J. Mol. Biol. (1977) 112, 377-397

Exciton Interactions and Chromophore Orientation in the Purple Membrane T. G. :EBREY, B. BECHER, B. MAO, P. KrLBRID~

De1~artment of Physiology and Biophysi~, University of Illinois Urbana, Ill. 61801, U.S.A. AND BARRY Ho~rm

Department of Physical Chemistry, The Hebrew University Jerusalem, Israel (Received 28 October 1976) I t has previously been shown (Henderson & Unwin, 1975) that the pigment molecules of the purple membrane of Halobacterium halobium are arranged in clusters of three within a two-dimensional crystal having P3 symmetry. We have shown that an exciton interaction exists between the retinal chromophores of these pig= ment molecules which causes a splitting of the three degenerate energy levels of the individual chromophores in a cluster into two new levels separated by an energy 3 Vg.~.A theoretical analysis of the consequences of this exciton interaction leads to three equations relating three measurable absorption and circular dichroism spectral properties to the three co-ordinates giving the relative orientations in space of the chromophores. These three quantities are the splitting, Vg.e, the relative absorption intensities of the exciton bands, D +/D-, and the rotational strengths of the exciton bands, R ~. These quantitites can be obtained from the optical properties of interacting (trimer) and non-interacting (monomer) chromophores. The degree of the chromophore interaction can be controlled by varying the percentage of the chromophore binding sites of the membrane occupied by the retinals, Accurate absorption and circular dichroic spectra of these samples were obtained, eliminating or minimizing a number of potential artifacts. An analysis of the data shows that the transition moments of the three chromophores of a cluster all lie approximately 19° out of the plane of the membrane, are pointed almost exactly toward the symmetry axis, and are very close together, about 15 A centerto-center. We have also deduced an energy level diagram of the chromophores with and without exciton interaction.

1. Introduction The purple membrane of Halobacterium halvbium is composed of a single protein to which a retinal is covalently bound via a protonated Sehiif base (OesterheIt & Stoeekenius, 1971; Lewis d al., 1974). Due to its spectral similarities to the visual pigment rhodopsin, the bacterial pigment has been called "bacteriorhodopsin". There is also evidence t h a t the photochemistry involved in the primary photoprocesses

377

378

T, G. E B R E Y

ET

AL.

._ __~vj

}0 0

~ (

i2ooX

C~

A2

,(30o c

0i O"-"O\"'A uo C (a)

axis

18 7" _

,,,~./~'

(b)

FIe. 1. (a) Schematic representation of a portion of the purple m e m b r a n e illustrating the P3 s y m m e t r y (after Henderson & Unwin, 1975). The distance between a n y 2 similar s y m m e t r y centers (the length of a u n i t cell) is approx. 62 A. The dimensions of each purple m e m b r a n e protein ("bacteriorhodopsin") u n i t in the m e m b r a n e is approximated in this drawing. The circles represent the helixes of each protein as indicated b y Henderson & Unwin. (b) Co-ordinate system used to specify chromophore transition moment, ~, direction, r is the distance from a s y m m e t r y center to a chromophore, r u ( = %/(3)r) is the distance between a n y 2 chromophores. A set of unit vectors was established a t each chromophore w i t h k perpendicular to the plane of t h e m e m b r a n e , j along the axis from the s y m m e t r y center to t h e ehromophore a n d i in the plane of the m e m b r a n e a n d perpendicular to j e n d k to form a right-handed co-ordinate system. 0 is the angle the chromophoro makes out of the plane of t h e m e m b r a n e , measured from k. ~ is the angle between the projection t h e chromophoro m a k e s on t h e / - j plane a n d the j axis. ¢, 8, a n d r specify the relative orientations of the ehromophores with each other.

EXCITON I N T E R A C T I O N S

AND CHROMOPHORE ORIENTATION

379

of the two pigments may be quite similar, for in both cases light apparently causes a geometrical change in the conformation of the chromophore (Rosenfeld e$ a~., 1977). However, there is abundant evidence that the structures of the two membranes containing the pigments are quite different. In contrast to the highly fluid visual pigment membrane (for a review see Ebrey & Houig, 1975), the purple membrane of H. halobium is an almost perfect two-dimensional crystal, with Ps symmetry (Henderson, 1975; Blaurock, 1975; Henderson & Unwin, 1975). A schematic diagram of a membrane with the protein arranged into clusters of three having P3 symmetry is shown in Figure l(a). Becher & Cassim (1975b) observed positive and negative bands in the circular dichroism spectrum of the purple membrane and suggested the possibility that these were due to exciton interactions between the retinal chromophores of different pigments. This hypothesis has been shown to be true in our study (Becher & Ebrey, 1976) as well as an independent one (Heyn et al., 1975; Bauer et al., 1976). It was demonstrated that the optical activity per chromophore was dependent upon the fraction of sites occupied (number of nearest neighbors of each chromophore) thus proving the existence of chromophore-chromophore interactions. Studies on the optical properties of the purple membrane are complicated by the fact that it can exist in two stable or metastable forms. There is a light-adapted form containing an all-trans chromophore and a dark-adapted form containing a mixture of 13-cis and all-trans retinal (Oesterhelt et aI., 1973; Pettei et al., 1977). We will be primarily concerned with the light-adapted pigment because of its homogeneous chromophore composition. In this paper we use a combination of absorption and CD~ spectra to study chromophore-chromophore interactions in the purple membrane. By varying the fraction of sites occupied by the chromophore we can control the extent of this interaction. When all of the sites within a cluster are occupied (trimers) the interaction is maximal, whereas at very low occupation levels, we are studying primarily single ehromophores within a cluster with little chromophore-chromophore interaction (monomers). Detailed analysis of the spectral data permits an unambiguous determination of monomer, dimer, and trimer energy levels with their corresponding dipole and rotational strengths. These are then used to fit the orientation of the chromophore with respect to both the plane of the membrane and one of its 3-fold axes.

Accurate spectral data for such detailed analysis are imperative. However, light scattering, which is significant in a membrane suspension like that of purple membrane, can induce serious spectral artifacts especially with CD measurements. Consequently, several methods were employed to reduce light scattering in the purple membrane samples including matching the index of refractions of the membrane and the suspending medium, and reduction of the distance between the sample and the photomultiplier tube. As a result, the CD and absorption spectra reported here are believed to be essentially free of the scattering artifacts which are present in previously reported spectra (Becher & Ebrey, 1976; Heyn et al., 1975; Bauer et al., 1976). An unexpected phenomenon we encountered was that the rate of dark adaptation is very strongly dependent on the fraction of sites occupied, with the monomers reverting almost an order of magnitude faster than the trimers. This suggests that Abbreviation used: CD, circular dichroism.

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in a m o n o m e r t h e a b s e n c e o f c h r o m o p h o r e s in n e i g h b o r i n g p r o t e i n s affects t h e conf o r m a t i o n o f t h e p r o t e i n w h i c h is b i n d i n g t h e c h r o m o p h o r e . B e c a u s e o f t h e r a p i d d a r k a d a p t a t i o n r a t e , e x t r e m e care m u s t be t a k e n in s t u d y i n g t h e s p e c t r a l p r o p e r t i e s o f the light-adapted monomer. 2. M e t h o d s (a) Mecsurements The absorption and CD spectra were recorded b y a Cary 118c spectrophotometer and a JASCO J-40A spectropolarimeter. The purple m e m b r a n e samples were prepared b y the m e t h o d of Beeher & Cassim (1975a). Bleached m e m b r a n e (used as a reference and for regeneration studies) was prepared b y exposing the purple m e m b r a n e in 0-5 M-hydroxylamine solution to intense (300 W projector) orange light (filtered through 3.2 cm of a 2"2~/o CuSO4 solution a n d Coming glass filter 3-68). The h y d r o x y l a m i n e solution was adjusted to p H 7 immediately before the s t a r t of bleaching in order to reduce its decomposition a t this p H . W i t h these procedures the membrane completely bleaches in approx. 90 min. The bleached samples were then washed free of hydroxylamine. The 100%, approx. 45~/o and approx. 17~/o regenerated purple membrane samples were p r e p a r e d b y successive additions of 1-~1 portions of 3 × 10 -a • all-trans retinal in ethanol to bleached membrane samples. Assuming r a n d o m binding to the chromophore sites in tlie membrane, a 17 % regenerated sample will have approx. 71 ~/o of the chromophores existing as single chromophores in a 3-site cluster (monomers), 27% as one of 2 (dimers), a n d 2~/o as one of 3 chromophores in a cluster (trimers). F o r a 45% regenerated sample, the corresponding numbers are 31~o as monomers, 50~/o as dimers and 19~/o as trimers, We have found t h a t carotenoids which are always present in the purple m e m b r a n e are p a r t i a l l y destroyed b y the intense light needed to prepare the bleached membrane. However, using completely bleached samples as blanks and then regenerating b a c k to 17% or 100~/o results in exactly the same carotenoid absorption in the sample a n d the reference. Thus it is possible to obtain extremely accurate absorption spectra of the purple m e m b r a n e alone in the range 475 to 750 nm. A t wavelengths shorter t h a n 475 nm, retinaloxime absorption begins to interfere. Purple membrane preparations are suspensions of m e m b r a n e sheets which scatter the light used in measuring absorption and CI) spectra, W e employed several techniques to reduce the light scattering (with its accompanying artifacts (Schneider, 1973)) a n d thus obtained reliable absorption a n d CD spectra. Suspending the purple membrane in 50~/o (w/w) sucrose or in 67~o (v/v) glycerol was found to reduce light scattering effectively b y closely matching the index of refraction of the solution to t h a t of the scattering membrane particles. The use of narrower pathlength cells was also found to reduce the scattering effect. I n addition, placement of the sample cell adjacent to the photomultiplier t u b e of the speetropolarimeter reduced scattering effects b y increasing t h e percentage of scattered light entering the photomultiplier. F o r absorption spectroscopy the use of opal glass placed between the cuvettes and tlie photomultiplier also significantly reduced scattering artifacts (Shibata et a l , 1954). W h e n recording absorption spectra of purple membrane, application of these methods resulted in slightly narrower bandwidths a n d small shifts in the absorption maxima. The use of the scatter-reduction techniques was most evident in the CD spectra. Figure 2, curve 1 is the CI) spectrum of purple membrane in water using a 1 cm cell in the normal cell posit i o n - 1 5 cm from the photomultiplier in the JASCO J-40A. Curve 2 is the spectrum under identical conditions except t h a t the solvent is 50~/o (w/w) sucrose in water. Curve 3 is the same sample in sucrose b u t immediately a d j a c e n t to the photomultiplier tube. Placement of the sample in a 0.5 em cell h a d an effect similar to t h a t obtained from the other methods. Generally, a n y two methods reduced the scatter effect to a point where use of the third did not change the CD spectrum. F o r the CD a n d absorption spectra measurements at room t e m p e r a t u r e presented here, the purple membrane preparations were suspended in 50~/o (w/w) sucrose solution. All CD spectra were recorded in 0.5-em cells with sample absorbances of approx. 1"0. F o r absorption

EXCITON

INTERACTIONS

8.0 "--1"

~

AND CHROMOPHORE

1

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1

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ORIENTATION

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440

480

520

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600

640

Wavelength(nm) FIG. 2. Reduction of light-scattering artifacts in the circular dichroic spectrum of 100% regenerated light-adapted purple membrane at 23°C. Curve 1, CD of the sample in water; curve 2, in 50% (w/w) sucrose; curve 3, in 50% (w/w) sucrose and with the optical cell adjacent to the photomultiplier tube. The spectrum of the sample in 50% (w/w) sucrose and in a 0.5 cm cell was the same as curve 3. A bleached sample which originally had the same absorbance was used as a reference in measuring all spectra. m e a s u r e m e n t s at l i q u i d n i t r o g e n t e m p e r a t u r e s , t h e m e m b r a n e was s u s p e n d e d in 67 % (v/v) glycerol which h a d t h e s a m e effect as sucrose in r e d u c i n g light scatter• A d d i t i o n a l a b s o r p t i o n s p e c t r a were r e c o r d e d using t h e opal glass t e c h n i q u e . Special care is r e q u i r e d for e x p e r i m e n t s a t r o o m t e m p e r a t u r e using l i g h t - a d a p t e d 17% r e g e n e r a t e d p u r p l e m e m b r a n e since t h e l i g h t - a d a p t e d f o r m of this p r e p a r a t i o n was f o u n d to r a p i d l y d a r k - a d a p t at r o o m t e m p e r a t u r e (see Fig. 3). To c i r c u m v e n t this p r o b l e m t h e 17 % r e g e n e r a t e d m e m b r a n e was l i g h t - a d a p t e d a n d t h e a b s o r p t i o n or CD s p e c t r u m r e c o r d e d for 5 s. T h e sample was t h e n l i g h t - a d a p t e d a g a i n a n d t h e process r e p e a t e d u n t i l t h e entire s p e c t r u m was o b t a i n e d . U n b l e a c h e d a n d 100% r e g e n e r a t e d p u r p l e m e m b r a n e d a r k - a d a p t e d m u c h m o r e slowly so this recording m e t h o d was n o t necessary w i t h t h e s e p r e p a r a t i o n s .

I-0 0.8

i

i

I

I

i

I

I

I

I

i

0.6 014

0.2

0.1

0

t

2000

4000 Time Is)

I

6000

FZG. 3. Comparison of the dark reversion rate of 100% regenerated and 5 % regenerated samples of purple membrane in 50% (w/w) sucrose at 23°C. Curve 1, 100%, t t = 2800 s I curv~ 2, 5 ~ , t t = 330 s,

382

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3. T h e o r e t i c a l

An analysis of the experiments reported below requires that we have expressions for the oscillator strengths, rotational strengths and energy levels for two or three interacting chromophores. General expressions for n identical chromophores interacting via a perturbation term V,j have been given b y Tinoco (1963). Because of the Ps symmetry in the purple membrane, all terms Vii are equivalent and we set V12 = V31 = V23. For the trimer there are three solutions to the perturbation matrix: a ( - } - ) state ~ith coefficients C1+ = C2+ = C3+ = 1/~¢/3 ( i n Tinoco's notation) and a doubly degenerate (--) pair of states ("a" and "b") with coefficients C~_ Ca -- 1/%/6, C~_ = --2/%/6, and C[_ Cb 1/%/2, C~_ 0. The degenerate states will be polarized perpendicular to one another and to the non-degenerate state. The excitation frequencies are given by u + ~ v~ ~ 2Vs.e/h and v~ = v~ -- Vs,e/h, where v~ is the absorption frequency of the individual trimer chromophores in the absence of exciton interactions. Since permanent dipoles can interact to shift monomer energy levels, v~ will equal the monomer absorption frequency, urn, only when the chromophores have no permanent ground or excited state dipole moments (~s,s or #o.e)" In the gromld state of a trimer the monomer levels are shifted by 3 Us.s, where Us. s is the interaction energy between two ground-state dipole moments. (The energy levels relevant to the absorption of a single photon are shifted (to first order) by the permanent dipoles by 2 Us, e -~ Uss, where Us. e is the interaction energy between the ground-state dipole on the chromophore with the excited-state dipole of another.) Thus, the permanent dipole interactions in a trimer shift the excited-state energy level relative to the ground state by 2(Us. e -- Us.g ) (see Fig. 9). The absorption frequencies of the trimer exciton states are now given by: +

~t = ~

~;

=

+ 2(us.~ -

.~

+ 2(us,~ -

Us,s)/h +

o

2rs,Jh

= + .t

2Vs.e/h (non-degenerate)

(la)

-- Vs,~/h (doubly degenerate).

(lb)

Us.s)/h - - r s , J h = + ~

The final splitting of the two energy levels will be 3 V s,e/h. The corresponding expressions for dimer states are: v~ : 'Jm + (Us.o -- Us.s)/h + r s . J h : v~ + r s . J h

(2a)

v ; = Vm -{- (Us.o

(2b)

--

Vs.g)/h

--

Vs.~/h = v~

-

-

Vs.Jh.

Using the coefficients given above, we obtain expressions for the trimer dipole strengths : D:

-t

= I~s,ol ~ + 2 ~ s . o . ~ , . ~

D ; - = 2l#,,ol ~ -

(3a)

2/xs.¢" #~.o -'

(3b)

and rotational strengths:

_e: R; -t

--t

= =

[,~I~3

[,~/c]

• ms.~ • ms,~ X /2/g.e)] [/Xs,e -' -' -~- 2/xs,e -' -' -~- ~;, " (/xs.o -' -' -' . , ~ o -.- , ~ J , [2/Xs,e " m- 's . e - - 2/~s.e

-' " (/~s.e × /2~,e)"

(4a) (4b)

/~s,e and ms. ~ are, respectively, the electric and magnetic transition moments for the chromophore i, and ~'¢ is the vector connecting them ; c is the speed of light. The term -( e • ms, - ' e leads to a weak intrinsic CD band and m a y be the origin of the band /zs, observed for the monomer of the purple membrane protein (see below).

EXCITON I N T E R A C T I O N S AND CHROMOPHORE O R I E N T A T I O N

383

The interaction energy between the transition moments of any pair of chromophores (i and j) is: v~.° = I~.el ~ n-~l':"l-~[~' " ~ -- 3(¢- ~'~) (~'" ~")], (5) where ~ is a unit vector along the direction of the transition dipole, i ~s is a unit vector along ~t¢ and n 2 is the dielectric constant for light, n being the index of refraction. A similar energy term arises from the interaction of the permanent dipoles. For essentially linear polyenes such as a l l - t r a n s retinal both the permanent dipoles and the transition dipole of the main absorption band are expected to lie approximately parallel to the polyene chain. (This is in fact found in model studies of protonated Schiff bases (Mathies & Stryer, 1976).) Thus, the angular dependence of the interaction term between the permanent dipoles should then be extremely close to that for the transition dipoles. So we can write:

u~.~ = Ip.~.,d 2 ~-~(,~,,)-~e¢. ~' - 3(¢. ~') (~,. ~'~)3

(6)

u,,.,, = I~,,.d I~..I ~-~(,~")-~E~'- ~' - 3(~'. ~',) (~'. ~")].

(7)

Equations (2) to (7) can be simplified b y making use of the P3 symmetry of the membrane. We define a rectangular co-ordinate system where the z axis is perpendicular to the plane of the membrane and the y axis intersects the axis of 3-fold symmetry (see Fig. l(b)). In polar co-ordinates the transition dipole /Xg,e _l of one of the three ehromophores is given by:

[#~,el(sinOcos¢i + sinOsinCj + cos0~),

-~

(8)

where g, j, and/c are unit vectors along the x, y, z axes, see Figure l(b). I f we assume that all sites are equivalent, then each chromophore has the same orientation with respect to the z axis so that each has the same value of 0. The angles ~bare related by the 3-fold symmetry of the system so t h a t ¢2 = ffl q- 120° and ¢3 = ~b~ A- 240 °. The vector ~t¢ lies in the plane of the membrane and has no z component. Hence e's = le'Sl(l/2~ + ,vi(3)/2j). v.,, = I~.ol ~- ,~-~l~"l-~g(o,¢) (9)

u:.: = i~:.,i ~ ,~-21e';I-~F(o,¢)

(lO)

U,.e = I~:.:1 I~.,I ,~-~le"l-~e(o,¢),

(11)

where F(0,¢) = [cos20 + sinZO(7[4 -- 3 eos2¢)].

(12)

D~ ----3]#g.ei2cos20

(13a)

D~ = 3]#~,ei%in20

(13b)

For a trimer:

(~rvtlc)[tx~. e ° -'

" m~.e-' -Jr- 21%.e-'" mS.e -t- (%/(3)/2)[rtSH/~,el 2 sin20cos~b]

R:

:

R~

= (~'vt/c)[21%.e° -' • m~.e-I __ 2/%. e _ ' . m,,e-t -- (%/(3)[2)[~¢]1p ~.~[2 sin20cos~].

(14a) (14b)

I t can be easily shown that _t

-S

_i

i~i

~,.o "~,.~ = I~,.el I ,.,111 + eos (,~ -- 60)1,

(15)

where A¢ is the difference in the polar co-ordinate ¢ between the magnetic and electric transition moments. Substituting into equation (4) we obtain R~+ = R ° + R exclt°n t

(16a)

R~- = 2R ° -- R~x¢lt°=,

(16b)

384

T.

G.

E B R E Y

ET

AL.

where ~e~c,to~ = 2Ro(1 ~_ cos (A¢ -- 60)) ~- - - l ~2 G' J I I ~ z

~t

'

~12sin20cos~

(17)

and

Ro

~_

(~) ~,~-' ~,-'._

-

-

. ...~

~.

(18)

6

Note that in addition to the familiar ~tj. (p, × ~j) contribution to the rotational strength of the exciton levels, the ~z.e-' " m~.e term can also contribute. This term, due to the interaction of the electric dipole of one ehromophore with the magnetic dipole of another, can be not greater than 4R °. 4. Circular Dichroism Curve Fitting The recorded elliptieity O(v) contains contributions from both exeiton (eqn (17)) and non-exeiton (monomer) circular diehroism bands. The circular dichroism due solely to the exciton interaction can be estimated quite accurately by taking the recorded spectrum and fulfilling the strict demand of theory (see e.g. Tinoeo, 1963) that the rotational strength, R = k S 0(V)dv, of the positive exeiton CD band equals D

that of the negative exeiton CD band. We first calculated the rotational strength of the positive and negative portions of the recorded CD spectrum. We found, in every case, that the rotational strength of the positive, shorter wavelength band was greater than that of the negative, longer wavelength band. Thus, there must be a source of positive optical activity which is added to the exeiton optical activity to give the observed CD spectrum. To find the true exeiton CD spectrum, we subtract out a positive band whose magnitude is chosen to make the remaining rotational bands sum to zero. This type of curve fitting is seen in Figure 6(b). The non-exeiton band is assumed to be the CD band of each monomeric pigment molecule. This positive CD band is probably due to the intrinsic circular dichroism, R °, of the pigment (see Discussion). A very good first approximation is that this intrinsic CD band has the shape and position of the pigment absorption spectrum. However, because of exciton splitting, the CD band should be split with two thirds of its intensity in the doubly degenerate (--) state and one third in the non-degenerate ( + ) state (see eqn (16)). In all the curve fittings presented below, we have assumed that the intrinsic CD band is the sum of two bands each having the shape of the monomer absorption spectrum, one centered at v- and contributing twice the intensity of the other centered at v +. While there m a y be some ambiguity in the precise shape and location of this band, the CD crossover of the pure exciton CD band is insensitive to these uncertainties. Shifting the intrinsic band or any other similarly shaped band 20 nm in either direction had little effect on the CD crossovers of the generated exciton CD spectra. 5. Results (a) Dark reversion Our analysis will be primarily concerned with light-adapted purple membrane. While the light-adapted form of the pigment is normally easy to work with because it reverts only slowly to the dark-adapted pigment, we have found that the rate of

E X C I T O N I N T E R A C T I O N S AND C H R O M O P H O R E O R I E N T A T I O N

385

reversion is a strong function of the percentage of chromophore sites occupied. Figure 3 shows t h a t 100% regenerated purple m e m b r a n e in 50% (w/w) sucrose a¢ p H 7 reverts as a single exponential with a hag-life of approximately 2800 seconds, while a 5 % regenerated sample (almost entirely monomer) reverts with a half-life of almost an order of magnitude faster, approximately 330 seconds. This difference in rate of d a r k adaptation demonstrates t h a t the presence of a chromophore in one binding site of the m e m b r a n e can strongly influence the properties of a chromophore at a presumably adjacent site. I n contrast to the direct ehromophore-chromophore interactions studied below, it is likely t h a t this influence is mediated through protein conformational changes in the one pigment induced b y the ehromophore binding to the neighboring proteins. (b) Absorptionspectra The room temperature absorption spectra of both the light- and dark-adapted forms of the 100% regenerated purple m e m b r a n e are shown in Figure 4. Because the samples were suspended in a 50 % sucrose solution and the opal glass technique was : .....

I.O

i

i

i

i

i

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i

i

i

(a)

I 0.8

0'6

///

0.4

1.0

s,/t

lb)

I

0-8

0"4

0

/

500

~

........550

600 650 Wavelenglh(nm)

7~3

FIe. 4. (a) Absorption spectra of 100% regenerated purple membrane in 50% (w/w) sucrose, at 23°C. Curve 1, after light adaptation (Am~x= 568 nm); curve 2, after dark adaptation (~m~x= 558 rim). (b) Absorption spectra of 17% regenerated purple membrane in 50% (w/w) sucrose, at 23°C. Curve 1, after light adaptation (~m~x= 571 nm); curve 2, after dark adaptation ()'max= 558 nm). Bleached samples identical to those regenerated were used as a reference. The lightadapted spectrum was normalized to an absorbance of 1.0 at the Amax-

386

T . G. E B R E Y

ET

AL.

employed, the spectra have minima] scatter artifacts. I n addition, carotenoid absorption was subtracted out as discussed above. The spectra of both the fight- and darkadapted forms of 100~/o regenerated sample are very similar to those of the native membrane, with the minor differences attributable to the lack of carotenoid absorption. The Amax of the light-adapted form is at 568 nm and the bandwidth is 3218 cm -1. The room temperature absorption Am,x of a 17% regenerated (essentially monomer with approx. 27% dimer) sample is at 571 nm, approximately 100 cm -1 to the red of the trimer Am,x. Its bandwidth appears to be slightly narrower, 3167 cm -1 (see Fig. 5(a)). The absorption spectrum of the dark-adapted monomer is shifted to the blue (Fig. 4(b)) in a manner similar to that in 100% regenerated membrane. At 77°K (Fig. 5(b)) the spectra of the light-adapted 100% and 17% regenerated samples are sharpened and the Am,x values shift to 575 nm and 578 nm, respectively. As in the case at room temperature, the 77°K absorption spectrum of a 100°//o regenerated sample is slightly broader (2760 cm -1) than that of a 17% regenerated sample (2640 cm-a) (Fig. 5(b)). We believe that the slight broadening of the trimer

(o) I,O I

o.8

o,6

0"4

0"2 8

~

o

0"8

0.6

0.4

0.2

0

I

500

I

I

550

I

I

I

600 Wovelength (nm}

I~

650

I

700

Fzo. 5. (a) Comparison of the absorption spectra at 23°0 of: curve 1, 100% regenerated (~max ~ 568 nm); and curve 2, 17% regenerated (~,~z = 571 nm) light-adapted purple membrane in 50% sucrose. (b) Comparison of the low temperature (77°K) absorption spectra of: curve 1, 100% regenerated (;~max= 575 nm); and curve 2, 17% regenerated (~max ~ 578 nn%) light-adapted purple membrane. Bleached samples identical to those regenerated were used as a reference. The spectra were normalized to an absorbance of 1-0 at their ~maxvalues.

EXCITON

INTERACTIONS

AND CHROMOPHORE

ORIENTATION

387

absorption spectrum is due to a weakly allowed exeiton band whose intensity is about 1 0 ~ that of the strongly allowed baud. The plausibility of this assignment will be shown below. (c) Circular dichroism spectra Figure 6(a) shows the CD spectra of the light- and dark-adapted forms of a 100~/o regenerated sample of the purple membrane protein under conditions where lightscattering artifacts are minimized. These spectra were identical with native membrane

"~

4"0'

¢

2-0

a,

I

t! -8-0

°

"\"\

E 4~o

."/,'7

'W '

4~o

'

5~o ' ~ ' Wavelength(n~)

6;o

' 6~o

'

(a)

4-0

'

t

I

3"0 ~

~.o

~_

I-O

go -~-0 ~. -2,0 -:3-0 -4,0 420

474

528

584

636

Frequency(vin s xI0~') (b)

FIG. 6. (a) Circular dichroism spectra of 100% regenerated purple membrane in 50% (w]w) sucrose with optical cell adjacent to the photomultiplier tube, at 23°C. Curve l, after light a d a p t a t i o n (~crossover = 562 nm); curve 2, after dark adaptation. Bleached samples identical to those regenerated were used as a reference. (b) Curve fitting of the 100% regenerated CD spectra, b u t now plotted as 0/v v e r s u ~ v. Curve 1, same as curve 1 above; curve 2, intrinsic CI) b a n d needed to be subtracted to give pure exciton (equal rotational strength) CI) spectra (see text) (~orossover = 560 nm); curve 3, pure exciton CD spectra; curves 4 and 5, positive and negative rotational s t r e n g t h bands having the shape of the 17% absorption spectrum, centered a t 550 n m a n d 570 nm, which a d d e d together give curve 6. The area under curve 5 = area under curve 4 = rotational strength = 2.3 Debye magnetons. The area under curve 2 = 0-05 Debye magneton.

388

ET

T. G. E B R E Y

AL.

light- and dark-adapted CD spectra taken under similar conditions (not shown). A curve.fitting analysis of the light-adapted form (Fig. 6(b)) indicates that there is a small positive intrinsic CD band, R ° N 0.05 Debye magneton. When this is subtracted, the crossover of the pure exciton CD curve is at 560 nm. Figure 7(a) shows the same set of spectra for a 17~/o regenerated sample. A curve fitting (Fig. 7(b)) similar to that of the 100% sample shows that the exciton CD crossover is now shifted

I.Z x

c

1

0-8 0"4

0 -0-4

~

-0'8 LU

/#/,

-I.2 4'~0

640 Wovelength(am) (a)

0.20 ~-_o

0.15 0,t0

"D

.c

0.05

•'.,

%

I

g - 0-05

--,.

-~ B. - O - t O uJ

-0.15 -0-20

420

474

528

584

636

Frequency (~ in s x I0 t2) (b)

FIQ. 7. (a) Circular diehroism spectra of 17% regenerated purple m e m b r a n e in 50% (w/w) sucrose with optical cell adjacent to the photomultiplier tube, a t 23°C. Curve 1, after light adaptation (Aoro,sov,r = 572 nm); curve 2, after dark adaptation. Bleached samples identical to those regenerated were used as a reference. (b) Curve fittings of the 17% regenerated CD spectra. Curve 1, same as curve 1 above; curve 2, intrinsic CD b a n d needed to be subtracted to give pure exciton (equal rotational strength) CD spectra (see text) (Acros,over= 567 nm); curve 3, pure exciton CD spectra; curves 4 a n d 5, positive a n d negative rotational strength bands having the shape of the 17% absorption spectrum, centered at 560 n m and 574 nm, which a d d together to give curve 6. The area of curve 5 = area of curve 4 = rotational strength = 0.5 Debye magneton. The area under curve 2 ----0.05 Debye magneton.

EXCITON

INTERACTIONS

AND CHROMOPHORE

ORIENTATION

389

7 n m to the red, to 567 nm. To emphasize the difference in optical activity per chromophore and hence the exeiton origin of the CD, the ellipicity/absorbance for the 100~/o and 17% samples are plotted on the same scale in Figure 8. Assuming random binding of the chromophores to the sites in the apomembrane during regeneration, a 17°/o regenerated sample will contain about 5% of the chromophores in trimers, 20 to 25°/o in dimers, and 70 to 75°/o in monomers. I t seems reasonable to suppose that the small amount of residual exciton optical activity is due to the 20 to 25°/o dimers since it can be shown that for a dimer the rotational strength per chromophore is three quarters t h a t of the trimer. Thus, the absorption spectrum of the 17°/o sample is essentially due to monomers but the CD spectrum is dominated by the contributions from the small numbers of dimers present (see Fig. 7(b), above). An exciton CD crossover (567 nm; spectrum not shown) similar to that for the 17°/o sample is seen in a 45~/o regenerated sample (15 to 20°/o of ehromophores in trimers'. approx. 50% in dimers) where the dominant CD contribution should also be due to dimers. i

i

i

i

i

i

'/

i

,

,

,

,

,

\

i

.... -'J I

. 440

.

. 480

.

.

. 520

.

,

. 560

600

,

,

640

Woveleflgth(nm)

FxQ. 8. Circular dichroism spectra of curve 1, 100% a n d curve 2, 17% regenerated purple m e m b r a n e with identical absorbances a t their Ama~. Spectra shown are light-adapted purple m e m b r a n e in 50~/o (w/w) sucrose with the optical cell adjacent to the photomultiplier tube a t 23°C.

6. A n a l y s i s o f E x p e r i m e n t a l R e s u l t s

Equations (9) to (14) m a y be used together with the experimental results reported in the previous section to determine (1) the sign and magnitude of the exciton splitting term Vg,e ; (2) the shift resulting from interactions of the permanent dipoles 2(Ug.e - - U,.,) ; and (3) the rotational strength, R~xcit°n. The analysis, though unambiguous, is somewhat complex because of the necessity of distinguishing between the contributions of the permanent and transition dipoles. The experimental data used include Ac°:, the crossover point of the exciton CD [v~° ---- (v+ -- v~)/2; A~° = c/vtc o ], the absorption maximum of the monomer (A~ s ---- c/v abs) and the absorption maximum of the trimer t /Aabs t = C/v[bs). In addition, we make the usual assumption, which should be rigorously true for strongly allowed transitions, that the positions of the exciton rotational bands coincide with the exciton absorption bands. W e will assume below t h a t the absorption differences between the monomer a n d the trlmer are due to energy shifts arising primarily from dipolar interactions r a t h e r t h a n changes in the binding site,

390

T. G. E B R E Y

c

/~x;°:s6o

ET

AL.

×/~ x~,°:56o /2(Ug,e-Ug,g) x

Cose I Vg,eT:>0

'

Case 2 I/g,e < 0

FIG. 9. TWO possible ways the splitting of the energy levels of a symmetrical trimer could give the correct ordering of the allowed absorption bands and circular dichroism crossover. Case 1, Vg.~ > 0 would make At- the lowest, allowed state. Case 2, V,.~ < 0 would make At+ the lowest, allowed state. In the text it is sho~n that only case 1 is consistent with the experimental results. Heavy lines represent positions of measured spectral features. Light lines represent inferred energy levels with the pair of light lines representing the doubly degenerate energy level. W e first o b s e r v e t h a t A~° m u s t lie h a l f w a y b e t w e e n t h e t w o e x e i t o n levels. Since A~bs lies 8 n m to t h e r e d o f A~°, one o f t h e e x c i t o n levels m u s t e i t h e r c o r r e s p o n d in :~abs e n e r g y t o ,,t or be a t longer w a v e l e n g t h s , while t h e s e c o n d e x c i t o n level m u s t be a t l e a s t 8 n m t o t h e blue o f A~°. I t is clear t (see Fig. 9) t h a t A° (defined from e q n ( l a ) ) m u s t be close to A~° a n d lie b e t w e e n A+ a n d A- b u t be h i g h e r in e n e r g y t h a n Ar,. This requires t h a t ( U g . e - U~.g) h a s a p o s i t i v e sign. Thus, we conclude t h a t t h e i n t e r a c t i o n b e t w e e n t h e p e r m a n e n t dipoles o f t h e r e t i n a l c h r o m o p h o r e s raises t h e e x c i t a t i o n e n e r g y of t h e t r i m e r r e l a t i v e to t h a t o f t h e m o n o m e r . Two possible e n e r g y level schemes can l e a d to t h e o b s e r v e d o r d e r i n g o f Aabs ~abs a n d A~°; t h e y are d e p i c t e d in F i g u r e 9. I n case 1 t h e e x c i t o n t e r m V,.e is p o s i t i v e a n d t h e d o u b l y d e g e n e r a t e s t a t e (A~ defined from e q n (lb)) m a k e s t h e m a j o r contrib u t i o n t o t h e o b s e r v e d a b s o r p t i o n m a x i m u m . I n case 2, w h e r e Vg.e < 0, A+ is t h e allowed s t a t e . To show t h a t t h e sign o f t h e r e s o n a n c e i n t e r a c t i o n V~.e m u s t b e p o s i t i v e , we n o t e f r o m e q u a t i o n (12) t l i a t i f F ( 8 , ¢ ) < 0, t h e n 8 m u s t b e g r e a t e r t h a n 42 °, e v e n for t h e o p t i m a l v a l u e o f ¢ = 0 °. B u t as s h o w n i n F i g u r e 9, w h e n Vg.e < 0, A~ is t h e lower, m o r e s t r o n g l y allowed, e x e i t o n level, i.e. D + > D - . F r o m e q u a t i o n (13), t h i s c a n b e t r u e o n l y w h e n 8 < 45 ° a n d so 8 is r e s t r i c t e d t o 42 ° < 8 < 45 °. H o w e v e r , t h e allowed b a n d m u s t be considerably m o r e i n t e n s e t h a n t h e w e a k e r e x c i t o n c o m p o n e n t t I t might seem conceivable that if the splitting were large enough, the lowest energy state could be below Am, and the 2 allowed bands at A+ and A- could still sum to give a band whose A~a. was at 568 nm. However, since there is very little broadening in going from the monomer to the trimer, and given the rather large level separation of at least 16 nm, the long wavelength transition must be strongly allowed. Consequently, the short wavelength transition must be only weakly allowed. Thus, these two bands could not sum to give both (a) Area ab8x a t 568 nm less than A~ and (b) a At° also less than Am.

EXCITON

INTERACTIONS

AND CHROMOPHORE

ORIENTATION

391

(see b e l o w ) a n d , i n c o n t r a s t t o t h i s r e q u i r e m e n t , i n t h e r a n g e 42 ° t o 45 ° t h e t w o b a n d s w o u l d be a p p r o x i m a t e l y e q u a l (D + ~ ] ) - ) . T h u s , w e c a n c o m p l e t e l y e x c l u d e case 2," w h e r e (V~.e < 0). W e c o n c l u d e t h e n t h a t case 1 i n l~igure 9 is a p p l i c a b l e a n d t h a t V , . . > 0. W e n o t e t h a t e q u a t i o n s (9) t o (12) n o w r e q u i r e t h a t b o t h U~.e a n d U ~ . , also b e g r e a t e r t h a n zero. I n o r d e r t o d e t e r m i n e t h e c o r r e c t v a l u e s f o r Vg.e a n d (Ug,e - - Ug.g) w e f i t t e d t h e 1.00 .

.

.

.

.

.

.

.

.

.

.

(o) 0.89 0-7~ O. 67 0-56

/...--

0.44

.

~.(

0.22 0.11

0'89

/ . "" ''..

o ~z i

i,/,- / 3

I /,,:= \ I // \\ r /y.. "/" ~ "+

o

4

soo

s4o

'%

~;o

o20 .......

6~o

'

7;o

Wevelenglh (nm)

Fie. 10. Curve fitting of the 100% regenerated absorption spectrum from two 17% regenerated absorption spectra symmetrically split about the circular dichroism crossover. (a) At 23°C, where the trimer CD crossover, A¢°~, is at 560 nm. A~- must be 5 7 0 i 2 nm and A + = 550+2 n m with D + / D - = 0.114-0.04. (b) At 77°K, where the CD crossover is assumed to be shifted by the same amount as the Area absx shifts, 7 nm. Then A~- = 557.5+2 and A+ = 577.54-2 with D + / D - = 0'1140.04. Curve 1 is the absorption spectrum of the 100% regenerated samples, curves 3 and 4 have the shape of the 17% regenerated absorption spectra but are located at A- and A+, respectively, and curve 2 is the sum of curves 3 and 4. Curve 2 is a very good fit to the experimen&aI absorption spectrum. Changing any of the variables, A+, A- or D +] D - , outside the lhnits given above, resulted in clearly unsatisfactory fits. •

26

392

T.G.

EBREY

ET

AL.

absorption spectrum of the trimer at room temperature to two bands (maxima at ~kt* and ~k~) both having the shape of the monomer absorption. I n this analysis the splitting about ~k~° and the relative intensities D + / D - are free parameters which must account for both the observed value of ~abst ( = 5 6 8 nm) and the shape of the trimer absorption band. We found t h a t the only satisfactory fits could be obtained in the range ~k~ = 570-4-2 nm, ~+t = 5 5 0 i 2 and D ÷ / D - = 0.11-+-0.05. A fit with those values is shown in Figure 10(a). F r o m equation (13), it follows t h a t 0 = 71-+-4 ° or 1 0 9 + 4 °, requiring t h a t chromophores lie close to, but not in the plane of the membrane. Taking the values ~k~ 570 nm and ~k~ + 550 nm, we find 209 cm -z and 1/u~ = A~ = 563.3 nm. Now from equation (1), vt - - Vm = 2(U~.. - - U g . , ) / h so t h a t Ug.e - - U,.~ = 120 cm -z. The energy levels for the trimer are summarized in Figure ll(a). We can substitute the value for (U~.e - - U~.g) and Vg.~ into equation (2) and obtain the corresponding diagram for the dimer case (Fig. ll(b)). We used the ratio D + / D - = 0.11 obtained at room t e m p e r a t u r e to fit the absorption spectrum of the trimer from the corresponding low temperature (77°K) monomer absorption spectrum. Because of the narrow bandwidths at low temperature, this constitutes a sensitive test of the parameters obtained from the room t e m p e r a t u r e analysis. As can be seen from Figure 10(b), the fit is quite good. Finally, to complete the analysis of the data, the rotational strength due to exciton interactions can be determined b y fitting the exciton CD spectrum (Fig. 6(b)) to two

=

=

o

V~,e~.~

X~ : 550

o=sto k0=563.3

.....

:)lUg e-Ug,g i 239 cm-I

3 ,e=626 cm-I

X; =570

Xm=571 Trimer

(o)

_• x~

(Ug,e-Ug,g) ; 120 crn-I

/~ =567.3

Xm=571

>,~°=567 ! ~"~ 2i,,,--417cm-'

Dimer (b)

FIo. I1. Energy level diagram of the purple membrane in going from a monomer, ~m, to: (a) a trlmer, (b) a dimer, with the energy shifts due to the interaction between the permanent ground and excited-state dipoles (U~.~, U~.e) and between the transition dipoles (V~.e) shown.

E X C I T O N I N T E R A C T I O N S AND CHROMOPHORE O R I E N T A T I O N

393

bands of opposite sign centered at A+ and A]~-. The exciton rotational strength is given b y R ~°lt°~t ---- k~t~(v) dr. We find ReXCit°nt: 2"3~0"2 Debye magnetons for U

the trimer. With R ° = 0.05 Debye magneton (Fig. 6(b)) then from equation (17) we obtain o

=~¢/(3)vt [~j[lg~.e[~ sin20cos~ ~ [2"3±0"2] -- [0.1(1 + cos(A~b -- 60))]. 2c

(19)

This equation and equation (9) are used below in fixing r and ~b. 7. Discussion An important result of this study t h a t is only indirectly related to the problem of exciton interactions is t h a t the rate of dark adaptation of the monomers is almost an order of magnitude faster than that of the trimers (Fig. 3). This finding suggests the existence of protein-mediated interactions between sites on different pigment molecules. We have found in the curve fittings for the 100% regenerated (Fig. 6(b)), 1 7 ~ regenerated (Fig. 7(b)), and 45°/o regenerated (not shown) samples t h a t the magnitude of the non-exciton CD band Ter chromophore is the same. This monomer CD band has a rotational strength of 0.05 Debye magneton, somewhat smaller than t h a t observed for visual pigments, 0.5 Debye magneton. Two mechanisms have been proposed to explain the optical activity of visual pigments, an intrinsic dissymmetry (due to twisting of the ll-cis retinal chromophore) or a dipolar interaction of the chromophore with some amino acid of the protein. The same two types of explanation for the monomer CD of the purple membrane protein are possible since an intrinsic dissymmetry resulting from ring-chain twisting of all-trans retinal can also lead to substantial rotational strengths (Honig et al., 1973). The values for Vs.e, ( U g . e - U~.~) and D+]D - that were determined from the experimental data m a y be tested in several ways. We first recall that D +/D--: 0.11 requires t h a t 0 ---- 71 ° (or 109°), a value consistent with the report of Blaurock & Stoeckenius (1971) that the chromophore lies approximately in the plane of the membrane. Second, an independent estimate of the (U~.e -- Us.s) can be obtained from the magnitude of the permanent dipoles of protonated Schiff bases of retinal reported b y Mathies & Stryer (1946). From equations (9) to (11), ( U ~ . e - Ug.g)] Vs.~ ---- [#s.~ • (/~.e --/~s.~)]/[/~.~[ 2" For the pigment we determined (U~.e -- Ug.~) ---120 cm -1 and Vg.e ---- 209 cm -1. Taking/~.e ---- 10.2 Debye which we calculate from the absorption spectrum assuming ¢ ---- 56,000 (Powers, Becher & Ebrey, unpublished results)t, we find ~g.~(/~e.e -- ~ . s ) ---- + 6 0 (Debye) 2. This value for the pigment is in reasonable agreement with t h a t determined for the model compound b y Mathies & Stryer (1976), A-96 (Debye) 2. Finally, another check of our analysis is obtained b y considering the extent to which the value of (U~.~ -- Ug.g) derived above from the trimer data can account for the experimental dimer CD spectra of Figure 7. I f we make the assumption that the relative positions of the chromophores in a dimer are the same as in a trimer, then the value of A~° for the ttlmer predicted from our analysis of the trimer data (shown in Fig. ll(b)) corresponds exactly to the experimental value of 567 nm. While so close an agreement is undoubtedly fortuitous, the ~fThis value is slightly smaller than that of Oesterhelt & Hess (1973). Their value of ~ ~ 63,000 would give/~r.e = 11 Debye.

T. O. E B R E Y E T A L .

394

}" C3 oxis is perpendicular to the poge (plone of the

T /.~

membrone)

f =85°

. . . . . . . . .

-/

,,

/

,

\\

"

"T.-" /

1 / ~ =8~0 ~kx.

i Plone of lhe membrone is perpendicular to the poge

l Cz,oxis I ~'(

r13~ . . . . .

r12 "

. . -

FIG. 12. Relative position of the transition moments of the chromophores in a t r i m e r in the purple membrane. The separation of the chromophores is about 15 A, they are oriented about 19° out of the plane of the membrane and are ~lmost paraIIel to a line radiating from the symmetry axis.

GD crossover p o i n t for b o t h the t r i m e r a n d the d i m e r does d e p e n d on the correct signs a n d m a g n i t u d e s of V~., a n d (U~. e - - U , . , ) . All t h r e e of t h e a b o v e i n d e p e n d e n t checks give us confidence t h a t our t r i m e r analysis is correct. W e n o w proceed to use the e x p e r i m e n t a l l y derived values of D+/D -, V~. e a n d R[ xclt°n to fit t h e position of the chromophore with respect to t h e 3-fold axis of F i g u r e l(b). As m e n t i o n e d above, D + / D - = 0.11_4=_0.05 requires 8-----71:l:4° (or 109:h4°). T h e n e q u a t i o n (9) a n d

~,,,_.v~ IP., ,l~lrU]sin20cos¢ 2C

= 1.9 to 2.5 D e b y e

magneton

as d e t e r m i n e d from e q u a t i o n (19) (the u n c e r t a i n t y allows for a n y v a l u e of ,t¢), c a n be solved s i m u l t a n e o u s l y for r a n d ~6. U s i n g 0 ---- 71 °, ~g.e ---- 10"2 Debye, Vg.e ---- 209 e r a - z a n d a s s u m i n g t n 2 ----2, we find r ~ 12 ~ a n d ¢ ~___85 °. W i t h t h e a l t e r n a t i v e v a l u e t n2 __ 2 is a reasonable estimate for the index of refraction of a protein and is also close to the macroscopic index of refraction of the membrane as determined by the sucrose concentration (50% (w/w), n= ----2) needed to minimize the Hghbscattering of membrane suspensions by matching the index ofrefrsmtion. I t is, however, only an approximation for the microscopic index of refraction due to the specific atoms in the space between a pair of chromophores. The asstunption n 2 ~ 2 is not critical and values of n2 ~ 1 to 3 will alter r by only 2 A.

EXGITON I N T E R A C T I O N S AND CHROMOPHORE O R I E N T A T I O N

395

of 0 = £09 °, ~ = 95 ° will also give the correct sign and magnitude of the rotational strength. Our data allow little deviation from these numbers. The only significant uncertainties are the assumption that the binding site determinants of chromophore color are unchanged in going from a monomer to a trimer (see footnote to page 389) and the assumptions associated with the use of the dipole approximation in the expression for the interaction energy, equation (5), when the dipoles are so close together. In order to test the validity of this approximation for small values of r, quantum mechanical calculations of the electron distribution of the chromophore were carried out (Honig et al., 1976; Honig, unpublished results). Monopoles obtained in the calculation were placed on each atom in the chromophore and the interaction energy F12 was calculated by summing the coulombic energy between all atoms on adjacent chromophores. The results indicate that the dipole approximation works remarkably well, even for small values of r. However, in the range ¢ ~ 90 ° the dipole approximation slightly overestimates V12 and we find t h a t a value of r ~ 15 A is obtained with the more accurate monopole estimation of the interaction energy. This value is close to minimum interchromophore separation consistent with the electron density maps of Henderson & Unwin (1975). We note that since ~ ~ 85 °, the ends of the chromophores must be nearly in contact with one another suggesting the possibility of steric chromophore-chromophore interactions as well as the exciton interactions we have described here. A set of three chromophores with the derived orientation is shown schematically in Figure 12. We cannot, of course, be certain about which of the three symmetry axes the chromophores are so oriented. The chromophores whose interaction leads to the exciton splitting could be located within a single cluster of three proteins, so that A 1 is the relevant 3-fold axis (Fig. l(a)). In this case, the distance r__~ 15 A is the separation between binding sites on a single cluster. Alternatively, the interacting chromophores could be at the outer surface of a cluster so t h a t / I 2 or As is the symmetry axis and the interacting units giving rise to the exciton splitting consist of chromophores belonging to different protein clusters. Given an exciton interaction of approximately 200 c m - 1, the rate of energy transfer between adjacent chromophores should be approximately 10 -is seconds (Kasha, 1963). This is an order of magnitude faster than the inferred fluorescence lifetime (Alfano et al., 1976) and suggests t h a t complete randomization of the excitation energy might occur before any significant photochemistry has taken place. However, a number of groups have reported that the purple membrane exhibits strong bleaching-induced dichroism (Razi-Naqvi et al., 1973; Cone, reported in Oesterhelt, 1974; Lozier & Niederberger, 1977) and highly polarized fluorescence (Lewis et al., 1976). These observations would be inconsistent with complete randomnization of the excitation energy over the three symmetrically arranged chromophores after light absorption. However, Robert Silbey (personal communication) has pointed out that the polarized light is absorbed b y only one of the two allowed degenerate energy levels. Each of these degenerate levels is polarized perpendicular to the other and absorption b y either one involves selective excitation of only certain members of a trimer set. The probability of a given chromophore being excited is determined b y the coefficients of the two degenerate energy (--) wave functions, which were given in the Theoretical section. After excitation, mixing of the two degenerate states could eventually lead to a random probability of excitation of any

396

T. G. E B R E Y

ET

AL.

one of the three chromophores. However, if the lowest excited state, formed after the absorption of the polarized light can be rapidly de.excited before mixing can take 1~/ace, then polarized bleaching (and emission) should, in fact, be observed. Given the short fluorescence lifetime (Alfano et al., 1976) and the rapid rate of photochemical conversion to the p r i m a r y photoproduet ( K a u f m a n n et al., 1976), this assumption seems quite reasonable, Supported in part by U.S. Public Health (EYO1323). One of us (B. B.) was the recipient of an Institutional National Research Service Award (USPH E Y e 7005) postdoctoral fellowship. Part of this work was done while another author (B. H.) was a Visiting Associate Professor of Biophysics at the University of Illinois. We thank Drs Fumio Tokunaga, Richard Henderson and Robert Silbey for m a n y useful conversations. We were first made aware of the importance of permanent dipole interactions in the purple membrane at a lecture by Arndt Kriebel of Cornell University at the Peter Leermakers Symposium, Wesleyan University. l~Iote added in proof: Recently an article by A. Kriebel and A. Albrecht on the exciton interaction in purple membrane has been published (Kriebel & Albrecht, 1976). While their equations are similar to ours, the experimental data they used included the absorption spectrum of the purple membrane in Triton X100, to represent the monomer spectrum, which is quite different from our monomer absorption spectra. We believe this spectrum is not that of the monomer, but rather that of a partially denatured species. Thus, the energy levels they infer, as well as the relative chromophore orientations, are quite different from our results.

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