chromophore

I

I

I

60 regenerotlon

I

80

4

I

(%I

Figure 3. (‘orrelation between extent of chromophore regeneration and quality of the X-ray diffraction pattern at low lipid to protein ratio. Samples were reconstituted either from purified fragment,s or from cleaved apomembrane solubilizrd in formic acid and transferred to SDS buffer. Halobacterium lipids t)o protein weight rabies varied from 0.7 : I to 1.2 : 1. Diffraction patterns were rated by visual inspection on a srale from 0 to 6 (see Materials and Methods). Samples whose diffract,ion pattern is illustrated in Fig. 2, or are otherwise mentioned in the text are identified. (0) Samples reconstituted from purified fragments according to the standard reconstitution protocol described in LVaterials and Methods. (h’os 267 and 272: large-scale samples (50 to 100 mg) used for neutron diffraction measurements; see Popot it al. (1986).) (0) Same as no. 396, but retinal was omitted from the reconstitution mixture; variable amounts of retinal were subsequently added to the reconstituted vesicles; resulting in samples with submaximal to maximal regeneration. ( x ) Samples reconstkuted from either purified fragments or from cleaved apomembrane, in the presence of’ retinal and taurocholate; experimental conditions for the preparatjion of the fragments and/or reconstitution deviated from the standard protocol. generally resulting in lower percentages of regenerat)ion. ( +) Same in the absence of taurocholat,e.

Time following

PDS precipitation

(days)

Figure 4. Stability upon storage of refolded BR fragments in Halobacterium lipid vesicles. Fragments from 3 different preparations were reinserted into Halobacterium lipid vesicles at 1 : 10 protein to lipid weight ratio, either separately or simultaneously, and stored at room temperature in K buffer for the time indicated. For each series of experiments, data are normalized to the initial level of regeneration averaged over the first 2 weeks. (m, 0) Fragments at equimolar ratio simultaneously refolded in the same vesicles; chromophore regeneration was measured 4 to 5 days following addition of retinal: initial levels of regeneration were 48 y. ( n ) and 58 y’ (a), (V, A, 0, 0) Fragments were independently refolded in separate vesicles in the absence of retinal; chromophore regeneration was measured 4 to 5 days following vesicle fusion in the presence of retinal at, various C-l to C-2 molar ratios; (V, A). excess C-l; initial levels of regeneration were 330~~(VI and 46O; (A); (Cl, 0) excess C-2; initial levels of regeneration were 38% (0) and 890/) (0).

666

,J.-I,.

Pvpot

from a mixt)ure of purified fragments gave t)he sa,me result Rccaunc there are variations in regeneration under the st,andard conditions (see Fig. 3). minor Pfkc~ts may have remained unnoticed. The (‘onsequences of varying the concentration of taurocholate, SI)S or KC1 are described in Materials and Methods. Taurocholate concent’ration is below its r.m.c.: upon dodecyl sulfate precipit,ation. taurocholate-containing vesicles should form. not mixed micelles. It is possible to suppress taurocholate entirely without obvious effects on the rxt,ent of chromophore regeneration and the quality of the lattice (Fig. 3). The renat.ured PM sediment.s rapidly. however; and becomes difficult to separat’r from the PIIS precipitate. High levels of chromophore regeneration can be obtained using various liljids. but Ifalobacterium lipids are necessary in order to obtain well-ordered lattices (see Materials and Methods: Fig. 2). Similarly. reformation of the (cl)

et al. lattice is much more sensitive t,han chromophore regeneration to variations of the lipid to protein ratio. 13est results were obtained at ratios of 0.75 : 1 to 14 : 1 (w/w) (see Materials and Methods). (b) Kenaturation (i) bimultaneou~s

%n the absence of retinwl

refoldiny

of the CT-1 nnd

P-% frayments

(Ieaved IW could be renatured using the same reconstitution protocol but omitting retinal from t~he reconstitution mixture. No lattice was observed in X-ray experiment’s (Fig. 3). However, upon subsequent’ addition of retinal, the chromophore was regenerated (Fig. 5(a)) and the P3 lattice could be observed (Fig. 3). Titration with retinal indicated an extinction coefficient similar to that of preparat’ions renatured in the presence of retinal (see Table 1). The quality of the lattice observed at

(b)

Cc)

0.5

l!!d 2000

400

600

800

Q 200

400

600

800

Wavelength(nm) Wavelength(nm) Wovelength(nml Figure 5. Regeneration of BR chromophore from fragments refolded either separately or simultaneously in the ahsencr of retinal. Purified fragments in SDS buffer were mixed with Halobacterium lipids (lipid to protein ratio 10 : I, M./W)in the absence of retinal and taurocholate and reconstituted by PDS precipitation as described in Materials and Methods. Following dialysis, the vesicle suspensions were clarified by a brief sonication. (a) C-l and C-2 in SDS buffer were mixed prior to PDi precipitation and simultaneously refolded in the same vesicles (top panel). Absorption spectra were recorded before (thin line) and after (thick line) addition of excess retinal (middle panel). Bottom: difference spectrum. (b) C-l and C-2 were reconstituted into separate vesicles (top panel). The vesicles were mixed (eyuimolar ratio of the fragments) and absorption spectra recorded before (thin line) and after (thick line) addition of excess retinal (middle panel). Bottom: difference spectrum. (c) C-1 and C-2 were reconstituted into separate vesicles. The vesicles were mixed (equimolar ratio of the fragments) and freeze-thawed (top panel) in the absence (thin line) or presence (thick line) of excaess retina1 (middle panel). Absorption spectra were recorded after clarification by brief sonication. The identical result was obtained if retinal was added after freeze--thawing. Bottom: difference spectrum.

Refolding of Bacteriorhodopsin various points on the titration curve was close to that observed for samples renatured to the same extent, in the presence of retinal (Fig. 3). The ability of reconstituted cB0 to regenerate the chromophore upon retinal addition decreased slowly with storage time. with an extrapolated half-time of about two mont’hs at room temperature (Fig. 4). We have compared the kinetics of chromophore regeneration following addition of retinal either to rhymotryptically cleaved apomembrane or to fragments renatured simultaneously in the absence of retinal (Fig. 6 and below; and see Table 2). Each of the kinetic curves can be described. to a good process, approximation. as a two-exponential although slight, deviations are nearly always present (Fig., 6). The kinetics of intact apomembrane regeneration is rapid (see Oesterhelt & Schuhmann, 1974; Rehorek & Heyn, 1979). We have not examined it, in detail but it can be fitted approximately by two exponentials with periods in the minute range (Table 2). The kinetics of regeneration of the cleaved apomembrane comprise a fast phase with a time-course similar to that observed with intact apomembrane and a much slower second phase with a half-time in t.he halfhour range. Wta have not investigated the cause of

I

0

I

I,

I

in Lipid

Bilayers

667

this difference. The time-course of chromophore regeneration for two preparations of fragments simultaneously refolded in the absence of retinal was very close to that observed wit,h cleaved apomembrane (Fig. 6 and Table 2). Tn order to determine whether the time-course of chromophore regeneration was diffusion-limited, the concenbrations of fragments and retinal in the lipid bilayer were varied by adjusting t,he protein to lipid ratio (from 1 : 10 to 1 : 1 in the reconstitution experiments; 3: 1 in cleaved apomembrane) and changing the protein to retinal ratio. Varying the [retinal] x [cBO] product over three orders of magnitude had no or very small effects on t’he kinetics of regeneration (see Fig. 8). (ii) Separate refolding of the C-1 and P-2 fragments (a) Keassoeiation of pre-refolded fragm,ents Studies were conducted on C-l and C-2 reconstituted separately into distinct populations of vesicles. When dodecyl sulfate was lipid precipitated from a mixture containing a 1 : 10 (w/w) ratio of either fragment and Halobacterium lipids, no chromophore was regenerated. Mixing the

I1

,

I

5

I

IO Time

I

,

I

I

I

15

Cmin)

Figure 6. Kinetics of chrornophore regeneration following either addition of retinal to renatured BO or fusion in the presence of retinal of vesicles containing refolded fragments. (a) and (b) Addition of retinal to pre-reassociated fragments. Samples were suspended in K buffer at 20°C. At time zero. retinal in ethanol was injected, the sample briefly shaken, and the increase of absorbance at 560 nm monitored as a function of time. Each data point is the average of 8 experiments in which the retinal to BO ratio was varied from sub- to super-stoichiometric. The concentration of retinal does not significantly affect the kinetics (see Fig. 8). For each experiment, the observed increases of absorbance at, time t (4,) were normalized to the maximal increase of absorbance measured after 8 days (A,). Continuous curves are d-exponential fits. The amplitudes and periods used are given in Table 2. Arrows indicate tymi” (see Fig. 8). Curve a (a), an equimolar mixture of fragments reconstituted in separate vesicles in the absence of retinal and subsequently mixed. frozen, thawed and sonicated prior to adding retinal (protein to lipid ratio 1 : 10, w/w). Curve b (O), an equimolar mixture of fragments simultaneously reconstituted in the absence of retinal (protein to lipid ratio 1 : 10, w/w). Curves c and d. fusion of vesicles containing separately refolded fragments in the presence of retinal. Two vesicle preparations suspended in I( buffer, containing, respectively. the C-l and the C-2 fragments at, 1 : 10 protein to lipid weight ratio, were mixed, suppietnented with a molar excess of retinal, frozen, thawed at O”C, and their temperature raised to .PO!O”C at f = 0 by a brief sonieation as described in Materials and Methods. Chrornophore regeneration was followed at 560 nm. (!urve c ( x ). preparation B: equimolar ratio of C-1 and C-2; average of 6 experiments. Curve d (+ ). preparation C: the ratio of (‘-1 to C-2 varied from I : 2.3 t,o I :O.fi (mol/mol) wit#hout a significant effect on t,he kinetics (see thr text,); average of 4 experimrnts

./.-I,.

t%iH -

Popot et al.

Table 2 Kinetics of chromophore reyrneratiorl upon (I) addition of retinal to native or reconstituted preparations or (2) fusion in the presence of reti’ml of qjesl;clrscontaining separately refolded fragments Experimental

Qast hxv (min)

(min)

condit,ions

(I ) .4tldition of‘ wtinai to. (a) Satire apornernbrant~ (- 18°C’) (h) (‘leaved apomembrane (y 18°C’) (c) ((‘I +(‘I)HLl (-18°C’) (d) ((‘1 +C2) HLIO (20°C:) (P) Nrached ((‘1 +(‘4+ret)HI,l (20”(‘) (f) Prresti~~thawed ((‘lHLlO+(‘ZHLlO)

A&S,

( ‘y, of AA,,,)

No. of expts

(20°C’)

(2) Fusion in the presenw uf retinal of vesicles containing separately refolded fragments. (il) Preparation A (ryimolar: - 18°C‘) (b) l’rrparation Ii (excess (‘1; 20°C) (equimolar; ZOY’) (PX(‘CHS(‘2. 2OY~) (t,) Preparation C’ (variable: ho(‘) (1) (a) and (b) Natiw I’M was (a) bleached and (b) c4eaved as described in Materials and Methods. ltrtinal oximr was not extracbed. (c) Equimolar mixture of fragments simultaneously refolded in the absrnw of retinal (1 : 1. protrin to lipid weight ratio). (d) Same experiment with a different preparation of fragments (1 : IO. protein to lipid weight ratio). (P) A sample (no. 396) reconstituted in the presence of retinal awording to thth standard protocol for lattice reformation (see Materials and Methods) and suhseyuentlv bleached as described for PM. (f) Two samples (nos 581 and 626) prepared by freezethawing eyiimolar mixtures of fragments separately refolded at I IO (w/w) prot,rin to lipid ratio in the absrnw of retinal: retinal was added to samples of each sample either 1 h or I, IO to 11 or 30 days after fusiotl; the 8 kinetics did not noticeably differ from one another and were pooled for t,he analysjs. In all other rxperiment,s included in this Table, frngment preparations were less than 2 weeks old. (2) (a) to (CL)Prrparations A. 1%and C: 3 independent pairs of fragments separately refolded in Ilnlohnrferiunt lipid \:rsi&s (1 : 10 (w/n) protein to lipid ratio) in the absenw of retinal. Thr molar ratio (‘-1 to (‘-2 is indicated (SW Fig. 6). I%, time-cwwsr of chromophore regeneration is described b,v titt,inp 2 rxponrntials with thr illtlicat.rd pwiods to thv plot of A,4,/Adri,,,. A&,,, was determined by repeating absorption mrasurements I to X days after adding retinal or 3 to 8 days after fusion. The unwrtainties on thr wtimatt~s of the fast and slow periods are about +I!?“,, and +500+,. rrspwtively. In fusion rsprrimrnth. the data did not allow the period(s) of the slo\~ phase(s) to he rstimatrd with an) precision: most tits used Tslow= 1 h. The uncertainty on the relative amplitude of each phase is about f IO”,,. Note that most kiwtivh ticvin.tr t,o some extent from a simple 2.Pxponcntial procc’ss (SW Fig. 6). The numtwr 01’ kiwtic c*xprriments pooled for rwh analysis is indicated in the last. column

two vesicle populations, following dialysis, in t,he presence of retinal did not’ result m any regeneration, even after several days at room temperature (Fig. 5(b)). When the same mixture was subjected to freeze-thawing and brought back to room temperature, however. a purplish co101 developed and t.he visible absorba.ncr spectrum, rc>corded after brief sonicat’ion to diminish lightscattering, gave evidence that the BK’ chromophore had regenerated (Fig. 5(c)). When retinal was absent. during freeze-thawing. t.hc subsequent, addition of retinal resulted in chromophore regeneration (Fig. 6, curve a). We shall refer to the separate vesicles as containing refolded fragments and to the freeze-thaw procedure as vesicle fusion. Titration of each refolded fragment by t,he other castablished which fraction of each peptide was able to contribute to chromophore regeneration. (We assume here that t*he extinction coefficient, of renatured cRR was the same whrt.her the fragments were refolded simultaneously or separately.) This fraction varied significantly from one preparation to the next. For the two preparations that we studied in most detail, average regenerat,ion over the first, t)wo weeks of storage, in thr presence of an

excess of both retinal and the other fragment’, was 33 and 460,b of Cl and 38 and S9’!<, of (‘-2, respectively. IJpon storage of the refolded fragments at room temperature, the ability to regenerate slowlgr diminished (Fig. 1). The hall’times for the inactivation of both fragment~s were similar (rxtrapolatjed tt x 2 months) and c4osr to that, observed for fragments tnixed before renaturation and stored under the same c,onditions (Fig. 4). At,tempts t.o refold BK f’raament.s into DMPC vesicles yielded lower percentages of’ overall regeneration following vesicle fusion (not. shown). fragmnts Far ultraviolet c*.d. spectra of two samples of fragments refolded into IIaloDnctwiurr(t lipid vesi&s at a protein to lipid ratio of 1 : 10 (w/w) were recorded (Fig. 7(a)). Analysis of thr spectra indicated a high cont)rnt of a-helix, 60 to 650/,, and x5 to 90(g) for the V-1 and (1-2 preparations, respectivel_v. Upon freeze-thawing of an equimolar rnixture of the refolded fragments in the presence of retinal, 58 9;) of the chromophore regenerated. The c:.d. spec*tra recorded after fusion in cit8hrr the

Refolding of Racteriorhodopsin

-30’

’ 190

’

’ 210

’

’ 230

’

’ 250

Wavelength

’

’ 270

’

’ 290

in Lipid Rilayers

’ -30’

’ 190

’

’ 210

(nm)

’

669

’ 230

’

Wavelength

’ 250

’

’ 270

’

’ 290

I

(nm)

(b)

(a)

Figure 7. C’ircular dichroism spectra of separate or reassociated BR fragments in Halobacterium

lipid vesirles and of lipid native RR in DMW vesicles. (a) Refolded C-l (continuous line) and C-2 (broken line) fragments in Halobacterium vesicles at 1 : IO protein to lipid ratio. Samples in K buffer without azide were sonicated briefly before c.d. spectra were recorded at 27°C a,x described in Materials and Methods. Amino acid concentrations (1.24 and 1.07 g/l, respectively) were calculated from the [3H]leucine content of the samples and the specific activity of the fragments. (b) The same P preparations of refolded fragments were mixed in equimolar ratio, supplemented with retinal (1.5 : 1 mol/mol) and the vrsirles fused by freeze-thawing. After incubating for 1 day at room temperature. the sample was sonicated briefly and the rd. spectrum recorded at 27°C (continuous line). An identical sa,mple but without retinal showed exactly t,he same spectrum (not shown). The U.V. c.d. spectrum of native BR dispersed into small unilamellar DMPC vesicles (from Mao & li’allacr.

1‘984)is shown for comparison (dott,ed line).

were absence or the presence of retinal indistinguishable and nearly identical with that of native BR incorporated into small lipid vesicles, exhibiting 75 to 80% a-helix (Fig. 7(b): and see Mao & Wallace, 1984: Wallace & Teeters, 1986). ((a) K~l’netics of chvvmophore regeneration following vesicle ~usiorr

To determine whether the reassociation of refolded fragments involves any separately structural changes, the time-course of regeneration of t)he absorption band at 560 nm following vesicle fusion in t,he presence of retinal was examined (Fig. 6(c) and (d)). Although the kinetic curves could again be approximately fitted by two exponentials, they differed from those observed when fragments were recombined prior to adding retinal in three respects: (1) about two days were required for maximum regeneration; (2) the relative amplitude of the fast process was smaller (Fig. 6 and Table 2); (3) the fast process itself was slower (see Fig. 8 and Table 2). We have not studied the slow phase(s) in detail, nor the factors that determine its amplitude relative to the fast one. Within experimental error, the kinetics of the fast phase, as characterized by the time tf3mi” required for AA to reach half of its value 13 minutes after fusion, did not vary over a storage period of three weeks to a month (not shown). The periods of the slow phases(s) are too long to be determined with any precision from available data. Those for the fast phase are about threefold longer than the period of the fast phase that follows addition of retinal to simultaneously refolded fragments. When vesicles containing separately refolded fragments were fused in the absence of retinal

t,o permit

prior

interaction of the fragments, the faster kinetics were observed upon addition of retinal (Ta,ble 2). In order to determine whether diffusion-limited processes were responsible for the slower kinetics observed upon vesicle fusion, the concent,rat,ions of the refolded fragments and of retinal in the lipid bilayers were varied by: (1) reconstituting the isolated fragments at protein to lipid ratios varying between 1 : 40 and 1 : 2; (2) varying the C-l to C-2 ratio in molar the freeze-thaw mixture: (3) supplementing the mixture with protein-free FiaZohacterium lipid vesicles; (4) adding the retinal in sub-stoichiometric quantities. Varying eit,her the [fragments] x [retinal] (Fig. 8) or the I(‘-l] x [C-2] concentration products (not shown) over more than three orders of magnitude had no rff’ect on the kinetics of regeneration. Freeze-thawing at pH 4.8 or incubating vesicles at this pH prior to freeze-thawing completely blocked chromophore regeneration (not shown). This probably precludes the use of most fusogenic viruses as an alternative to freeze-tha,wing (see White et al., 1983). Regeneration was considerably slower at 4°C. At this temperature, less than lO?h of the maximal extent was reached one hour after fusion. After three days at 4”C, however, regeneration was about 80% of the final level rra,ched at room temperature (not shown). 4. Discussion (a) Renaturation experiments with bacteriorhodopsk Studies

of the

renaturation

of prot,eins

are of

interest on two grounds. From a technical viewpoint. they offer new opportunities to investigate

Popot et al.

J.-L.

670

100 _ c -1 - \ _ \

\

IO 2.E E loo1 -2

-* -

.

\

\

\

\

\

\

\

\

\

\

\

lo...+..i ‘ ‘,. . s‘\$ \

-..,.......,.,...,...,..,...,...............\.................~dk

\

\

0 \

x AX \

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l

\

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10-B

lo-’

[retmal] x [BO] (n-101”kg-“)

Figure 8. Absence of effert of reactant concentration on the rate of chromophore regeneration following either reassociation of fragments by vesicle fusion or addition of retinal to pre-reassociated fragments. The rate of the fast phase of regeneration has been characterized by the time ti3 min needed for AA,,, to reach half its value at t = 13 min (see Fig. 6). All experiments were performed at 18 to 20°C in K buffer. The log of tf’ mi” (min) is plotted as a function of the log of the concentration product [retinal] x [HI]. When one fragment was in excess over the other. [BOJ has been taken as being equal to the concentration of the limiting fragment. All components of the system being highly insoluble in water, the relevant concentrations are those in the lipid phase and are expressed in mol/kg lipids. The slope expected for a diffusion-limited bimolecular reaction retinal + (C-l/C-2) --t cleaved BR is indicated (broken line). Concentrations were varied as indicated .in the text. A plot of log (tf’ min) against log([C-1] x [C-2]) gave the identical result, (not shown). Filled symbols: rate of chromophore regeneration following reassociation of pre-refolded fragments by vesicle fusion in the presence of retinal; (0) the protein to lipid ratio was varied in the presence of excess retinal; (m) retinal concentration was varied from sub- to super-stoichiometric at constant protein concentration; (A) fragments were tit~rated one by the other in the presence of excess retinal. Open symbols and crosses: rate of chromophore regeneration following addition of retinal to: (0) intact apomembrane; (0) cleaved apomembrane; (A) a sample reconst’ituted from fragments at 1 : 1 protein to lipid weight ratio in the presence of retinal and subsequently bleached; ( x ) fragments simultaneously refolded in the absence of retinal at 1 : 1 or 1 : 10 protein to lipid weight ratio; ( +) fragments separately refolded at 1 : 10 protein to lipid weight ratio and reassociated by vesicle fusion prior to adding retinal. the structure and function of a protein. For instance, the possibility of renaturing BR from its two chymotryptic fragments (Huang et al., 1981; Lao et al., 1983) has been used to show in a definitive manner that migration of the retinal’s Rchiff base from Lys216 to Lys41 plays no rble in light-driven proton pumping (Huang et al., 19826; Abercrombie & Khorana, 1986). We have recently applied the reconstitution procedure described in t,his paper to the localization of the same two fragments in the structural map of BR by neutron diffraction (Popot et al., 1986; Trewhella et al., 1986). From the viewpoint of protein folding, examination of the conditions under which renaturation takes place contributes to understanding the native state of a protein and testing ideas about folding paths. The renatured preparations described here represent developments of the work of Khorana and colleagues in two directions: (1) we have established conditions under which renatured BR or cBR reform molecules the hexagonal lattice

characteristic of PM, which permits structural studies of the renatured molecules using diffraction methods?; (2) we show that separate chymotryptic fragments can be stably reinserted into lipid bilayers, and can subsequently interact with one another to reform cBR. In our studies, as in the earlier ones. renaturation is initiated by removing dodecyl sulfate from a solution of the chymotryptic fragments in SDS. In Huang’s protocol, SDS removal was achieved by dilution into a large excess of lipid/bile salt mixed micelles (Huang et al., 1981; Liao et al.. 1983). In our experiments, dodecyl sulfate is removed from a protein/SDS/lipid mixture by precipitation of its t London & Khorana (1982) have observed that HR renatured by dilution of SDS-solubilized PM into Triton X- 100 oligomerizes following removal of detergent, It is not known whether the material was crystalline. The procedure cannot be applied to cBR because of the inhibitory effect of Triton X-100 on renaturation (Liao et al., 1983).

Refolding of Bacteriorhodopsin potassium salt (PDS). The procedure is selective, efficient and very rapid. At a lipid to protein ratio of about 1 : 1 (w/w), the PM lattice can be reformed. The presence of a non-solubilizing concentration of the conjugated bile salt taurocholate. while not required for refolding or crystallization, facilitates the separation of PDS precipitate from the reconstituted sample. To achieve separate refolding of the fragment,s, a lipid to protein ratio of 10: 1 (w/w) is preferable, and no taurocholate is added. In both cases, residual dodecyl sulfate and taurocholate, if present, are removed by dialysis. We will successively discuss t!he products of t,hese two renaturation protocols rmat,ured cBR and refolded fragments. (h) Renaturation

of cBR

The main properties of native PM and of the products of the two reconstitution protocols are compared in Table 3. We have characterized renatured cBR by biochemical fractionation and analysis, optical spectroscopy, X-ray and neutron diffraction and, to a preliminary extent, circular dichroism, limited proteolytic digestion and electron microscopy. The reconstituted preparations exhibited the polypeptide and amino acid compositions expected from a 1 : 1 (mol/mol) mixture of the two chymotryptic fragments, and

671

in Lipid Hilayers

the phosphorus content expected from a 1 : 1 (w/w) lipid to protein ratio. They equilibrated on sucrose gradients at the density expected from this ratio, with no change in composition wit,h respect, to the original reconstituted samples. Electron microscopy after negative staining showed unilamellar vesicles 400 to 800 A in diameter. From a biochemical point of view, the reconstituted material appears essentially homogeneous. After orientation by controlled drying, the samples showed X-ray and neutron diffraction patterns characteristic of crystalline purple membrane. As shown elsewhere, the dimensions of t,he unit cell, containing one BR trimer, are identical wit,h those of PM to within 1 O/o (0.6 8). and the relative intensities of the neutron and X-ray reflections are indistinguishable from those generated by PM to at least 7 A resolution (Popot et al., 1986). Limited proteolysis suggested a predominantly inside-out orientation (MI. E. Dumont, personal communication). Diffraction studies probe the entire structure of the molecule at medium resolution. The chromophore, on the other hand, is a sensitive reporter of minor perturbat,ions in its immediate environment (see Nakanishi et al., 1980). The visible absorpt’ion maximum of reconstituted samples, &,,, = 554 (+2) nm, was identical with that of dark-adapted CUR in PM. &,,, changed by less than 3 nm when the temperature was varied from 2°C to 35°C. A

Table 3 kqorneproperties of native (cleaved) bacteriorhodopsin versus cBR reconstituted from fragments Native (cleaved)” Physical state

Sheets’~“

\‘esic,les

Protein to lipid ratio (w/w) Extent, of regeneration Stability of refolded intermediates temperature (t:): ( ‘- 1 ( ‘-2 rU() An,, (I)A)

3 : I’,’ n.d.

I:1 53 (HIS)“,

n.a. n.a. n.d. 556 nm

-2 - 2 - 2 554

+X nm 45.000

+2-3 nm 44.500

Latticek

Partial aggregation Lat,tire

Shift upon illumination E,,,

(M-l

cm-‘)

Mixed micelles 850/,

Vesicles

20 ming -7 hs -3 hg -555 nm: 45G-500 nmh f5 nm’ 47.000

n.a. n.a. n.a. n.d.

Monomersc.j

n.d

*,.a.

n.d.’

-Cl:100 > 30 9(,

at room

Aggregation state: In suspension After partial dehydration

Reconstituted (Liao et al. 1983)b

Reconstituted (present work)

months’ months’ montjhs’ nm

n.d. n.d.

n.a. not applicable; n.d. not determined. “(:lraved apomembrane regenerated with retinal; data from the present work except where otherwise indicated. bData from Liao et al. (1983), except where otherwise indicated. ‘Determined on uncleaved material. d131aurock & Stoeckenius (1971). ‘Storvkenius & Kunau (1968). ‘Lipid to protein ratio 10: 1 (w/w); extrapolated tt (see Fig. 4). gKinetics are not exponential. “TWO species in temperature-dependent equilibrium; see also Abercrombie t Khorana (1986). ‘Abcrcrombie & Khorana (1986). ‘London & Khorana (1982). kS. V. Katre & M. Stroud (unpublished results quoted by Wolber & Stoeckenius, 1984). ‘No lat,tice expected (present work, see Materials and Methods).

67%


much larger variat.ion of’ jLmax(25 ntn) over t)he same temperature range has been observed in mixed micelles. indicative of an equilibrium between two forms of renaturcd cBR (Liao rt al.. 1983: Abercrombie &, Khorana. 19%); our observations suggest that, detergent binding is involved in the t,ransit,ion. The exGrrction coefficient of renatured cI%R in vesicles was identical wit,h that of tlarkadapted cRR in I’M and close to that of renatured c*I
The near absence of red shift upon light adapt,at,iort of reconstituted satnples was unexpected. cHR in purple membrane does exhibit a reduced but signiticant red shift (Table 3). While this discrepanqv could be indicative of imperfect renaturation, another explanation appears plausible. Dispersion of native KR into lipid bilayers is known to result’ in a diminution of the extent of the red shift ((:asadio & Stoeckenius. 1980). Depending on experimental csonditions. this effect ranges from a tnarginal decrease (Lind rf nl., 1981) to nearly c*omplete abolition of t)he red shift (Wencher rf nl.. 1981). We have therefore examined the evidence for the existence of a lattice under the conditions where absorpt,ion spectra were recorded. The visible e.d. spectra recorded on a reconstituted satnple in suspension fkat ured a very lopsided biphasic shape, the positive peak much larger than the negative one. Such a spectrum would be expected from a mixture of monomeric and oligomeric or crystalline BR. with onlv a fraction of t#hr molecules being associated. k-ray scattering from suspended samples exhibit’ed no diffraction pattern, indicating that, the associated molecules themselves do not form extended lat’tices. Crystallization therefore does not take place concomitantly wit,h rcconstitution, but. during orientation of the samples, prior to X-ra? crystallography. (Jrystallizat~ton appears to oc(aur in two steps. The IZR latticse is present in oriented satnples t.hat art highfy.h\drat,ed (flushed with 9ci to 100~~orelative humtdtty helium), where the lipids form a bil,aytbr. Why a lattice is observed under these condtt~tons and not in suspension is not entirely clear. as the average number of RR molecules per vesicle (about a thousand) would be sufficient to form lattices 15 to 20 unit cells wide. large enough to generate sharp Bragg reflections. Among possible driving forces (partial dehydration, close apposition of the bilayers, etc.}, removal of t.he mechanical constraint. imposed by vesicle curvature on t.he planar latticbes is perhaps-the most likely. (It should be noted that freezr+hawing of the reconstituted vesicles did not

et: al sufice to induce lattice formation; the incrcasc in radius of curvature caused by fusion might however. be litnited.) A second step in lattice formation wa,s revealed i)y neutron diffraction experiments. When relative hutnidity was decreased by st,eps from !#7’?{, to 32 yi, ) the amplitude of the low-angle reflect,ions progressively diminished relative to the rest of the pattern, until it, matched t’hat observed with n&ivft I’M (Zaccai’ & I’opot. unpublished results: and see T’opot. rf uI.. I9%). This most likely means thaf at high rrlativcx humidity not. all rrfoldrd t~~olec:~rles arp in well-ordered lat~tices. Small aggtqatrs or very disordered lattjices arc probably present and contribute only to the lower-angle reflections. lhtritig dehydration. purt’ I/nlohfr.rf~ri~rvr~ lipids undergo a phase tjransitjiotr from a typicaal I,, bil:~~c~t structure to a hexagonal, presumably H,, phast (1Srigelmatt 8r T’opot. unpublished s -lx\ observations)?. As rei~otistitut~ed satn~‘l~Y ii I’(’ progressively dehydrated and more lipids arc’ tlrivc~t~ int’o the hexagonal phase. the c~onc~entratiott of’ cl3R in t.he retrtainin~ bilayer tnust) rise. Obsrrsations on 0t1~w prot&tjlipid synt#ftms (see l)t~vans & Seigneurrt, 1985) suggest t’hat this drivtas the recruit~ment of HR monomers and loose aggregates by the lattice. The identity of the X-ray and neutron diffraction patt,erns from dc~h~draltrd samples with those frotn PM (I’opot (I/.. 19%) st’rongly suggests that 1hc, lattIicSc,incSorporatc~s(Ill!? those molecules that have properly rt~foltit~t!. The same phenomenon appears to ot’cur upon st~lrctivc~ solubilization of the reconstit,uted satnpk~s in taurocholate: most lipids and improperly rc~folded protein are removed. yielding preparations with thcl same drnsit.\r as PM a proport,ioti of’ ;tc*tiv{, protein close to 100 ‘/o. The limited aggregation of (+13RSin suspt*nded Halohnctrri~um lipid vesicles accounts salisfact orilh for t)hr minor differences in spectroscopic propertics compared with native cRR in PM I3oth our spectroscopic and diffraction data t,hrrrfortA indicate that the native strucature has been regained ver> accurately. This supports a previous proposal (Huang ccl., 1981). that the structure of the (‘-I/ (‘-2 ret,inal complex in lipid bilayrrs does not depend on t,hr biosynthetic history of the pept.ithbs. but lies at a free cneqp tninimum. rt

illld

Pt

t The interactions of polar lipids from Halobncterimr with nrutral lipids and with cations are ver.v caomplex(l’lachy et al., 1974: Latqi et al.. 1974). (‘hen ul ol. (1974) c~onclttdedfrom osmomrtric and tnic~roscqir observations that polar lipids form sealed vesicles onl! at low ionic strength (
cutirubruvt

Refolding of Racteriorhodopsin Correct refolding was observed in the absence of retinal (see Results) and is therefore entirely determined by the interaction of t,he polypeptiden with the lipid and aqueous phases. In order to define its mechanism further, we have examined the refolding of individual fragments.

U’hen PDS was precipitated from mixtures of each individual fragment with SDS and flabbu&4um lipids, the fragments inserted into lipid vesicles and adopted highly stable structures (the half-time for inactivation at room temperature was about, 2 months). When the two populations of vesicles were fused, t’he fragments recognized each other. assembled, bound retinal and regenerated the chromophore. This suggest’s that the structure favored by each fragment in isolation must, be close to what it adopts in the intact molecule, or in equilibrium with it. liltraviolet c.d. spectra of separat,ely refolded fragments recorded in collaboration with B. A. Wallace indeed indicat$ed a high proportion of a-helical structure. These observat,ions are similar to those reported by Lao et al. (1983), except that in the earlier experiments reassociation of the fragments took place in t’he presence of det,ergent, and their stability in the mixed micellrs was limited. (e) The renaturation

pathway

In an at,tempt to determine whether a conformational rearrangement was required for the fragments to assemble, we have examined the kinetics of chromophore regeneration under various cbonditions. (1) [Jpon addition of retinal to preparations in which the fragments had been allowed to interact, namely bleached RR in PM, bleached cBR in PM, and fragmenbs either refolded simultaneously or refolded independently and brought together by vesicle fusion. (2) Upon fusion in the presence of retinal of vesicles contjaining independently refolded fragments. Tn all cases, kinetics were complex, exhibiting a fast phase in the minute range and one or several slower phases. We have concentrated on a comparison of the fast processes. Upon addition of retinal to pre-mixed fragments, the fast phase of chromophore regeneration developed with a period lfas, of 1 to 1.5 minutes. Kineics were the same for native c*BO and for refolded fragments. ~~~~~ did not depend on either retinal or protein concentrations, and probably represents an internal rearrangement, of the (‘-1 /(“-‘t/retinal complex. Such a rearrangement has been observed spectroscopically (at 0°C) upon addition of retinal to native apomembrane (Hchreckenbach et al., 1977). London 8 Khorana (1982) also observed retinalindependent kinetics when renaturing intact BO in mixed micrllrs. They favored the idea of a rate-

in Lipid

Bilayers

673

limit’ing step preceding retinal binding, but a rearrangement subsequent to it would fit t,heir data. When vesicles containing separately refolded fragments were fused in the presence of retinal, the fast phase of regeneration occurred with markedly slower kinetics (qast = 4 to 5 min)t. This cannot be accounted for by diffusional processes, since tlast remained constant when the (C-l] x ((‘-21 or 1protein] x 1retinal] concentration products were varied over more than three orders of magnitude. Fragments that had been refolded separately and 1atc.r allowed to interact exhibited the faster kineticas characteristic of cH0. They had therefore reassociated in the absence of ret,inal. The difference in kinetics most probably means that the quilibrium conformation of at least one of thus fragment,s is different’. depending on whether it exists in I he bilayer on it,s own or as part of cB0. The fact that, 7fas,does not depend on the (‘-1 to C-2 ratio in the fused vesicles indicates that, t,he ratelimiting conformational change is after reassociation (unless it affects both fragments). We note t,hat its rat,e and temperature dependence (see Results) are compatible with those of proline imide isomerization, a frequently postubond cis-tram lat’ed limiting st)ep in t,he renaturation of soluble prot,eins (Rrandts et al.: 1975: e.g. see Osterhout & Sal]. 1985; Schmid Pt al., 1986; and references therein). Wr cannot rigorously exclude the possibility t,hat “isolated” fragments actually autoass0ciat.e. and that the rate-limiting step is the slow dissociation of these aggregates. Such a hypothesis, however, is not supported by the observation t,hat qast is not affected by varying either the (‘-1 to C-2 molar ratio over an eightfold range or the protein t,o lipid wright. ratio over a 20.fold range. In the first case, one would expect the kinet,ics of regeneration to depend on the concentration of t well be due at least in part to such a process.) A word of caution about bhe fusion procedure is in order. Thawing takes several minutes and. whilst the samples are brought from 0°C to %)“(I in 30 to 60 seconds and chromophore regeneration is all but blocked at. 0°C’. we cannot exclude the possibility that some or all of the fragments actually associated before the temperat,urr was raised. However. any reassociation that started before the t,, of the fusion experiments should have resulted in artefactually faster kinetics. not, the slower ones we

t The kinetics of’ chromophore regeneration observed upon simultaneous renaturation of’ fragments in mixed micelles are also multiphasic, with a similar fast, phase (Abercrombie & Khorana, 1986). A close romparison is probably not warranted, however: in t,he mixed micelle ytem. refolding. reassociation and rebinding of retinal occur conc~omit~nntlyand are complicated by the simultaneous development of detergent inactivation of the fragments. so that similar kinetics rould r&ect different events.

671

J.-f,. Popot ct. al

ac~tuallv observe. Virus-induced fusion turned out’ to he difficult to implements because of the pH dependence of t,he reassociation process (see Results) or t,he poor solubility of cholesterol in S1)S (( krchman & Popot. unpublished results). Prcliminary attempts at ot,her fusion rnethods raised other difficulties. The following overall scheme for t)he reformation of vHR starting from t
Ey comparison with the scheme proposed by Lao et al. (1983) for renaturation in mixed micelles, we introduce the notion that the rate-limiting steps are isomerization steps that’ follow formation of thr transient C-l /C-2 and cBO/retinal complexes. All steps occur in lipid hilayers, in an environment that, must be similar t#othe natural plasma membrane of Halobacterium, and do not require the presence of detergent. Four of the six protein species in the scheme are now available as stable entities in lipid bilayers. accessible to structural analysis. Further study of the various steps of refolding should contribute to a better understanding of their energetics. They could provide experimental tests of t,he earlier proposal that BR structure is stabilized by polar interactions between transmembrane segments (Engelman & Zaccai’, 1980). (f ) Two-stage folding

mechanism mem,brane proteins

of integral

chymotryptic fragments were When KR reinserted into lipid bilayers they separately refolded under conditions unlike those prevalent during biosynthesis. Association with the lipids was brought about by a different mechanism (detergent, rernoval). Refolding occurred in the absence of sequence segments that are synthesized in viva, and possibly inserted, prior to their own synthesis (the leader peptide in the case of C2, the leader peptide followed by C-2 in the case of C-1). Our observations indicate that both fragments nevertheless adopted conformations similar to those they have in the native molecule. Local properties of the sequence appear more important in determing the final conformation than the presence and folding of t,he rest of the molecule. or biosynthetic history. This behavior is consistent with a two-stage folding mechanism for cl-helical integral membrane proteins. In a first stage, the number and position of t’ransmembrane helices would be established as a function of local interactions of the corresponding sequence segments with the aqueous and lipid phases. The energetics of this stage are dominated by the hydrophobic effect, driving insertion of the most apolar segments, and hydrogen-bond formaCon. forcing them into cc-helical con-

format,ion. In a second stage, helices would pack without extensive rearrangement. Possible driving forces arc polar interactions between helicaes. helix/ helix ~/~er.s~~,s helix/lipid packing, constraints on the length and conformation of extramembrane loops. interactions with covalent and rio,l-covalent ligands. etc. In t,his model, transmrmhrane helixes act as individual folding domains. c*omparat)le t.o those evidenced for instance in the refolding of 1htb P2 subunit) of the soluble enzyme tr,vptophan synthasr (Zetina & Goldberg, 1980). Because of’ the hydrophobic nature of t,heir environment t)htby art’ subject t)o st’ronger c:onst~raints than structural elementIs in soluble proteins. and the?; can he both smaller than folding domains (see Wetlaufer. 198 I ) and more sta,ble t’han secondary structure units of similar length (see Kirn $ Baldwin, 1984). A two-stage folding rnec*haniam is imptic in recent att,empts at predicting. on t)he basis of the distribution of hydrophobit residues in theit sequence. t,he topology of membrane proteins whose three-dimensional structures are poorly understood (for a review. see Engelman rt al., 1986). During the past few years this approach has been applied, to. for example, the red blood ~11 gluc~os~~transportrar (Mueckler et ~1.. 1985), band 3 (Kopit)o B Lodish. 1985), lrrc permease (Foster et al.. 1983), HM(X!oA reductase (Clhin et nl.. 1984). (‘a’+-ATPase (MacLennan of al.. 19X5), t,he nicotinic (for a review. see Pop& $ (Ihangeux, 1984) and rnuscarinic (Kuho et al., 1986) acetylcholine receptors. rhodopsin (see Ovchinnikor. 1982; Hargravcl rt rrf.. 1983); etca. In each of these cases. I)r(~tein~l)t’ot’t,irl cont,acts between transmembratie segments are certainly ext.ensive. while predictive schemes rely on the Interact,ion of individual helices with lipids. There arc fe\v instances in which tht> acc~urac~gof such predictionshas been testedexperimentally. However. available structural data indicate t,hat, in the Gridis three intrinsic proteins of Rhodopwudomonas photoreaction centers (Deisenhofer ~:tnl., 1985) and, as far as can be ascertained, in KR (see Engelmarr c~f nl.. 1982). trar~smemt~rane helices are near thch positions rxpr&ed from calculations of free t:nttrg\ (Michel it rcl., 1988; Engelman et al.. 19X6), as would be expected for a two-stage folding mechanism. Excepting bacterial out’er mernbranr proteins that. perhaps because of biosynt.hetic c*onstraint,s imposed by their localization. appear to bts composed mainly of P-strands, it remains an open question whether some proteins include hydrophilic or amphipathic transmembrane segments. whost, formation and packing would havt> to obtby differrrrt rules. In conclusion, we have shown that two denatured fragments of BR can associate with native lipids to reform a purple membrane very similar to the native structure. The fragments can be individually refolded t,o a stable form in lipid vesicles. and reassociate to form BR when thfb vesicles are fused to permit’ contact. Our observations support, the idea that BR folds in two basic stages: st~ablt~helices are independently formed in the bilayer: these

Refolding of Bacteriorhodopsin interact wit.hout folded protein.

major

rearrangement

to give

the

Particular t’hanks are due to B. A. Wallace (Columbia LTniversity. Xew York), M. E. Dumont (University of Rochester) and G. Zaccai’ (Institut Laue-Langevin. Grenoble) for permission to quote data obtained in collaboration with t,hem; to H. G. Khorana, M.-J. Liao and D. M. Abercrombie (MIT, Boston) for generously sharing their experience with BR fragment preparation and renat,uration; to ,J. F. Hunt (Yale IJniversity) for preliminary ultraviolet circular dichroism measurements: to B. Pirkos (Yale. &born Electron Mirroscopy Facility) for help with the preparation of samples for electron microscopy: to P. S. Goldman-Rakic and J.-P. Bourgeois (Yale) for LISP of a Jeof 100 S electron microscope; t,o tJ. E. Coleman (Yale) for access to the Car?; 60 spectropolarimet,er: to G. Davis and A. J. Lanzetti (Yale Membrane (‘ent#er) for performing the amino acid analyses: to R. J)oms and z% Helenius (Yale) for M gift of duck influenza virus; to M. S. Capel. P. 1). McCrea, T. W. Kahn and thts members of the WERMS group and of the laboratories of R. M. MaeNab and R. G. Shulman at Yale IIniversity for help and discussions; and to J. Barra. M. E. Dumont. I’. ,Joliot, A. Sobel and G. Zaccai’ for their critical reading of earlier versions of the manuscript. This work was supported bv grants AI20466 and GM 22778 from thr Xational Inst&utes of Health a,nd grant N’M 8216854 from the ICat.ional Science Foundation to D.M.E.. and by fellowships from t.he European Molecular and the Fondation pour la Biology Organization RrchercahtA Mbdicale to ,I.-L.P.

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Edited by A. Klug