Proc. Nati. Acad. Sci. USAVol. 88, pp. 149-153, January 1991Biophysics

Binding of a single divalent cation directly correlates with theblue-to-purple transition in bacteriorhodopsin

(purple membrane/blue membrane/bound cation/acdve site)

RoY JONAS AND THOMAS G. EBREYDepartment of Physiology and Biophysics, University of Illinois, Urbana, IL 61801

Communicated by Mostafa A. El-Sayed, September 28, 1990 (received for review June 11, 1990)

ABSTRACT We have characterized a unique divalentcation binding site on bacteriorhodopsin which controls theblue-to-purple transition in the purple membrane of Halobac-terium halobium. To identify this site we first showed thecorrelation between the binding of one Ca2+ per bacterio-rhodopsin and the amount of blue membrane converted topurple membrane. When the free Ca2+ was reduced below 1pM, and the pH was set below 5.0 with 0.5 mM citrate, onlybinding to this high-affinity site was observed, and we couldseparate its effect from the effect of other divalent cationsbinding to the membrane under other conditions. Second, thetitration ofpurple membrane showed that protons are taken upin two distinct steps, about 13 with a pK. of 4-5 and anadditional 2 protons with a pK. of 2.75, in 5 mM MgSO4. Thelatter is identical to the pK. for the purple-to-blue transition in5 mM MgSO4. Taken together, these observations stronglysuggest a direct role for cations in the regulation of thebacteriorhodopsin color under normal conditions. We havealso found that the intrinsic pK. for the purple-to-blue tran-sition is about 2.05, suggesting this is the pK. of the group orgroups that, when protonated, lead to the blue membrane.Previously published data can now be interpreted to suggestthat the cation regulates an active site near the retinal chro-mophore. A binding site for the divalent cation that includesAsp-212 and interactions with the protonated Schiff base,Asp-85, Tyr-57, Tyr-185, and Arg-82 is proposed.

Under conditions of oxygen deprivation Halobacteriumhalobium can continue its ATP production in the light byusing the purple membrane, which is capable of establishinga light-driven proton gradient. Bacteriorhodopsin (bR) is theonly protein found in the purple membrane. Biophysical,genetic, and structural studies have made bR perhaps thebest-studied ion pump (e.g., refs. 1-3).The purple membrane (absorbance maximum 558 nm in the

dark) turns blue (absorbance maximum 605 nm) at low pH (4).An essentially identical blue membrane is formed by cationremoval (5, 6). Dufiach et al. (7), using Mn2+ ESR todetermine apparent binding affinities of divalent cations topurple membrane, reported one very high-, three identicalhigh-, one moderate-, and five rather low-affinity sites. Thelatter were seemingly associated with the five carboxylicacids in the C-terminal tail of bR. All of these apparentaffinities are greatly altered by the surface potential for theside of the purple membrane to which they bind (8).

Several groups have proposed that the blue is due to theprotonation ofa negative charge near the chromophore (e.g.,refs. 9-13). There are two independent but related questionsinvolved in how cations control the color of purple mem-brane. First, What is the physical origin ofthe ability ofaddedcations to turn the blue membrane back to purple? Several

groups have suggested that the effect ofthe cations is via theirregulation of the surface potential; the surface potentialwould in turn control the local proton concentration near themembrane, which would, in turn, control the protonation ofthe color-regulating negative charge (e.g., refs. 5, 7, and 14).A somewhat more elaborate version of this hypothesis is thatthe protonation causes a conformational change which indi-rectly regulates the color of the membrane (15). The secondquestion, related to the first, is, How are the cations asso-ciated with the membrane? Is there specific binding or are thecations just trapped in the double layer? We have previouslysuggested that the cations act to change the surface potentialboth by acting as free Gouy-Chapman ions and by loweringthe surface charge density of the membrane through specificbinding, which also would change the surface potential andthus the local proton concentration (14).Here we provide evidence that the cations have two

distinct roles in regulating color and maintaining the properionization state ofkey amino acids in the membrane. One rolewould be in regulating the local pH, as suggested before.However, here we also provide evidence for the directinvolvement of a cation in a special binding site. This cationwould aid in maintaining the ionization state of key aminoacids in the active site in the face of the local protonconcentration, although eventually a low enough local pHcan force the protonation ofeven the acidic groups associatedwith this special cation.

MATERIALS AND METHODSCation Binding to Dark-Adapted Purple Membrane. Purple

membrane was isolated from the Halobacterium halobiumstrain S-9. Dark-adapted purple membrane was incubated in5 mM CaCl2 to fully occupy the divalent cation binding siteswith Ca2' (6) and then washed six to eight times in about 25ml of 0.5 mM citric acid buffer (pH set from 3.25 to 5.00 withNaOH; Beckman J2-21 centrifuge, JA-20 head, 20,000 rpm,200C; 35 min). After the final sedimentation a few ml of thesupernatant was removed and saved for Ca2' analysis; thenall but 4-5 ml of the supernatant was removed and discarded.Finally, the purple membrane pellet was resuspended in theremaining supernatant and saved for Ca2' and spectralanalysis. The purple membrane in these suspensions was -75.uM. The membrane solutions were sonicated to homogene-ity, and spectra were taken of 0.2-ml aliquots that werediluted 1:10 with their specific buffer solutions. The variousspectra were normalized at 594 nm (see below) and therelative concentration of purple membrane was estimated bythe increase in absorbance at 550 nm, compared to that of thesample at pH 3.25, which appeared fully blue. Ca2+ wasquantified by inductively coupled argon plasma emissionspectroscopy using 0.5 mM citrate/Ca2+ standards. Ca21binding stoichiometry was determined by subtracting the

Abbreviation: bR, bacteriorhodopsin.

149

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

150 Biophysics: Jonas and Ebrey

supernatant Ca2l from the suspension Ca2", using an extinc-tion of63,000 M-1-cm-1 to quantitate the bR concentration ofan aliquot after its full conversion to the purple membraneand light adaptation. Residual Mg2' was also measured; itwas insignificant.

Absorption Spectra During the Acid-Induced Formation ofthe Dark-Adapted Blue Membrane. Spectra were obtained byusing an Aviv 14DS UV-visible spectrophotometer (AvivAssociates, Lakewood, NJ). Samples were kept darkadapted to avoid an additional light-dark transition, whichtakes place faster than the spectra can be recorded at acidicpHs (16). The anion used in all solutions was sulfate toeliminate complications from the anion-specific formation ofacid-purple membrane from the blue membrane at very lowpH values (10). When Na2SO4 was used, the membrane wasfirst incubated twice in a 500 mM solution of the salt todisplace divalent cations from the membrane and thenwashed four times by sedimentation in the appropriate con-centration of the solution (centrifugation as above). WhenMgSO4 was used, the membrane was incubated in a 10 mMsolution of the salt before washing. Preparations werewashed in twice the desired final salt concentrations andresuspended in a few milliliters of the solution after the finalwash, and then 3 ml of this suspension was added to 3 ml ofspectrophotometric-grade glycerol. The glycerol helped tomaintain the membrane in suspension and minimize aggre-gation effects. The photomultiplier was positioned close tothe measuring and reference beams. This maximized thequality of the spectra of the aggregating samples. Only saltconcentrations which were sufficient to maintain the purplecolor before acid addition were chosen. After titrations withmicroliter quantities of 0.5 or 2.5 M H2SO4, 2.5 ml of thesuspension was removed for a spectrum, then returned forfurther acid addition, pH measurement, and the next spec-trum.pH Titration of Dark-Adapted Membranes. Approximately

4 ml of a 50% (vol/vol) glycerol/aqueous salt solution,containing 1 or more umol of dark-adapted bR, was titratedwith microliter quantities of H2SO4. The suspensions wereprepared by pelleting the membrane samples in twice thedesired final salt concentrations at least four times, resus-pending them in 2 ml of the solution after the final wash, andthen adding 2 ml of glycerol. Identical solutions without thepurple membrane were also titrated, and the differencebetween the two titrations was quantifiable in terms of thenumber of additional protons necessary to reach a particularpH per bR in the suspension, or the number of protons takenup by the membrane at a particular pH within the range of thetitrations.

RESULTSBinding of One Divalent Cation Correlates with the Spectral

Transition. Fig. 1 shows the number of moles of Ca2+ boundper mole of bR versus the pH of the purple membranesuspension, set by 0.5 mM citrate buffer. Spectra of theCa2+-enriched membranes, well washed in 0.5 mM citratebuffers (pH range 3.25-5.00), were normalized at 594 nm (seebelow). Membranes were washed so that the free Ca2+ in thesupernatants was less than 1 AM. This, together with thelower pH values and monovalent salt concentration, elimi-nated the binding of several lower-affinity cations that do notappear to directly affect the color. Fig. 1 shows an excellentcorrelation between the relative conversion of blue mem-brane to purple membrane at the various pH values and thebinding of one Ca2'. The titration of both the color changeand the binding ofone Ca2+ suggests cooperative, two-protonevents as shown by the curve in Fig. 1 (n = 2-type behavior).

Intrinsic pK of the Purple-to-Blue Transition Is 2.05. Fig. 2shows the apparent pKa of the acid-induced color change

0

+

C)0e

4.00Buffer pH

.

01-0

f

L)LO

U)

c

D;2en

-D0U)

._

a)COcoa)U)U)C:

FIG. 1. Moles of Ca2W bound per mole ofbR versus the pH of the0.5mM citrate buffer it has been washed in (-), and relative increasein absorbance at 550 nm as the membrane goes from blue (pH 3.25)to purple (pH 5.00) membrane (0). The curve is drawn for the caseoftwo protons, suggesting that both the color change and the bindingof the cation are cooperative events (n = 2). (Inset) Spectra of themembrane suspensions at each pH normalized at 594 nm.

from purple to blue as a function of the negative logarithm ofthe concentration ofmonovalent (pNa) or divalent (pMg) salt.The pKa values were determined by the relative changes inabsorbance at 550 nm, the loss ofpurple membrane during thetitration (see, for example, Fig. 2 Inset). As the salt concen-tration increased, the pKa decreased. At the intersection ofthe lines for monovalent and divalent cations, where theapparent pKa is about 2.05 + 0.05, monovalent and divalentcations have identical effects on the pKa. Thus, this shouldbe close to the intrinsic pKa for the formation of the bluemembrane. Although the monovalent and divalent cationsmay have different direct effects on the membrane, theresults appear nearly linear over the concentrations used, andthis seems to justify our conclusion to a first approximation.A similar experiment was done by Kimura et al. (5), but theirslopes were half as steep and their intercept was at about pH1. The apparent pKa of the titrations in Fig. 1 is -4.3 andwould be consistent with a 0.1 mM Na' concentrationaccording to the data in Fig. 2.During the titrations, four closely spaced but distinct

isosbestic points were observed, depending on the salt con-ditions. The spectral transition in most of the MgSO4 solu-tions had largely, but not exclusively, one isosbestic point at594 nm, and this result was used above to normalize thespectrum in Fig. 1 Inset. One reason for multiple isosbesticpoints is that there appear to be two stable forms of the bluemembrane. One of these blue membranes shows an isosbesticpoint at 594 nm and can be transformed under certainconditions to the blue membrane with the 584 nm isosbesticpoint by light or further acid addition (unpublished observa-tions).Two Protons Are Taken Up by the Purple Membrane at

Exactly the pH Where the Purple Membrane Turns Blue. Fig.3 shows the difference between the number of proton equiv-alents that were added to reach each particular pH in a

Proc. Natl. Acad Sci. USA 88 (1991)

Proc. Natl. Acad. Sci. USA 88 (1991) 151

4

cD 20 ]

223

0.

I- ~ ~ P

0

z01

0

-22 3 4

pKa

FIG. 2. pKa of the H2SO4-induced purple-to-blue spectral titra-tion as determined by the relative loss in absorbance at 550 nm versuspNa (n) or pMg (e). (Inset) Titration spectra in 5 mM MgSO4 at pH6.2, 3.97, 3.68, 3.47, 3.19, 2.85, 2.56, 2.28, 2.07, and 1.99.

suspension ofpurple membrane in aS mM or 200mM MgSO4solution and in the MgSO4 solutions alone. This differencegives the number of protons taken up per bR molecule. The

a:CDco

._

+

I1513

1

C

6.0 5.0 4.0 3.0 2.0pHbulk

FIG. 3. Number of protons specifically taken up by the purplemembrane in a 5 mM MgSO4 solution as a function of pH. The pKaobserved under identical conditions for the acid-induced spectraltitration from purple to blue membrane is shown by the dashedarrow. (Inset) Similar titration in 200 mM MgSO4. Titrations: a, thesolution plus purple membrane; b, the solution only; c, a - b, thepurple membrane-specific titration.

data shown for 5mM MgSO4 are the average offive titrations,so the exact points are not given. As the protons are addedand the pH decreases, approximately 13 protons per bR areinitially taken up by the membrane in either 5 or 200 mMMgSO4 with apparent pKa values between 3 and 5. Theseprotons are probably taken up by the protonation of surfacecarboxylates on bR (13-15 available).As additional protons are added to the sample in 5 mM

MgSO4, 2 more protons per bR are taken up by groups witha much lower apparent pKa, 2.75. This pKa coincides withthat of the purple-to-blue transition seen during spectraltitrations in 5 mM MgSO4 (Fig. 2). Thus the purple-to-bluetransition is associated with the protonation of two acidicgroups. A difference of 1 or 2 mol of H' per mol of bR wasreported previously (9).When these titration experiments were done at much

higher salt concentrations (e.g., 200 mM MgSO4), as noted,the uptake of the 12-13 protons per bR was observed,followed by a clear plateau where no more protons weretaken up over a significant pH range (Fig. 3 Inset). Unfor-tunately, at these higherionic strengths the uptake ofprotonsby the salt solution itself became too great at the lower pHvalues to obtain accurate differences between the membrane-and non-membrane-containing solutions. For this reason, thedata could not be extended down to where the color titrationtook place in 200 mM MgSO4 (e.g., pKa = 2.35). Because ofthe purple membrane's large surface potential, the lowerionic strength of the 5 mM MgSO4 solution shifted theapparent pKa of the groups involved in the color titration upto higher pH values which were more easily studied (pKa =2.75) and also reduced the proton uptake by the salt solutionitself.

DISCUSSIONRole of the Divalent Cation Site. The above experiments

suggest that the binding of one divalent cation is directlyinvolved in the blue-to-purple transition and that this cationis associated with two acidic groups having intrinsic pKavalues of about 2. First, the correlation between the blue-to-purple change and the binding of one divalent cation is clearwhen the binding to other, lower-affinity, cation binding siteson the membrane is excluded by low free divalent cationconcentrations and pH regulation. Next, below pH 6 uptakeof protons added to a purple membrane suspension can beascribed to two types of groups. Although not entirelyhomogeneous, the pKa of one of these types of groups isabout 4-5 in 5 mM MgSO4. This type probably is composedof surface carboxylates whose charge could influence thelocalpH ofthe color-regulating amino acids. The second typeinvolves two acidic groups that take up protons at the samePKa as that ofthe color transition, about 2.75 in 5mM MgSO4.The latter are involved in the binding ofa special cation to thepurple membrane which can be distinguished from othercations that bind with lower apparent affinities and with ahigher intrinsic pKa. The identification of this direct cationeffect on the color can be used to assimilate many recentresults into a model which includes the cation in an active sitein bR (see below).One role ofthe special cation may be to maintain the purple

color by keeping the Schiff base counterion in its unproto-nated state; however, it is possible to have a purple mem-brane without the cation being present, if other adjustmentsare made to the natural state of the membrane which wouldkeep the localpH near the chromophore high enough that thecolor-controlling acidic group could be unprotonated even inthe absence of the cation (see, for example, the discussion ofdelipidated purple membrane, below). However, normally,the membrane uses the cation to maintain the color.

5 mM MgSO4

Color pa

IIII

I

Biophysics: Jonas and Ebrey

If

II

152 Biophysics: Jonas and Ebrey

Nature of the Cation Binding Site. Hill plots of the colortitration (17) show that the blue-to-purple change is cooper-ative with n slightly below 2, and this is confirmed in Fig. 1and by the proton uptake stoichiometry at the pKaofthe colortransition (Fig. 3) in this report. These data suggest that thecolor titration and the binding of the special divalent cationinvolve two protonatable groups.The binding of this cation at a location outside the active

site in bR but with a direct effect on the active site is possible,although it seems unlikely not only from our results but alsofrom the studies of bR mutants. The color of retinal proteinssuch as bR is thought to be primarily regulated by a negativecounterion near the positively charged, protonated, Schiffbase, while other charge interactions along with steric and/orisomeric changes are also important (e.g., see refs. 18 and19). The interaction of the protonated Schiff base with itscounterion has been suggested to be particularly weak (20).The bR mutant studies have suggested that certain amino acidresidues such as D85, D212, Y185, and R82 (using thestandard one-letter symbols for the residues; i.e., D = Asp, Y= Tyr, and R = Arg), which are believed to be in the protonchannel near the Schiff base and involved in proton pumping,are also closely related to the purple-to-blue transition.Several groups (e.g., see refs. 21-23) have suggested that theprotonation of a specific amino acid, D85, is responsible forthe purple-to-blue shift. The protonation state of this aminoacid is thought to be influenced by other nearby chargedgroups. A metal ion in the active site of bR should interactwith these and perhaps other ionizable groups, helping toregulate their state of protonation. The cation could alsofacilitate changes within the photocycle. The near lack ofspecificity for a particular cation suggests a very open andflexible binding site.

Certain bR mutants appear to show changes in the coop-erativity of the color titration. Subramaniam et al. (23)showed that the D85E, D85N, R82A, and D212E mutantsremain blue-like up to pH 6 or 7. While the D85E and R82Atitrations displayed what appears to be cooperative, n = 2,behavior, the D212E mutant showed noncooperative, n = 1,behavior. D212N stayed purple between pH 5 and 10, and atlower pH values, instead of turning blue, it developed a peakat 440 nm which was attributed to a loss of chromophore-protein interactions. A loss of cooperativity could indicatethe direct liganding of the particular group to the cation, anindirect perturbation of an actual liganding group by themutation to prevent its interaction with the cation, or theseparation of the counterion from another group that nor-

mally influences its pKa. The large shift in the pKa seen withseveral of the mutants suggests a close interaction betweenthe mutated groups and the Schiff base.

Structural Description. Henderson et al. (3) have nowextended our understanding of the bR tertiary structure. TheC-terminal side ofthe proton channel is described as tight andhydrophobic, and therefore ion-restrictive, while the N-ter-minal side, the side of proton release, is more open andhydrophilic. On the N-terminal side, the model of Hendersonet al. places D85 and D212 4-5 A below the Schiff base whileY57 and Y185 are about 6 Afrom the Schiffbase. D212 is nearY185, W86, and Y57, all polarizable groups that could add tothe relative stability of the anionic forms of the aspartate or

the tyrosines. R82 is in the proton channel, =12 A below theSchiff base. Because the charged guanadinium group of R82is at the end of a flexible carbon chain it may be somewhatmobile. The Schiff base, D85, D212, Y57, Y185, and R82approximately form an octahedron, where the positivelycharged Schiff base and R82 are at the vertices (see Fig. 4).The structural model suggests a pocket on the N-terminalside, with many charged and polarizable residues, mostlylocated on separate a-helices. If the special cation bindingsite is in this region, as the color-affected mutants suggest,

this certainly would help to account for its somewhat unusualnature.

Protonation State ofIonizable Groups of bR. Carboxyls, andprobably tyrosines, undergo significant changes in the courseof the proton-pumping process. Mutant studies have sug-gested that D85, which is normally deprotonated, accepts a

proton from the Schiff base during the photocycle; D212 isalso normally deprotonated (24). The unusually high frequen-cies ofthese groups' infrared vibrations suggest that theirpKavalues in the M state are about 2.1 and 2.8 for D85 and D212,respectively (25).

Tyrosines also seem to be important for the photocycle,and at least one tyrosinate may exist in bR, but the role oftyrosines is controversial. The Y57N mutant (21), and theY185F mutant at pH 5 (26), like the blue membrane, do notform the M photointermediate or pump protons. It has beensuggested that Y57 may be important for the isomerization ofthe chromophore to the all-trans conformation (21, 27).Braiman et al. (27) proposed that Y185 undergoes protonationchanges during the photocycle. Further supporting the exis-tence of tyrosinates, Harada et al. (28) used UV resonanceRaman spectra to show that at least one tyrosinate is presentin light-adapted purple membrane. In contrast to these re-sults, Thompson et al. (29) used NMR spectra of [4'-13C]tyrosine-labeled bR to conclude that a tyrosinate is notpresent in light-adapted bR or in the M photointermediate,but that unusual tyrosine interactions or hydrogen bondingmay take place.Model for a Cation in an Active Site. A model for the

placement of a divalent cation in its binding site is proposedin Fig. 4. As indicated by the Fourier-transform IR data, thebinding site has both D85 and D212 deprotonated. We are

proposing a very interrelated environment for the Schiffbase,D85, D212, Y185, Y57, and R82; and that D212, Y57, andY185 are in more polarizable environments than D85, similarto the proposal of Henderson et al. (3). We suggest that D212and D85 are protonated during the formation of the bluemembrane.

Reevaluation of Previous Data. The cation in the active siteis presumably identical to the site of highest apparent affinity(Ka = 26 p.M` at pH 5) reported by Dufnach et al. (7).Probably we did not recognize this was an unusual site in a

previous report (figure 2 in ref. 12) because our methods werenot sufficiently sensitive. The protein concentration used forcation analysis in this report was 5 times greater than in ourearlier report, and the instrument used for the cation analysiswas more sensitive.

Although the results presented here can be understood inthe context of many recent structural observations, thesedata may seem to disagree with several earlier conclusions.First, Ariki and Lanyi (30) suggested that there are twohigh-affinity sites in bR and that the second site, which hasa lower affinity than the first, is responsible for the color

bR558 blue

5;D85 HY57 H-D85

D212E) Y185 2H+ M2 D212-H Y185

FIG. 4. Two-dimensional model ofthe binding ofa divalent cation

within an active site in bR and ofthe blue membrane. -, Polarizable

amino acids such as tryptophans, serines, or tyrosines.

ll%%~~R82

Proc. Natl. Acad. Sci. USA 88 (1991)

/ R82

Proc. NatL. Acad. Sci. USA 88 (1991) 153

change. Although intriguing, their data are actually quitedifficult to evaluate because the pH during the measurementsis unspecified and not regulated. Thus the complex bindingthey report may be due to the interaction of changes in pHwith the binding of the cations. The results are similar,however, with respect to the concept ofcolor- and non-color-regulating cation binding sites.

Szundi and Stoeckenius (15) reported findings from whichthey concluded that cation binding affects the formation ofpurple membrane only by changing the surface pH. Theyfound that in lipid-depleted membrane, which had an absor-bance maximum at 560 nm, deionization did not lead to theformation of the blue membrane. However, lowering the pHwith HCO did shift the absorbance maximum to 585 nm witha pKa of about 1.4. From their experiments, they concludedthat cations are not necessary for the formation of purplemembrane. In our model if the surface pH is not too low bRcan maintain its purple color-i.e., keep D85 deprotonated-even in the absence of a cation in the special site. Byremoving most of the acidic phospholipids Szundi and Sto-eckenius have increased the local pH relative to that of thebulk in addition to potentially causing conformationalchanges in the active site.Again in contrast to our results, Dufiach et al. (7) reported

that all five of the higher-affinity divalent cation binding sitesare ofequal importance in the spectral titration when divalentcations are added back to blue membrane in deionized water,This is probably because under their conditions (very lowsalt) the titrations of the color-regulating site and the surfacepotential-regulating sites were not well separated because ofchanges in the apparent affinities of the sites with changes inthe surface potential (8). Even in 5 mM MgSO4, in ourexperiment, the titration of the surface groups and the colortitrating groups nearly overlapped (Fig. 3).

We thank M. El-Sayed, B. Honig, W. Hubbell, R. Renthal, J.-L.Rigaud, M. Seigneuret, and C. Wraight for their careful reading ofthemanuscript and B. Jonas for isolating the purple membrane. Wethank the National Science Foundation (Grant DMB-8815824) andthe Department of Energy (Grant 88 Ek 13948) for support.

1. Khorana, H. G. (1988) J. Biol. Chem. 263, 7439-7442.2. Oesterhelt, D. & Tittorj J. (1989) Trends Biochem. Sci. 14,

57-61.3. Henderson, R., Baldwin, J. M., Ceska, T. A., Zemlin, F.,

Beckmann, E. & Downing, K. H. (1990) J. Mol. Biol. 213,899-929.

4. Oesterhelt, D. & Stoeckenius, W. (1971) Nature (London) NewBiol. 233, 149-152.

5. Kimura, Y., Ikegami, A. & Stoeckenius, W. (1984) Photochem.Photobiol. 40, 641-646.

6. Chang, C.-H., Chen, J.-G., Govindjee, R. & Ebrey, T. (1985)Proc. Nat!. Acad. Sci. USA 82, 396-400.

7. Dunfach, M., Seigneuret, M., Rigaud, J.-L. & Padros, E. (1987)Biochemistry 26, 1179-1186.

8. Jonas, R., Koutalos, Y. & Ebrey, T. G. (1990) Photochem.Photobiol. 52, 1163-1178.

9. Mowery, P. C., Lozier, R. H., Chae, Q., Tseng, Y.-W., Tay-lor, M. & Stoeckenius, W. (1979) Biochemistry 18, 4100-4107.

10. Fischer, U. & Oesterhelt, D. (1979) Biophys. J. 28, 211-230.11. Muccio, D. D. & Cassim, J. Y. (1979) J. Mol. Biol. 135,

595-609.12. Nakanishi, K., Balogh-Nair, V., Arnaboldi, M., Tsujimoto, K.

& Honig, B. (1980) J. Am. Chem. Soc. 102, 7945-7947.13. Warshel, A. & Ottolenghi, M. (1979) Photochem. Photobiol.

30, 291-293.14. Chang, C.-H., Jonas, R., Melchiore, S., Govindjee, R. &

Ebrey, T. G. (1986) Biophys. J. 49, 731-739.15. Szundi, I. & Stoeckeniusj W. (1987) Proc. Nat!. Acad. Sci.

USA 84, 3681-3684.16. Ohno, K., Takeuchi, Y. & Yoshida, M. (1977) Biochim. Bio-

phys. Acta 462, 575-582.17. Renthal, R. & Wallace, B. (1980) Biochem. Biophys. Acta 592,

621-625.18. Lugtenburg, J., Muradin-Szweykowska, M., Heeremans, C.,

Pardoen, J. A., Harbison, G. S., Herzfeld J., Griffin, R. G.,Smith, S. 0. & Mathies, R. A. (1986) J. Am. Chem. Soc. 108,3104-3105.

19. Spudich, J. L., McCain, D. A., Nakanishi, K., Okabe, M.,Shimizi, N., Rodman, H., Honig, B. & Bolgomoni, R. A. (1986)Biophys. J. 49, 479-483.

20. de Groot, H. J. M., Harbison, G. S., Herzfeld, J. & Griffin,R. G. (1989) Biochemistry 28, 3346-3352.

21. Soppa, J., Otomo, I., Straub, J., Tittor, J., Meessen, S. &Oesterhelt, D. (1989) J. Biol. Chem. 264, 13049-13056.

22. Otto, H., Marti, T., Holtz, M., Mogi, T., Stem, L. J., Engel,F., Khorana, H. G. & Heyn, M. P. (1990) Proc. Nat!. Acad.Sci. USA 87, 1018-1022.

23. Subramaniam, S., Marti, T. & Khorana, H. G. (1990) Proc.Nat!. Acad. Sci. USA 87, 1013-1017.

24. Braiman, M. S., Mogi, T., Marti, T., Stem, L. J., Khorana,H. G. & Rothschild, K. J. (1988) Biochemistry 27, 8516-8520.

25. Bellamy, L. J. (1968) Advances in Infrared Group Frequencies(Chauser Press/Bungay, Suffolk, U.K.).

26. Duflach, M., Berkowitz, S., Marti, T., Mogi, T., Khorana,H. G. & Rothschild, K. J. (1990) Biophys. J. 57, 362a (abstr.).

27. Braiman, M. S., Mogi, T., Stemn, L. J., Hackett, N. R., Chao,B. H., Khorana, H. G. & Rothschild, K. J. (1988) ProteinsStruct. Funct. Genet. 3, 219-229.

28. Harada, I., Yamagishi, T., Uchida, K. & Takeuchi, H. (1990)J. Am. Chem. Soc. 112, 2443-2445.

29. Thompson, L. K., McDermott, A. E., Farrar, M. R., Griffin,R. G., Winkle, C., Lugtenburg, J., Brown, R. S. & Herzfeld,J. (1990) Biophys. J. 57, 364a (abstr.).

30. Ariki, M. & Lanyi, J. K. (1986) J. Biol. Chem. 261, 8167-8174.

Biophysics: Jonas and Ebrey