Optical and electrical properties of bacteriorhodopsin Langmuir-Blodgett films

,. ~ i ' ~ ' ~

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ELSEVIER

Thin S~litl I:ilm.,, 312 (I908) 3()h-312

Optical and electrical properties of bacteriorhodopsin Langmuir-Blodgett films Howard H. Weetall "~, Lynne A. S a m u e l s o n D, t,

' Nutiomd ht.~titut¢ ql Stanchtrds am/Teclmnh~gv. Biolerhmdo.~,y I)iri.~ion. Gaither.dml~,. Mi) 20,~99.00t11, I/SA I/.S. Arm)' A'atic'k Rexem'rh. ih'tehgmr,nt aml l:'tLeim'erink' ('e,tet, iliotechmdo.~,y I)iri.~,,,. NaticL MA OI 7fiO-5021L I/.~,4 Received I I March lot)7: accepted 27 Augu~,l 1007

A bst tact

Langmuil-Blodgett films prepared from purple membranes dissolved ill pure hexane were characterized for M-ntate decay kinetics and photocurrent production. These studies indicated (hal the films are preferentially oriented with the cyloplasl]lic side of the I~acteriorhodol~sin molecule.facing a w a y . l i m , the .s'lil.[itl'e t!] the lilt oxidu c'ottt('d glct,~'S eh'ctrodu.s. C h a r a c t e r i z a t i o n o f Langmuir-Blodgett films in high salt strongly indicate.~ that the transient photoctu'rent.s observed are directly due to proton release and uptake and rlol to charge separation. Published by Elsevier Science S.A. Ke~ umd+w Bacleriorht+dot+ni n: I.ar]~rnuir-I$lodgelt

li hI+s: ()lifo-electrical

proPcrlien

I. I n t r o d u c t i o n

Bacteriorhodopsin (bR) in a light-sensitive protein produced by the bacterium Haloba~'leritun sa/intlritm~. This transmembrane protein is located in a structural]y distinct area of the plasma membrane known as "purp]e membrane" [I]. When this protein is activated hy yellow light, it undergoes a photocycle whereby a proton is transported from the cytoplasmic side of the membrane It) the periplasmic space. Retinal. the chromophore thai is bound to Lys-216 of the protein by a protonated Schil'fhase. in responsible for tile absorption that occurs at the maxirnum of approximately 570 nm. A quantum of yellow light induces a trans-cis isomerization of the retinal, followed hy transitions of the protein-retinal complex thr¢,ugh a number of intermediate states (], J, K, L, M, N, and O) lhat are characterized by different protonation states of the Schiff base and of seven,', amino acid residues within the protein [2,3]. The M-state of the photocycle is formed when, as a result of isomerization o1" retinal, the proton on the Schiff base is transferred to Asp-g5, and suhsequently a proton l'rona another arnino acid, possibly Arg-82. in released into the solution on the periplasrnic side of the membrane [4]. Renrotonation of the Schiff base is from the ('~1~I'1¢~,polld JJl~ iltl|hOl', ()()4l)-ffflqO/()O/,%lUJ)() i'11 S()()4(l-6(19()({)7

Puhli',hcd t~', Ifl~,u'~ icr Science S.A, )t)()7 I I -fl

Asp-9fl thai in reprotonaled from the cytoplasmic solution, The hR, like other nlemhrane-bound proteins normally operates at an interface between high dielectric (water) :rod low dielectric (mernbrane) phases. The vectorial nature of the proton transport demands that the protein in the purple membrane he oriented at this intel'face. Hwallg et al. [5] prepared Langmuir-Btodgell ( L - B ) films containing bR membrane fragments by combining the bR purple membrane fragments with soybean phosphatidylcholine (soy PC). These L - B films were examined hy adsorption spectroscopy and electron microscopy using shadowing and freeze fracturing techniques. In addition, surface pressure and surl'acu potential vs. area isoll~ern]s of the purple membrane intert'ace films using 7:I hR/soy PC n]onolaycr indicated the presence of a net negative charge in the films or the introduction of permanent dipoles or both. Since bR has a large permanent dipole [6], lhis could ¢orltribute to the change in the observed potential, if the molecules were oriented at the interface. Using electron microscopy, it was observed that the purple illenlbralle Iragments were not completely dissolved in the hexane solvent used Io prepare the films, but randomly distributed and separated by s,nooth spaces. The authors assurnetl that tile Slllooth areas w~2re ~ccupicd by soy PC aml purple men(brahe lipids in a monolayer. [)sin~ freeze fracture preparations, they concluded that more than 85~?t of the purlfle mcmhrane t'rag-

tt, H, We,,tall, L,A, 5"amu~'ls.n/ 77fin S.lid Film. 312 f i 998t 306- 3 / 2

i'flenls were attached to tile glass surface with the inner membrane (cytoplasmic side) lowal'd tile glass sub-aqueous phase. Recently. it has been suggested [7] that L - B films prepared on indium-tin oxide modified optically transparent electrodes (ITO-OTE) were oriented with the cytoplasmic side oriented to the electrode surface. Their data. however suggests that tile filnm are oriented cytoplasmic side up rather than down. These authors report that the displacement cun'ent caused by a dipole a n d / o r protoq transport in the bR molecule corresponds I.o a displacement of a positive charge toward lhe electrode surface and is inverse to the direction of proton transport [8], This response is maximal when ihe cyloplasrnic side is oriented to the electrode surface. The results obtained by Li el al. [7] cannot distinguish between displacemem of a positive charge in the inverse direction or proton pumping. It is our belief that what is observed is indeed proton transport to the tin oxide electrode surface. We have undertaken to examine the opto-electrical and kinetic properties of these l'ilms in more detail using the wild-type (WT) bR, We have also attempted to carry o u t experiments to distinguish between proton release and charge displacement by perforrning experiments in high sail concenlralions where the effect of charge displacement should be minimized,

2. Materials and methods 2. i. Piw~aration ~!/"L a . g m u i r - B h M k , en ,fihns

Langmuir-Btodgelt filrns were prepared as ptvviously described [9] by dissolving the purple membrane fragments in reagent grade hexane. The films were all prepared on a Lauda MGW Filmwaag Balance. Millig water was used as the subphase and maintained at a constant telnperature of 30°C. The AT() slides were prelreated with Malinklodl Chem-Solv from Malinkrodt Chemicals (Paris. KY). water rinsed and then lowered into the subphase prior to spreading of Ihe bR solution. The bR spreading solution was prepared by adding approximately I rng of bR (fl'Ol'n 4 - 5 m g / m l waler stock solutions) to 2 ml of hexane. The mixtures were then sonicaled for approximately I rain to get a b R / h e x a n e suspension. This entire spreading solution was then dropped OiltO the water surface using a glass pipet. The material was left io stand for approximately 10 rain belbre compression, Filnls were then cornpressed !o Jill annealing target pressure of 20 m N / m . In all cases the I'ilms were lelt to anneal for tit least 20 rain until constant pressure was maintained, The ATO slides were then slowly withdrawn I'rom the subsl)hase at a speed of 10 mlal/min and then left to completely dry before characterization, Since there were no lipids added to these spreading solutions, much of the bR went into the subphase. However, the bR that did remain 011 the water surface formed a very steep compression curve and was found to be very stable.

3()7

since films could be held at constant pressure for hours with little observable loss of area. In each case. when the ATO slide was withdrawn from the subphase, tlae barrier slowly compressed indicating direct transfer of the bR from the water onto the ATO glass electrodes. Two batches of L - B films were prepared. The first batch was prepared by applying the spreading solution and then withdrawing the electrode through the stabilized purple membrane film to produce a single monolayer. The second batch was a multilayered film prepared by repeatedly lowering and withdrawing tl~e same electrode through the Langmuir m~ugh for a total of nine layers. The final films were air dried and stored under refrigeration. 2.2. E l e c t r . c h e m i c a l set-up

The electrode set-up has been previously described in detail [10] and consisted e r a flow-through system prepared from a electrochemical cell (BAS CC-4 Flow Cell) modified to hold the working tin oxide elecu'ode. The metal body of the electrode served t's the counter electrode and a silver/silver chloride electrode as the reference. The electrochemical cell cover was of black nylon with a hole 5 mm in diameter directly over the L - B filrn. The spot was illuminated with a mercury-arc lamp (LEP HBO 100), The cell was connected to a potentioslat consisting of an operational amplifier (OPA222) with a I M[I resistor in the feedback circu,t. The voltage across the resistor was amplified by an instrumentation amplifier (AD524) with gain = I. The entire cell was shielded by placement in an alurninum box. The oulpul of the electroehL~mical cell was connecled to an oscilloscope (Tektronix TDS 410)capable of giving a response time for the system of approximately 4 ms. The polarity of the signal was adjusted so that a pH decrease caused an increase in observed current while increased pH produced a decrease in observed current in Ihe downward direclion, The photocurrents were determined al pH 7.0 and 3.0 in 0,005 m o l / L phosphate buffer. 0,05 i n o l / L KCI. For high sail the KCI was increased to 0.5 m o l / L . 2,3. Spe,'trosc'~qLv

Spectrophotorrt.~'lry wan carried out oil an on a diode array spectrophotometer (HP-8452A). The fihns ,,,.,ere irradialed with 25 m W / c m ~ of yellow light from a 300 W halogen Kodak projeclor lamp through a 530 nm long-pass filler (Oriel 59500). T h e L - B films were exarnined spectropl~olornelricall), in the dry slate,

3. Results

Specll'opholonlelrJc examitlalion of the 'single nlonola):er" I . - B dried film in light and dark showed the presence of a p h o t o c y c l e as indi,,:aled by the ,',mall dil'fereno~" in tile absorptio11 sl'|ectra produced under these conditions.

3()8

H, II. +ig,,,t.ll. L.A. S.,m;..l.,,+,,~t/ 77d. S+;li,tl l.'il,,;,,.., 312 119t),~,) 306+o312

0.24500 - -

O~

0.24300 - -

el_ 0 m e~

.<

0.24100

- ~ ' ~ ' x ~ ~

.

0.23900--

0.23700 - -

0.23500

i I0

i

J

20

30

==

i

=:l

40

I

50

60

Se~opd~ Fig. I. Kinetics of M-stale decay of dry multi layer Larlgnmir-Bh+dgclt fi]m monitored at 410 nm ip Ih¢ dark al+terfl()n of yellow light.

The spectra, however, did not show specific peaks tit the expected wavelengths o f 570 nm and 410 rim. but showed a difference across the entire wavelength range examined. Attempts to determine the kinetic half-times ( T a / 2 ) for the

conversion of the M-state intermediate to the were unsuceessl'ul with the 'monolayer" L - B ics studies on the nine-layer sample (Fig. i) 410 nm using yellow light for excitation

ground state films. Kinetmonitored at gave a T~,,,

'x"+

o

200 m s Fig. 2. l)ht~h'l~:urrenlIr~'n,'.,i~.'nt,',ul the 'm
H. ti. Weelall, L.A, S , mm'l,wm /

wdttes t'or the M-state decay rates of 1.35 s and 5.40 s for the first and second exponential using a two ,-xponential decay model [11]. Repeats of this same experiment on the St|lliiC electrode gave very poor reproducibility. The d~ta was considered only as proof that the L-B films containing the bR were active. The transient photocurrents produced by the "single monolaye," and the nine-layer L-B films at pH 7.0 are shown in Figs. 2 and 3 respectively. The tran.,~ient light-on photocurrents are in the direction of pH decrease (proton release) while the light-off transients after a I-s light exposure art; in the opposite direction (proton uptake). The nine-layer films produced a rnaximum photoc~lrrent of 80 nA + 6 nA while the 'monolayer' film produced a st'nailer current of 37 nA _+ 4 hA. The experiments were repeated at pH 3.0 (Figs. 4 and 5) with the observed F,hotocurrent transients now in the opposite direction and smaller in size. Increasing the salt concentration to 0,5 mol,/L at pH 7.0 with tile 'monolayer' L-B film had no effect on the size or shape of the signal.

771i,Solid

t.'ilm.~ 312 f 199~ll 3lh5-.¢ 12

399

4. Discussion The results of the spectral and kinetic determinations on the dried L - B films indicates dmt they are active even though we were unable to obtain an M-state decay rate for the 'monolayer' fih'n. The spectra of the 'monolayer" L - B films lacked specific features but showed a difference between the sample exposed to yellow light vs. the sample in the dark. These spectra did not show any maxima at 410 nm or 570 nm. Since the 'rnonolayer" was prepared with the hydrophilic side facing the electrode surface, it is very unlikely that it is a true monolaver. Since the lipophilic side ot' the film would be facing the aqueous solution, it is possible that the film has lblded into, at least, a bilayer or possibly liposome like fragments, The nine-layer L - B films on the tin oxide electrode is made oF multiple layers with the last layer lacing the aqueous phase being hydrophobic in nature. The spectrum of the nine-layer sample was sirnilar to the 'monolayer', again suggesting that some readjustment may occur when the bR L - B films are placed

;

...',:"~-

_-._- -_~_=¢-_-~--,%.~.-~_-;_~,-

200 m s

I:ig. 3. Photo,tun'rent Irannicrlt.,, Lff the mullihtyer I.tm~muir-Iflodgcll film iu oml~¢l ~ itl'l 1.).{~15m,~l,,t pota,,,,,ium I~h~sphale. O.ll5 m~d/I K('I at pll 7.0. The.' 5cllow lighl wa.,, maimaincd l'~i I ,,. The inilial l"u'sl'jq~1|s¢ I'cl'qt'scftl.', ll~,u' light-on tral;si¢|U (pll dcclva~,cL The light-~lf qt~.tlsk'ltll is il~ Ihe t~Pl~v,ite dinzclio~ (pH i11¢ru'~lsu').

3 IO

H.ll. lVeet.lt, L.A. S..tttu/.so. / Thi. Solid i.'ilm.~' 312 I /(t9,",'1 306 312 ,,

,

.11

..

i

L

11

__

_ _ m

--

i,

,

,

!!

T

i

<=

200 m s

Fig. 4. F'ho~t~ctlrrel~l lrall,,it.,nl,, of Ihc "mt~llOta} or' l.;,~l|~llltlh'~J~hMg¢tl film il~ c~mt;~u~, v, ith I.U.II~5 tool/i polasnitinl j~]l~sphalc, I).D5 |llt~l/~ K('] al pl[ 3.11. The )t.'ll~x~ light ~;~,, mai~hfined for I ".,. "('hc ilxi',i;d t',.'~,potl',c tt'pl'CX~'lll~ l~lc tiglll-~?,ll Icaa,,ieut tiallt~¢d t'~, Ih¢ light-ell' h;tn~,ictm II~th r~,h~,'fl,,:,¢t.lrlt..'lll Iil';.tll,.,,iCilts ;.llC ill the Ol',r,~,,,ile dil'ecti~ul IiOlll 111c I~hl.lhlt.'lllTClll ll'ili'l'.,iClll'-, OI",'-,¢'l'%',2t.[;,1I, }all "7,11.

in ¢onll.tcl will1 but'lcr. When examined by difference spcclra, it did shoe.' the expected peeks, ulll~ugh vcith a great deal of scauer. The M-state deeply rate of Ihe ninelayer film wax obserxablc (Fig. I) yieldin,# a hallCtimcs (7"~ _,) withil~ 1110 .~]011¢I'l.t} 1"(111~¢ I)l" ~,'l.lllteS observed in gelatin films Jill. Tt~c ~eiutin film.,. :.t,, with ihe I.--B films, sllowed slowed M-stere decay fete.,.,. It is believed thztt the M-.,,I;tlc dcczty r;.lle.s ;ire greatly ;.tftk.'ctetl by the limilcd Vealer c',)lltelll and llae h~x~. prt~t~n nu~bilit.v. Water in necessary inside rthc pr~t.ein Ik~ the reproto]l~ltion o1" the Schiff base becau,,e ti'tc larL,,est part ,~1" Ihe barrier in the e~ll~ull~y ~1" .scpal;tlin~ Ihc pi'~l~m Ir~mt Asp-t)6 i;q~proximu|ely 40 k.l/mol in the u,ild-lypc bRI [12l In the cane of the geh.flin I'ilrn,,. ihc preneuce ~1' lhe polymer binder in fl'~e mutrix ~d" tl'~c film resuhed in uddid~ma( ~.l¢iwdi.'~Wllor lhc bR cycling ;~ctixitv. In tile C;.tse ~i" ~.hc I.-B I'ilms. the resull in ralhcr ,,urprisii~,~.in lhal tw~c would expccl lhe prc.,,cncc nf lhe \,v~Icr ~ppt~,,ite the film v,~)uld perrMt sufik'ien! byth';m~n since the I.-B film t~rwc cI~sely resembles a tmlural membrane. IIv,;m~ et ~I. ~51. iudicatcd thai Ihe kinetics ~1" decay of the inlermediale ere slower than in atlt]eoun sttspt, nsit>n,, of pro'pie membrane.

Althotigh I11e aLIthol'.,, ~a\¢ Yto x'al',t~2~ |'¢w tb.~ M-st.altr decay r'atc, the Ctu've they prcser~led, rnonittwetl ~.|l 410 nrn over a

33) ,,, time ,,putl, shov,'cd .'.,Iov,'~d M-slutc dct.'ay. The figure .~[l~tY,,Vxthe M-.,,;tate decay still decreasing ut 3.0 s. ,.,.,l~ert tile exPcrimel~l eroded, l'Tfis d;tta nu,2,,o,,I,, thai 1lie bR is m~l .~. ,. fully hydrated in d'~¢ I . - B films evctl in Ihe pt'esent.'e o1" ;.| I;.lllt~lk2excess or WIltOI'. The Ot'lsCI'VCd light-on pll~locurre111 tl'~.itlSi¢IIlS ~,/(" ~,;t.ll" "m(molayer" and tl-~e multiluver h R - A T O films shov,,ed peak currenus ~ff 37 p.A and 8(1 nA respectively. These d~lta ;~l.'.,u nuggcs, t that the trutlsicnl i)htfl~curlcnl in title t.o proton release. It in very likely lhal Ihe mullihLvered I.--B film tiled ;.uld filled in .,,paces ~u ~,l'~e AT() hurlace th;.tl would haxe I~eev~ lef; hare ~t~ the 'll,,llohtver'. Since Ihe differeu~.'e helvveen the nine-layered I . - B film and Ille sin~-,lelayered I.- ['t film.,, is less 1hen a I;.t¢tor of 3. it ~ppear.s that ~ml,, q'te bR next I~ II~e AT() is i~rt~tlucii)g the tr~lllsje|ll pholoct~rl'enl. The photocurrent trctl~sienl.,, ~N~norvcd i~ our 1.-1-1 I'ilmx v, erc similar to Ih~.,,e observed by P,ol~erlsoll ;arid I,'tlk;.~.'.;hev [131 u.',inS elcctn~ph~wclicully depo.,,ited t~riented l'ilms on AT(). l'l~cs¢ electro,des sht~u,, thai Ihe transienl oplo-electl'i•

,.. C...~

H.tl. Wuulctll. L.A. Sem,r,l~n, / 77~i, ,Solid I.'ilmx .t12 I 1 ~ t

3#6 312

311

o ..ii

200 m s

l:i~.5. |~II~h~CLIM'CIIIIrillinienI.,oI the muhil:iver l.;ili~illtlh'lllotlgeIll'illllin c.~IIl:|cl~.~.itb (l.lr)5,m~I/l l',m,,,,ium ph~,nph:~le.1).I)5mol/l K('I al pll 3.If.Th~ .~.'lh~ li~hl ~a~, m~dnl~d,ed I~,r t ,,. Thu' imlml r~.'.,,lmm, e l¢lll~.'senls Ille li~h~-tm II'~l~:.ni¢lll Followed by lhc IIShI/,',IT Ir:ll~.,..i~.-Ill. I]~th ph~.ll~.~u'llrrellf Ir~|llsi~.'nl~ III'L' ill the ,pp~siu: dii'u'cliol~ Ilx'~ill I1~,' l'~tloloeLIrl'etll Irunnienls ~fl~nersvtl ILl fql 7.1). F.~L~.'h t~o\ hI lhc .\" dh'e¢li, m rel'~resenls 2OII m,,. I~ach I'~t~x ill the I" diru'u'liot~ iVl'~l'¢.,.enls 20 P.A,

cal response observed on irr:~tlialion wilh yellow !i.~h[ is due II) lhe chan~e it1 ptl al lhe etecllx)de surli~ce cuused by pl'olol'~ I'elea,~e. Ar, lirnony w~ls .subsliluled I'~r h'~dium i'~ecause Ihe ]TO elecll'odes I'estmnded to yellow li~hl produchl~ ~,i current. Responses similar l,a Ihose observed in lhe b R - A T O elecirodes were generaled by inlrodtlu'lion of ditule I ICI usin~ a I'h~w-injcclior~ system with II~e Ixu'e AT() electrode,s. "['he,se data strongly sugLze.,,1thttt the I'flmtocurrent re.sptmse,s observed with irradi~ttion is caused hy pl'OtOlt release at the surl'ace of the electrode, not uptake. Rel+mval of the visihle light SOUl'Ce tm the film gi~,es a reverse signal. This, is due Io an in~.'rease in pll at the electrode stll'l~lce, rel'~reselltin ~ proltm Ul'~lake. Addilional eXl~erirnent,s ,showed thai hy cl'ml'l~in~z tile pl'evailin# ptl from "7.() m 3.(). one observed a reversal ill the direcli~m of lhe opm-eleclricul respon.se on in'adiation, indicalin,g pro_ ion upLake, This ou'cu,'s heeause tl~e n~eclmnism involved in protein I'eleane is shul down or greally slov,ed by the decreased ptl, It i,s reasonable Io ussume Ih~l Ihe pK:l of Ihe a.rrlirlo acids involved in release (l~l'ol'~al'flv ul Ihe .,\rg-~2 and/or Asp-~I5 po.silitms)remains pl'OlOlxlted al low pH

and lherel'~re doen nol release a prolon 1o II1¢ exlel"n~,il envh'onmenl or releases the pl'olon al a ral,~ lh~.ff our illMrLIIllClll[|tiOll CIIllIIlOI detect. However, 011 relllOvLll of the li~:tll source, detectable uptake does occur and protons are rem~>ved from the surface of the electrode, These d~lt~.l stroll~ly sU.~.~esls Ih~.it there nl~,|ybe some misunderstanding of lhe experimelll~.d d~lla previously oh.served [7,B]. !1 is also Imssihle Ih:|l file :|ddilioll of file so) PC Io Ihe ITO-I'~rel'mralion induu'ed resuhs urdike Iho,sc observed by Roberlson and l.ukashe~ [13] on ATO eleelrodes usin~ e leclrophorelically delmsiled orienled purple palches mid tLv Weel~,lll el ~d. [10] in a more del~iled stud.V o1" lhe efl'ects of pH on t'~hotocun'ellt pr~duced in AT() elecltx'jdes. "l'hi,, slud~~ w a s Ulldet'taken to I'e-ex;.illlil|¢ lhin q u e n l j O I | using. I.-t-1 films prepared ~,vilhoul Ihe addilion cd" soy PC. I-or this slud~,', ihe purrde nleml'q'mle has been suspended in pure hexane, wilhoul the atldilion ~l" any pho.spl'wdipid, for i'~relxU'alion of d'le hR I.an~l'nu i t - ltlod~eu filn'ls. Their data st|'orl,,t',;+, su~esls that the films are oriented with the cytoplasmic side of the membrane a~',a) flX~lllfile Slll'l~lee of lhc eleclrode.

3 t2

H.H. Weelall,

I..A. SamtwL++,+/

Thin S.,!id Filmy 312 +I~J98) .10(~+.412

Another factor that mt.+:,;I be considered is the difference i~ the tin oxide electrodes. We observed that ITO gave transient photoct, rrent when irradiated with yellow light. while ATO showed no response under identical conditions. The differences in the nature of these two electrode lypes may also play a role in the observed differences between our results and the previously data. Data collected on these L - B films under acid conditions showed no release or ',,cry slowed release of protons at a rate undetectable by our instrumentation. It did not prevent proton uptake that was observed on irradiation with yellow light (Figs. 4 and 5). The transient photocurrent uptake signals, under acid conditions, were decreased by approximately one third, suggesting that, at least, two thirds of the bR molecules are oriented with the cytoplasmic side away from the electrode surface. The addition of 0.5 mol/I KCI to the system did not effect the photocurrent transients in any way. Thi:~ strongly suggests that the observed photocurrent transients are duc to proton release and uptake rather than charge separation. The high salt concentration should eliminate any ob,+ervable signal due to charge separation within the bR L - B film. It is possible that charge separation contributes to the overall response to irradiation. However, this contribution is too small or too slow for our system to detect.

It is possible thai by nlll adding any soy PC. our films are riot directly comparable. The ~tuthors state in Illeir repori. that more than 85~h of fracture faces appear rough, that is. corresponding to the inner inell~branc half, Since the freeze fracture is that of the membrane h a l f attached to the glass support, the electron n~ic+"ographs indicate that, in the interface film, over 85~3~ of the purple membnmc fragments are oriented with their cytoplasmic surface towards the aqueous subphase. Since the freeze fracture data were generated nn fresh L-B films, it is possible that they were indeed its described. However, when placed in an aqueous environment, where the lipophilic side would face the aqueous phase, they could reform into bilayers or multilayers with the cytoplasnlic side away from the electrode surface. This could explain both our data and the previously published data [5,7]. In order to prove this hypothesis, we plan to carry out thrther experiments using the mt,'tant bR $35C. This mutant has a cysleine substittltcd in one of the loops, outside of the lipid bilayer. By placing a reporter group on the cysteine we should be able to determine its location after we prepare the I.-B fihn and determine whether the I'ilm changes conl'i~uration al'ter re-exposure to water.

5. Conclusions

Acknowledgements

on

T h e data presented here argues that the bR o r i e n t a t i o n a A T O electrode prepared under those condilior,,s is

predominantly with the cytoplasmic side tip rather than down. As prepared, one would expect the hydrophilic side of the membrane to be next to the surface. However, it is most unlikely that the hydrophobic portion of the monolayer would remain in the presence of water in the photochemical cell. it is possible that the L - B film has reoriented into a bilayer or multilayer structure with the hydrophilic portion facing out rather than toward the electrode surface, it is also possible that we are observing only that portion of the bR oriented cytoptasn~ic side up. However, it this case, one would expect to observe a larger proton uptake signal. We did not observe such a signal. In addition, the experiment in 0.5 s o l / ! KCI, strongly suggests that the transient photocurrent observed on irradiation with yellow light is due entirely to a pH decrease at the surface of the electrode caused by proton transport to the electrode surface. If there is a photoclcclrical effect duc to charge separation, it is either too fast Ior our system to detect or it is below the detection limits of our instrumentati'an. We must also consider the possibility that some signal is generated by the use t~l IT() electrodes that do respond to yellow light. We believe, lhat the orientation o f our hR I.-B films

are not as previously described [5]. Unlike the L - B films described hy H w a n g et al. [5], we did not add :my soy PC.

We acknowledge Baldwin Rohertson of the National Institute ol" Standards and Technology for his helpful su,~gestions d u r i n g the preparation o f this manuscript.

Rel'erences [1] I). (]¢,,lerhell, W. Sh~eckelfiu.,,. Nmure New Bit~l+ 237, (It.l?ll 14tl. [2] T.(]. Ebre), ill: M.B. ,|ack.',tm ( I!d. ). Thcrnlt~dyn:uuien o1" Mt.,Int'~rane Receptors ~.11~.1('hillll|els, (.'l/,(' i)l'e:,s. ('lcveland. It)t)3, p. 352. [3] R.A. Mathics, S.W. I+il~..I.B. Ames. W.T. l)o11:u'd, Aml. Re',. I~,iol'~hy.,,. ]:liol~llw,. ('hem. 20 ( I~)1 ) 491. (4] ,"i.I:~ Ilal:znht)v. R. (IOVindj,t2k', M. K~t)na. I'L l)~tm,,hcxa. I!. I.llk:rt.,dlt..'x, T.(L tGbrcx. R.K. ('touch, D.Rr Mcnick, Y. I:cl+g. I+iou'hcn~istr.~ 3" { Itjt)3) 10331. [5] S.I-i. fl,,vang, j.I. Ktwt'nhrtll. W. ,",ilueckcnitts. J. Mcnlhr. Biol. 3I> hy,,. 65 (19Th) 4575. 17] B-I:. I+i. J-R. [.i. Y-('. "iLm~, I.. jiun~. Mmcr. 5~.i. Iqlg. C?, (It)9.':i) 2 ~t), [~'~I J.I. Korunbn~l. Mclhod I!n,'ynlol, x~, (l~)~2J 45, [IOI H,II, Wu¢lall. A, I)rushk~. A,R. du I.cra. R, Al~,:iI¢/, B. Rt~b~i'lsol|. in preparation. I l l ] T. l)yukm, a, ll.lt. Wcctall, B. !{,,+l'l,u'rls~+n, Iht~,~)stcms I~j97, itl [121 Y. ('u~. (i. Vary+. M. (.'han+. I+. Ni. P,. NccdlemmL IL l.:myi. B it,u" he m i ,,tI'.,,+ 3(} ( I ~t} 3 ) I (It.~72. [13] !]. R,~bcl'lsoll. I'ZP. l.uk:lnhe~.. I:~i~pll)s. J. h~, (I~)t)5) 15()7.