J. Mol. Biol. (1977)
117, 607-620
Transient and Linear Dichroism Studies on Bacteriorhodopsin : Determination of the Orientation of the 568 nm All-trans Retinal Chromophore M.P. HEYN Department
of Biophysical Chemistry, Biozentrum CH-4056 Basel. Switzerland
It. J. CHERRY AND U.
M~~LLER
Laboratorium fiir Biochemie, ETH Zentrum. CH-8092 Ziirich, Switzerland (Received 8 July 1977) The orientation of the 568 nm transition dipole moment of the retinal chromophore of bacteriorhodopsin has been determined in purple membranes from Halobacterium halobium and in reconstituted vesicles. The angle between the 568 nm transition dipole moment and the normal to the plane of the membrane was measured in two different ways. In the first method the angle was obtained from transient dichroism measurements on bacteriorhodopsin incorporated into large phosphatidylcholine vesicles. Following flash excitation with linearly polarized light, the anisotropy of the 568 nm ground-state depletion signal first decays but then reaches a timeindependent value. This result, obtained above the lipid phase transition, is interpreted as arising from rotational motion of bacteriorhodopsin which is confined to an axis normal to the plane of the membrane. It is shown that the relative amplitude of the time-independent component depends on the orientation of the 568 nm transition dipole moment. From the data an angle of 78” + 3” is determined. In the second method the linear dichroism was measured as a function of the angle of tilt between the oriented purple membranes and the direction of tile light beam. The results were corrected for the angular distribution of the membranes within the oriented samples, which was determined from the mosaic spread of the first-order lamellar neutron diffraction peak. In substantial agreement with the results of the transient, dichroism method, linear dichroism measurements on oriented samples lead to an angle of 7 1’ h 4”. No significant wavelength dependence of the dichroic ratio across the 568 nm band was observed, implying that the exciton splitting in this band must be substa.ntially smaller tha.n the recently suggest,ed value of 20 nm (Ebrey et al., 1977). The orientation of the 568 nm transition dipole moment, which coincides wit11 the direction of the all-kalzs polyene chain of retinal, is not only of interest in connection with models for the proton pump, but can also be used to calculate the inter-chromophore distances in the purple membrane.
1. Introduction in the purple membrane of Halobacterium
Bacteriorhodopsin a light-driven rhodopsin
proton absorbs
pump. at
568
In the light-adapted nm.
When
light
GO7
state is absorbed
halobium
the chromophore a photochemical
functions
as
of bacteriocycle
is
608
M. P. HEYN,
R. J. CHERRY
AND
U. MULLER
initiated which has been investigated in great detail. In this cycle and probably in the proton transport as well, the retinal chromophore which is bound to the protein moiety (bacteria-opsin) eia a Schiff base linkage plays a central role (for recent reviews see Oesterhelt, 1976; Henderson, 1977). Little is known about the position and orientation of the chromophore within the protein. In formulating possible models for the mechanism of the proton pump, it is desirable to have information available on the spatial localization and orientation of the chromophore. In the structural investigations using X-ray diffraction and electron microscopy, the chromophore has not yet been resolved (Blaurock & Stoeckenius, 1971; Henderson, 1975; Blaurock, 1975; Unwin & Henderson, 1975; Henderson & Unwin, 1975). The bacteriorhodopsin molecules are arranged within the membrane in a rigid hexagonal lattice. Within this lattice the protein is immobilized (Razi Naqvi et al., 1973). Since a tight and highly specific retinal-protein complex is formed, we will assume that the chromophore has a fixed position and orientation with respect to the protein moiety. This assumption is supported by experimental evidence which will be discussed later. A well-defined angle 0 thus exists between the direction of the chromophore transition dipole moment and the normal to the plane of the membrane (Fig. 1). In the light-adapted state retinal is in the all-trans form (Oesterhelt et al., 1973) and its transition dipole moment coincides with that of the all-trans polyene chain. Previous measurements showed that this direction is preferentially parallel to the plane of the membrane (Blaurock & Stoeckenius, 1971). It was first pointed out on the basis of circular dichroism measurements that the 568 nm transition dipole moment cannot be exactly in the plane of the membrane (Heyn et al., 1975). Direct evidence for exciton interactions between the 568 nm transition dipole moments of adjacent bacteriorhodopsin molecules has been obtained from circular dichroism spectra (Heyn et al., 1975; Bauer et aE., 1976; Becher & Ebrey, 1976). Knowledge of the chromophore orientation is important for the interpretation of the exciton effects, since in combination with circular dichroism and absorption measurements it enables the chromophore-chromophore distance to be calculated. The wavelength dependence of the linear dichroism of oriented purple membranes is of particular significance in connection with the magnitude of the exciton splitting. In this paper we report the determination of 0 using two different methods : transient dichroism of bacteriorhodopsin incorporated into large phosphatidylcholine vesicles and linear dichroism of oriented purple membranes. The method, described in this paper, of determining the orientation of a membrane-bound chromophore from the decay of the anisotropy of the flash-induced transient absorption is new and may find application in other membrane systems. It is therefore of additional interest to test the validity of this approach by comparing its results with those obtained by means of linear dichroism. Some preliminary results of this investigation and related linear dichroism studies have recently been briefly reported (Heyn & Cherry, 1977; Bogomolni et al., 1977).
2. Materials and Methods (a) Incorporation
of bacteriorhodopsin
into lipid
vesicles
Purple membranes were isolated from H. halobiuna (strain RIM,) as described by Oesterhelt & Stoeckenius (1974). The membranes were solubilized by suspending 1 mg in 4 ml of O-1 M-acetate buffer (pH 5.0) containing 0.1% Triton Xl00 for 24 to 30 h in the dark at room temperature. Membrane reconstitution followed a method proposed by
RETINAL
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Henderson (1977). After adding the requisite quantity (1 to 5 mg) of phosphatidylcholine (either dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine), Triton was buffer (pH 5.0) containing O.OZ’$/b removed by prolonged dialysis against 0.1 M-a.W3tate sodium azide. %ollowing dialysis, the sample was purified by centrifugation through a sucrose density gradient (4.5% to 40%) to remove any non-recombined lipid and protein. The method yields predominantly unilamellar lipid-protein vesicles with diameters t,ypically in the range 0.3 to 0.5 pm. Further details of the procedure will be presented in preparation). Protein was determined by tilt> c!lsewhere (Cherry et al., manuscript method of Lowry el al. (1951) and lipid by phosphorus analysis (Chen et al., 1956). Proteill : pllospholipid ratios (w/w) in the reconstituted membranes used in this st,udy were iu t,hc rangt~ 0.6 to 1.7. (b) Preparation of oriented membrane samples Suspensions of purple membranes in 0.2 mM-acetate buffer (pH 4.7) were dried on a flat quartz plate in a desiccator in the presence of silica gel. Under these conditions tllc rigid flat membrane patches, which have an average diameter of 0.5 Pm, orient themselves parallel to the quartz plate by stacking. Well-oriented purple membrane samples havcl been previously prepared in a similar way for X-ray diffraction measurements (Blaurock & Stoeckenius, 1971; Blaurock, 1975; Henderson, 1975). The angular distribution of t,hr orientation of individual membrane stacks within the sample with respect to the plane of the quartz support was determined from the width of the mosaic spread of the first-order lamellar neutron diffraction peak (Worcester & Franks, 1976). 2H,0 was used to enhance tlrr contrast. For the thickest layers used in the linear dichroism measurements (0.1). 0.6 at 568 nm), the half-width of the angular distribution at half-height w&s 7.5”. The Bragg spaciug of the membrane system varied from 49.2 if for completely dried out samples to 70.1 -4 for samples in a saturated 2H,0 atmosphere. Over this range of hydration tlrtb diffraction measurements \V~IY. change in the mosaic spread was only I”. The neutron performed at the Institut Lane-Langevin (Grenoble). (c) Transient
dichroism
measurements
The flash photolysis apparatus used in these experiments is described elsewhere (CllerrJ& Schneider, 1976). The application of the method to the investigation of rotational diffusion of bacteriorhodopsin has also been reported (Cherry et al., 1977a; Razi Naqvi et al., 1973; Heyn et al., 1977). In brief, ground-state depletion of the 568 nm absorption band is detected following laser flash excitation at 540 nm. Transient absorbance changes (A,,(t), A.,(t)) at time t after the flash are simultaneously measured for light polarized parallel and perpendicular with respect to the polarization of the exciting flash. The allisotropy parameter r(t) is calculated from the expression r(t)
=
A,,@)-- A,(t) + 2 A,(t) .
4(t)
In all measurements bacteriorhodopsin the source of the measuring beam. (d) L&ear
was light-adapted
dichroism
by the tungsten
lamp
used as
measurements
Linear dichroism measurements on oriented purple membrane samples were performed with a modified Rehovoth Instruments CD-HC attachment designed for use with the Cary 15 spectrophotometer (Jaffe et al., 1967). The optical system is shown schematically in Fig. 2. The horizontal plane is defined a~ the plane of Fig. 2. The undeviated horizontally polarized beam produced by a Rochon prism is incident upon a waveplste of quartz set, with its optic axis at 45” to the horizontal plane. The vertically polarized extraordinary ray is blocked by a knife-edge. The precise state of polarization of the light emerging from the waveplate depends on the thickness of the plate (d), the wavelength (A) and the birefringence of quartz (An(h)). The relative retardation produced by the waveplate is given by 2nddn(X) A
*
610
M. P. HEYN,
R. J. CHERRY
FIG. 1. Sche~tic ~pre~~tation of the plane of the membrane. The 2 axes labelled by D,, is perpendicular to the to the plane of the membrane and the
‘c’. HOLLER
the orientation of b8c~riorh~opsin (BR) with respect to labelled with D, lie in the plane of the membr8ne. The 8xis plane of the membrctne. 0 is the angle between the normal direction of the 668 nm transition dipole moment,
Oriented metnbrones an quartz support
Quartz waveplate with optic axis set at 45”
\
Polar ixer (Rochon prism)
AND
I
Depolarizer
Knife edge Goniometer angle a variobke
3%. 2. Schematic diagram of the optical system principle of operstion is described in the text. The black patch on the quartz support. The plane of the plane of the Figure. In the reference beam an instead of purple membranes.
used for linear dichroism measurements. The oriented membranes 8re represented by the the oriented membr8nes is perpendioular to identical unit ~8s used with apomembranes
Since the retardation is a function of h, and since the birefringence of quartz is of the order of 0.01, the retardation will ohange within 8 few nm by an amount of the order of V, if the waveplate is sufficiently thick. When the retardation equals an even multiple of x the emerging beam will be horizontally polerized, when it equals an odd multiple of GTthe beam will be vertically polarized. Thus 88 the wavelength region is scanned the state of polarization of the be8m alternates between horizont&l and vertio8L If 8 line&rly dichroic sample is put in the beam after the waveplate, one records at those wavelengths where the be8m is horizontally polarized the absorbance in that st&e of polarization. At those wavelengths where the beam is vertically polarized, one records likewise the absorbance for vertically polarized light. In this w8y an absorption spectrum is obtained with superimposed oscillations due to the linear dichroism. Connecting the points belonging to the
RETINAL
ORIENTATION
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BACTERIORHODOPSIN
611
same state of polarization by interpolation, we obtain directly the 2 absorption spectra. The linear dichroism is thus obtained from a single scan. The advantages of this method are that no polarizers need to be moved and that the beam passes for both states of polarization through the same area of the possibly inhomogeneous layer of orient’& membranes. The attachment was modified in 2 ways. An extra 4 mm thick quartz waveplate was added for increased resolution in the visible region. The cell-holder was replaced by a goniometer which allowed the variable angle CLbetween the plane of the oriented membranes and the direction of the incident light beam to be accurately determined (Fig. 2). An identical unit was used in the reference beam. A layer of oriented chromophore-fret> apomembranes of the same thickness was used as a reference (Oesterhelt et al., 1974). Matching in terms of the same number of bacteriorhodopsin molecules per unit> area was verified by comparing the near ultraviolet absorption spectra of the oriented sample nrhd reference. Since the purple membrane and the apomembrane have the same densit) (Oesterhelt eb al., 1974) the refractive indices of the 2 layers are expected to be very similar. The reflection coefficient of a layer is different for the 2 states of polarization and depends on the refractive index and on G(.If a significant mismatch in refractive index were present. 0110 would expect to observe an apparent dichroism in regions where no absorption occurs. Above 650 nm the dichroism goes to zero however (see Fig. 4). By keeping c( idenbical in sample and reference compartment, and by using layers of the same thickness and conposit,ion on both quartz plates, the reflection problem could be solved, since well-orierrt’efl layers of large thickness could be prepared. With samples of opt,ical density O-6 at 568 1~. the error in the absorbance due to differential reflect,ion of bhe 2 polarization stat)rls uxs less than 1 “/o for a = 30”. ‘I’lrt~ anomalous dispersiou within the 568 nm absorption band leads to a small diff‘erenct, in refractive index between the apomembrane and thr purple membrane in t,his wavesl(,ngt,ll region. Thr dichroism of this band leads to a ~~avelnngtlI-dependent hirefringf~rrr~~ whictl could in turn, via t,he difference in reflection coefficient, cause an apparelIt wa\~lrngt,h dependence of the dichroic ratio. Such birefringence with the overall shape of thcl anomalous dispersion curve has recently been observed in the related system of orientrltl rod cjuter segments of Rana piypielzs, in which the retinal transition dipole moment is oriented almost parallel to the membrane (Liebman et al., 1974). The maximum birrfrirr. genccl difference across the retinal absorption band was about 0.001. A similar value cat) be estimated for the present system from the absorption spectra using dispersion relations. \I-e conclude therefore that anomalous dispersion effects can be neglected. All measurements uTere made with bacteriorhodopsin in the light-adapted state. itI wlricll the chromophore is in the all-tram form.
3. Results (a) Determination of 8 from transient dichroism measurements with reconstituted vesicles (i) Principles
of the method
ideas of the method as applied to the present system can be understood Excitation of bacteriorhodopsin results in a negative absorbance change at 568 nm (ground-state depletion) due to excited molecules entering the photochemical cycle. By means of a short (2 ps) flash of linearly polarized light an anisotropic distribution of 568 nm transition dipole moments is photoselected from the initial isotropic distribution present in the vesicle suspension. Hence the ground-stabe depletion signal observed immediately after excitation is highly dichroic. The absorbance change decays in a few milliseconds, the lifetime of the 412 nm intermediate. If rotational motion allows complete randomization of the 568 nm transition dipole The
basic
as follows.
612
M. P. HEYN,
R. J. CHERRY
AND
TJ. MijLLER
moment directions within this time, then the anisotropy of the transient absorbance will decay to zero. If, however, rotational motion is restricted, no complete randomization can occur and the anisotropy will attain a constant non-zero value. Intuitively it is clear that the amplitude of the residual anisotropy depends on the kind of motional freedom allowed. It will be shown that this amplitude depends on the angle B (Fig. 1) if we assume that the bacteriorhodopsin molecules incorporated into lipid vesicles are restricted to rotational motion around the normal to the plane of the membrane and are unable to tumble across the membrane. A general theoretical treatment of protein rotational diffusion in membranes which takes into account both the anisotropy of the protein and of the membrane has so far not been presented. However, as pointed out previously (Cherry et al., 1976), it is probably a reasonable approximation to suppose that anisotropic effects are dominated by the anisotropy of the membrane itself. Rotational motion may then be characterized by two diffusion coefficients, D,, for rotation about an axis normal to the membrane and D, for rotation about axes lying in the plane of the membrane (Fig. 1). With this simplification, the description of the rotational motion of the protein becomes formally equivalent to that of a body possessing an axis of symmetry immersed in an isotropic solution. Existing theoretical treatments (Ehrenberg & Rigler, 1972 ; Chuang & Eisenthal, 1972) then give the following expression for the time dependence of the anisotropy parameter r of the flash-induced transient dichroism : r(t)
= i
Ai emEli,
(1)
i=l
where
A, = (6/5) (sin2 0 cos2 0); A, = (3/10) (sin4 0); A, = (l/10) (3 cos2 8 -1)2; E,=(5D,+D,,);E2=(2D,+4D,,);E,=6D,,
0 is the angle between the transition moment for absorption and the normal to the plane of the membrane (Fig. 1). It is assumed that the same transition moment is used for excitation and measurement. Now suppose that the ra,te at which the protein can tumble across the membrane is negligibly small, i.e. D, m 0. Then equation (1) simplifies to give r(t)
= A, eeDllt + A, em4Qt + A,.
(2)
The above equation predicts that r does not fall to zero, but after an initial decay reaches a time-independent value determined by the coe&ient A,. A curve of this general shape has been experimentally observed with band 3 proteins in the human erythrocyte membrane (Cherry et al., 1976), with bacteriorhodopsin in the cell membrane of H. halo&m under conditions where the usual crystalline lattice is not formed (Cherry et al., 1977; Heyn et al., 1977) and with bacteriorhodopsin incorporated into lipid membranes (Cherry et al., 19773). The conclusion that the shape of the curve in these cases is due to restriction of rotation to a single axis is supported by arguments based on membrane structure which also indicate that many and possibly all integral membrane proteins are unable to tumble across the membrane (Bretscher, 1973; Singer, 1974). Now let m be the ratio of the time-independent component A, to r0 (the value of r at zero time). From equation (2) ??h=: A,/?-, = A&i,
$ A, + A,) = (l/4) (3 COS2
e -
1)2,
(3)
RETINAL
ORIENTATION
IN
BACTERIORHOUOPSIN
6I3
i.e.
Hence determination of m enables 8 to be calculated. It should be noted that in practice measured values of r are smaller than theoretical predictions because of instrumental effects. These may be taken into account by multiplying the right-hand side of equation (1) or equation (2) by a constant factor. This factor does not appear in equation (3) because it is the ratio of the coefficients which is of importance.
(ii) Time dependence of the anisotropy for bacteriorhodopsin phosphatidylcholine
incorporated
into
vesicles
Figure 3 shows typical plots of r against time obtained with bacteriorhodopsin incorporated into dipalmitoylphosphatidylcholine vesicles. At 45”C, above the phase transition of the lipids, r reaches a time-independent value after an initial rapid decay. As discussed in detail elsewhere (Cherry et al., 1977a; Cherry et al., manuscript in preparation), this fast decay is attributed to the rotat,ional motion of bacteriorhodopsin in the membra.ne. Since the diameter of the vesicles is of the order of 0.4 pm. rotational motion of the whole vesicles would lead to a decay characterized by a rotational correlation time of the order of 10 ms. In order to determine m, we need to measure both r0 and the time-independent value of r. The latter can be read direct’ly from the experimental curve but the determination of r0 is not completely straightforward. We have used two methods to determine rO. In the first method we obt,ain r,, by extrapolating the experimental curve back to zero time. The disadvantage of this n&hod is that at short times the transient absorbance change is a composite signal.
24°C
45oc
0.02
-
I
I
I
0.5
I.0
IL5
I
2-o
1
2.5
Time hs)
FIG. 3. Time dependenceof the enisotropy
r of the 668 nm depletion signal for beoteriorhodopsin incorporated in dipdmitoylphosphatidylcholine vesicles, measured above and below the lipid phase transition (protein to phospholipid ratio, 0.81: 1; 0.1 M-sodium acetate buffer, pH 6.0).
614
M.
P. HEYN,
R. J. CHERRY
AND
U. MijLLER
At room temperature the 568 nm depletion signal is distorted for times up to about 650positive transient (Lozier et al., 1975; Lozier & Nieder50 ps by the overlapping LLA berger, 1977). As pointed out by Rigler & Ehrenberg (1973), r(t) can no longer be assumed to be independent of the signal lifetime when more than one component is involved. Indeed, we have observed variations in r of up to 5% during the first 50 ps of signals obtained from purple membranes (Cherry, unpublished observations), although bacteriorhodopsin is certainly immobilized in this case. Lozier t Niederberger (1977) have also detected a similar effect with purple membranes at 620 nm and 5°C. Because these factors introduce some uncertainty in the extrapolation method, we have also used an alternative method of determining rO, which is based on the observation that bacteriorhodopsin is immobilized when the vesicles are cooled below the phase transition of the lipids (Cherry et al., manuscript in preparation). Circular dichroism, X-ray diffraction and electron microscopy evidence indicate that the hexagonal lattice is reformed under these conditions. Thus below the lipid phase transition r does not decay from its initial value of T,, and is therefore easily measured (Fig. 3). Hence m can be obtained from measurements made with the same sample both above and below the lipid phase transition. When determining m in this way, it is necessary to be careful that the fraction of bacteriorhodopsin molecules excited is the same at the two temperatures, since this fraction is sufficiently high in our experiments for r,, to vary with flash intensity. Within the experimental accuracy, we obtain the same value of m using either of t#hetwo methods outlined above. We also find that m is the same for bacteriorhodopsin
t
0.11,
,
450
500
, 550 Wavelength
,
,
600
650
(nm)
FIG. 4. Linear dichroiem of oriented purple membranes in the 568 nm absorption band. CL,the angle of tilt in Fig. 2, is 30’. In the lower curve (. . .) the state of polarization is horizontal with respect to the plane of Fig. 2, in the upper curve (---) the polarization is vertical.
RETINAL
ORIENTATION
IN
BACTERIORHODOPSIN
615
incorporated into either dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine vesicles. The value which we obtain is m = 0*19&0.03. Substituting this value in equation (4) then gives 0 = 78” &- 3” if we take the negative sign in front of the square root. If we take the positive sign we obtain the alternative solution 0 --= 38” & 1.5” which may reasonably be rejected since it is totally incompatible wit,h bot,h the present, and previous static linear dichroism measurements (Blaurock & Stoeckenius, 1971). (b) Determination
of 0 from the linear dichroism
of oriented purple
membranes
A typical linear dichroism spectrum of the light-adapted oriented purple membrane layers, reproduced directly from the recorder paper, is shown in Figure 4. The minima and maxima in absorbance are connected together to give the absorption spectra for the two polarization states. The photochemical cycle is still functional in the driedout layered samples. The cycle is considerably slowed down, however. Light-dark adaptation can also be observed with these oriented samples. The dichroic ratio D, defined as the ratio of the absorbance for horizontally and vertically polarized light, can be calculated for each value of a from the spectra. Figure 5 shons the dependence of D - 1 on cos”a at 568 nm. The experimental points lie on a straight line going through the origin. At normal incidence (a = n/2), no linear dichroism was observed. This lack of dichroism shows, as expected, that the transition dipole moments are randomly oriented around the normal to the plane of the membranes. For such axially symmetric samples an expression for the dependence of D - 1 on cos2a was previously derived for the case of non-interacting chromophores (Cherry et al., 1972). Rearranging their equation and correcting for refraction at the air/layer interface, we obtain the following expression 1 D-1=-(2cot20-l)cos%, n2
(5)
-0.4
D-l
-0.2
FIQ. 5. Dependence tilt a.
of the dichroic
ratio at 568 nm on the square of the cosine of the an.@ of
616
M. P. HEYN,
R. J. CHERRY
AND
U. MifLLER
where 0 is the angle between the 568 nm transition dipole moment and the normal t,o the plane of the membrane (Fig. 1) and n is the refractive index of the oriented purple membrane layer. In the presence of chromophore-chromophore interactions (exciton coupling) equation (5) needs to be modified. We will assume, as was done in all previous discussions of exciton effects in the purple membrane, that exciton coupling occurs amongst bacteriorhodopsin trimers within the hexagonal lattice (Bauer et al., 1976; Kriebel & Albrecht, 1976; Ebrey et al., 1977). This assumption appears to be justified since the lattice is built up of trimers with inter-trimer distances larger than intratrimer distances, and since exciton coupling was also observed when no lattice is present (Cherry et al., 1977a). With exciton coupling between the three chromophores of the trimer, the transition dipole moments belonging to the three new stationary states are linear combinations of the old ones and have directions which differ from those of the uncoupled chromophores. Due to the coupling the threefold degenerate level splits into two levels. One level is twofold degenerate with transition dipole moments in the plane of the membrane. The other level is non-degenerate and its transition dipole moment is perpendicular to the plane of the membrane. Since the two split levels have their transition dipole moments parallel (0 = 90”) and perpendicular (6 = 0’) to the plane of the membrane, one may expect a considerable variation of D - 1 with wavelength. Let X- be the wavelength of the absorption maximum for the twofold degenerate level and let X, be the corresponding quantity for the nondegenerate level. The exciton splitting Ah is then defined by AX = h- - X,. The average wavelength x is defined by 1 = (h, + X-)/2. Assuming Gaussian band shapes for the two split bands with the same bandwidth A, it can be shown (Heyn, unpublished results) that in this case the dichroism at wavelength A is given by 1 D - 1 = _ (2 cot2 (j e (CIA/d) (‘;i-l)/d _ 1) ~082c(, n2 Equation (6) reduces to (5) in the limit Ah/A + 0. The observed linear dependence of D - 1 on cos2 u at a fixed wavelength is thus in accordance with theoretical predictions whether exciton coupling occurs or not. According to equation (6) the slope of the straight line of Figure 5 contains information on the chromophore orientation and on the chromophore-chromophore distances, since it depends on cot2 8 and on the exciton splitting AX. Ah is itself a function of 0, of the chromophore-chromophore distance in the trimer and of a third geometrical variable. In principle both 0 and Ah can be determined if, experimentally, D depends on h. Figure 6 shows, however, that for two representative values of u, D is, within experimental accuracy, independent of h over approximately one bandwidth on either side of 568 nm. One way to reconcile the wavelength independence of D - 1 with a large value for AX is to assume that 0 w 90” so that cota 8 w 0. This case can be excluded however not only on the basis of the large exciton circular dichroism amplitude (Heyn et al., 1975) and the present transient dichroism measurements but also on the basis of the linear dichroism data themselves. With cot2 0 PV0, the slope of Figure 5 is according to equation (6) equal to -l/n2. Since experimentally the slope equals -0.335, the refractive index of the sample would have to assume the unrealistically high value of 1.73. In view of the wavelength independence of the linear dichroism the only other
RETINAL
ORIENTATION
I.0
I
I
I
IN
BACTERIORHODOPSTN I
I
I
I
I
T
I L
61 i
I
~~
II-
0.9
--~---p---p--s--B--~---~--~---~---~--
x. 0
. '4.
04
'\
'\ '-.
T
.
T r
!
. Z-
y
@ *-.
T f
r
-.-.-. -.-.
-.-._
0.7
0.6
I
520
I
I
540
I
L
I
I
580 560 Wovelengthkm)
t
I
600
I
620
FIG. 6. Wavelength dependence of the dichroic ratio L). (0) c( = 30”; (0) CL= 60”. wavelength-independent dichroic ratio is indicated by the horizontal lines (---, a : 30”; ----, 60”). For other values of OLsimilar results are obtained. The predicted wavelength dependence AX -: 20 nm and 0 = 71.2” is shown for G(= 30” (-.-.). For smaller values of o! the deviation the observed wavelength independence is larger than at 30”.
The c( with with
possible interpretation is that Ah/A
117, 607-620
Transient and Linear Dichroism Studies on Bacteriorhodopsin : Determination of the Orientation of the 568 nm All-trans Retinal Chromophore M.P. HEYN Department
of Biophysical Chemistry, Biozentrum CH-4056 Basel. Switzerland
It. J. CHERRY AND U.
M~~LLER
Laboratorium fiir Biochemie, ETH Zentrum. CH-8092 Ziirich, Switzerland (Received 8 July 1977) The orientation of the 568 nm transition dipole moment of the retinal chromophore of bacteriorhodopsin has been determined in purple membranes from Halobacterium halobium and in reconstituted vesicles. The angle between the 568 nm transition dipole moment and the normal to the plane of the membrane was measured in two different ways. In the first method the angle was obtained from transient dichroism measurements on bacteriorhodopsin incorporated into large phosphatidylcholine vesicles. Following flash excitation with linearly polarized light, the anisotropy of the 568 nm ground-state depletion signal first decays but then reaches a timeindependent value. This result, obtained above the lipid phase transition, is interpreted as arising from rotational motion of bacteriorhodopsin which is confined to an axis normal to the plane of the membrane. It is shown that the relative amplitude of the time-independent component depends on the orientation of the 568 nm transition dipole moment. From the data an angle of 78” + 3” is determined. In the second method the linear dichroism was measured as a function of the angle of tilt between the oriented purple membranes and the direction of tile light beam. The results were corrected for the angular distribution of the membranes within the oriented samples, which was determined from the mosaic spread of the first-order lamellar neutron diffraction peak. In substantial agreement with the results of the transient, dichroism method, linear dichroism measurements on oriented samples lead to an angle of 7 1’ h 4”. No significant wavelength dependence of the dichroic ratio across the 568 nm band was observed, implying that the exciton splitting in this band must be substa.ntially smaller tha.n the recently suggest,ed value of 20 nm (Ebrey et al., 1977). The orientation of the 568 nm transition dipole moment, which coincides wit11 the direction of the all-kalzs polyene chain of retinal, is not only of interest in connection with models for the proton pump, but can also be used to calculate the inter-chromophore distances in the purple membrane.
1. Introduction in the purple membrane of Halobacterium
Bacteriorhodopsin a light-driven rhodopsin
proton absorbs
pump. at
568
In the light-adapted nm.
When
light
GO7
state is absorbed
halobium
the chromophore a photochemical
functions
as
of bacteriocycle
is
608
M. P. HEYN,
R. J. CHERRY
AND
U. MULLER
initiated which has been investigated in great detail. In this cycle and probably in the proton transport as well, the retinal chromophore which is bound to the protein moiety (bacteria-opsin) eia a Schiff base linkage plays a central role (for recent reviews see Oesterhelt, 1976; Henderson, 1977). Little is known about the position and orientation of the chromophore within the protein. In formulating possible models for the mechanism of the proton pump, it is desirable to have information available on the spatial localization and orientation of the chromophore. In the structural investigations using X-ray diffraction and electron microscopy, the chromophore has not yet been resolved (Blaurock & Stoeckenius, 1971; Henderson, 1975; Blaurock, 1975; Unwin & Henderson, 1975; Henderson & Unwin, 1975). The bacteriorhodopsin molecules are arranged within the membrane in a rigid hexagonal lattice. Within this lattice the protein is immobilized (Razi Naqvi et al., 1973). Since a tight and highly specific retinal-protein complex is formed, we will assume that the chromophore has a fixed position and orientation with respect to the protein moiety. This assumption is supported by experimental evidence which will be discussed later. A well-defined angle 0 thus exists between the direction of the chromophore transition dipole moment and the normal to the plane of the membrane (Fig. 1). In the light-adapted state retinal is in the all-trans form (Oesterhelt et al., 1973) and its transition dipole moment coincides with that of the all-trans polyene chain. Previous measurements showed that this direction is preferentially parallel to the plane of the membrane (Blaurock & Stoeckenius, 1971). It was first pointed out on the basis of circular dichroism measurements that the 568 nm transition dipole moment cannot be exactly in the plane of the membrane (Heyn et al., 1975). Direct evidence for exciton interactions between the 568 nm transition dipole moments of adjacent bacteriorhodopsin molecules has been obtained from circular dichroism spectra (Heyn et al., 1975; Bauer et aE., 1976; Becher & Ebrey, 1976). Knowledge of the chromophore orientation is important for the interpretation of the exciton effects, since in combination with circular dichroism and absorption measurements it enables the chromophore-chromophore distance to be calculated. The wavelength dependence of the linear dichroism of oriented purple membranes is of particular significance in connection with the magnitude of the exciton splitting. In this paper we report the determination of 0 using two different methods : transient dichroism of bacteriorhodopsin incorporated into large phosphatidylcholine vesicles and linear dichroism of oriented purple membranes. The method, described in this paper, of determining the orientation of a membrane-bound chromophore from the decay of the anisotropy of the flash-induced transient absorption is new and may find application in other membrane systems. It is therefore of additional interest to test the validity of this approach by comparing its results with those obtained by means of linear dichroism. Some preliminary results of this investigation and related linear dichroism studies have recently been briefly reported (Heyn & Cherry, 1977; Bogomolni et al., 1977).
2. Materials and Methods (a) Incorporation
of bacteriorhodopsin
into lipid
vesicles
Purple membranes were isolated from H. halobiuna (strain RIM,) as described by Oesterhelt & Stoeckenius (1974). The membranes were solubilized by suspending 1 mg in 4 ml of O-1 M-acetate buffer (pH 5.0) containing 0.1% Triton Xl00 for 24 to 30 h in the dark at room temperature. Membrane reconstitution followed a method proposed by
RETINAL
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Henderson (1977). After adding the requisite quantity (1 to 5 mg) of phosphatidylcholine (either dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine), Triton was buffer (pH 5.0) containing O.OZ’$/b removed by prolonged dialysis against 0.1 M-a.W3tate sodium azide. %ollowing dialysis, the sample was purified by centrifugation through a sucrose density gradient (4.5% to 40%) to remove any non-recombined lipid and protein. The method yields predominantly unilamellar lipid-protein vesicles with diameters t,ypically in the range 0.3 to 0.5 pm. Further details of the procedure will be presented in preparation). Protein was determined by tilt> c!lsewhere (Cherry et al., manuscript method of Lowry el al. (1951) and lipid by phosphorus analysis (Chen et al., 1956). Proteill : pllospholipid ratios (w/w) in the reconstituted membranes used in this st,udy were iu t,hc rangt~ 0.6 to 1.7. (b) Preparation of oriented membrane samples Suspensions of purple membranes in 0.2 mM-acetate buffer (pH 4.7) were dried on a flat quartz plate in a desiccator in the presence of silica gel. Under these conditions tllc rigid flat membrane patches, which have an average diameter of 0.5 Pm, orient themselves parallel to the quartz plate by stacking. Well-oriented purple membrane samples havcl been previously prepared in a similar way for X-ray diffraction measurements (Blaurock & Stoeckenius, 1971; Blaurock, 1975; Henderson, 1975). The angular distribution of t,hr orientation of individual membrane stacks within the sample with respect to the plane of the quartz support was determined from the width of the mosaic spread of the first-order lamellar neutron diffraction peak (Worcester & Franks, 1976). 2H,0 was used to enhance tlrr contrast. For the thickest layers used in the linear dichroism measurements (0.1). 0.6 at 568 nm), the half-width of the angular distribution at half-height w&s 7.5”. The Bragg spaciug of the membrane system varied from 49.2 if for completely dried out samples to 70.1 -4 for samples in a saturated 2H,0 atmosphere. Over this range of hydration tlrtb diffraction measurements \V~IY. change in the mosaic spread was only I”. The neutron performed at the Institut Lane-Langevin (Grenoble). (c) Transient
dichroism
measurements
The flash photolysis apparatus used in these experiments is described elsewhere (CllerrJ& Schneider, 1976). The application of the method to the investigation of rotational diffusion of bacteriorhodopsin has also been reported (Cherry et al., 1977a; Razi Naqvi et al., 1973; Heyn et al., 1977). In brief, ground-state depletion of the 568 nm absorption band is detected following laser flash excitation at 540 nm. Transient absorbance changes (A,,(t), A.,(t)) at time t after the flash are simultaneously measured for light polarized parallel and perpendicular with respect to the polarization of the exciting flash. The allisotropy parameter r(t) is calculated from the expression r(t)
=
A,,@)-- A,(t) + 2 A,(t) .
4(t)
In all measurements bacteriorhodopsin the source of the measuring beam. (d) L&ear
was light-adapted
dichroism
by the tungsten
lamp
used as
measurements
Linear dichroism measurements on oriented purple membrane samples were performed with a modified Rehovoth Instruments CD-HC attachment designed for use with the Cary 15 spectrophotometer (Jaffe et al., 1967). The optical system is shown schematically in Fig. 2. The horizontal plane is defined a~ the plane of Fig. 2. The undeviated horizontally polarized beam produced by a Rochon prism is incident upon a waveplste of quartz set, with its optic axis at 45” to the horizontal plane. The vertically polarized extraordinary ray is blocked by a knife-edge. The precise state of polarization of the light emerging from the waveplate depends on the thickness of the plate (d), the wavelength (A) and the birefringence of quartz (An(h)). The relative retardation produced by the waveplate is given by 2nddn(X) A
*
610
M. P. HEYN,
R. J. CHERRY
FIG. 1. Sche~tic ~pre~~tation of the plane of the membrane. The 2 axes labelled by D,, is perpendicular to the to the plane of the membrane and the
‘c’. HOLLER
the orientation of b8c~riorh~opsin (BR) with respect to labelled with D, lie in the plane of the membr8ne. The 8xis plane of the membrctne. 0 is the angle between the normal direction of the 668 nm transition dipole moment,
Oriented metnbrones an quartz support
Quartz waveplate with optic axis set at 45”
\
Polar ixer (Rochon prism)
AND
I
Depolarizer
Knife edge Goniometer angle a variobke
3%. 2. Schematic diagram of the optical system principle of operstion is described in the text. The black patch on the quartz support. The plane of the plane of the Figure. In the reference beam an instead of purple membranes.
used for linear dichroism measurements. The oriented membranes 8re represented by the the oriented membr8nes is perpendioular to identical unit ~8s used with apomembranes
Since the retardation is a function of h, and since the birefringence of quartz is of the order of 0.01, the retardation will ohange within 8 few nm by an amount of the order of V, if the waveplate is sufficiently thick. When the retardation equals an even multiple of x the emerging beam will be horizontally polerized, when it equals an odd multiple of GTthe beam will be vertically polarized. Thus 88 the wavelength region is scanned the state of polarization of the be8m alternates between horizont&l and vertio8L If 8 line&rly dichroic sample is put in the beam after the waveplate, one records at those wavelengths where the be8m is horizontally polarized the absorbance in that st&e of polarization. At those wavelengths where the beam is vertically polarized, one records likewise the absorbance for vertically polarized light. In this w8y an absorption spectrum is obtained with superimposed oscillations due to the linear dichroism. Connecting the points belonging to the
RETINAL
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611
same state of polarization by interpolation, we obtain directly the 2 absorption spectra. The linear dichroism is thus obtained from a single scan. The advantages of this method are that no polarizers need to be moved and that the beam passes for both states of polarization through the same area of the possibly inhomogeneous layer of orient’& membranes. The attachment was modified in 2 ways. An extra 4 mm thick quartz waveplate was added for increased resolution in the visible region. The cell-holder was replaced by a goniometer which allowed the variable angle CLbetween the plane of the oriented membranes and the direction of the incident light beam to be accurately determined (Fig. 2). An identical unit was used in the reference beam. A layer of oriented chromophore-fret> apomembranes of the same thickness was used as a reference (Oesterhelt et al., 1974). Matching in terms of the same number of bacteriorhodopsin molecules per unit> area was verified by comparing the near ultraviolet absorption spectra of the oriented sample nrhd reference. Since the purple membrane and the apomembrane have the same densit) (Oesterhelt eb al., 1974) the refractive indices of the 2 layers are expected to be very similar. The reflection coefficient of a layer is different for the 2 states of polarization and depends on the refractive index and on G(.If a significant mismatch in refractive index were present. 0110 would expect to observe an apparent dichroism in regions where no absorption occurs. Above 650 nm the dichroism goes to zero however (see Fig. 4). By keeping c( idenbical in sample and reference compartment, and by using layers of the same thickness and conposit,ion on both quartz plates, the reflection problem could be solved, since well-orierrt’efl layers of large thickness could be prepared. With samples of opt,ical density O-6 at 568 1~. the error in the absorbance due to differential reflect,ion of bhe 2 polarization stat)rls uxs less than 1 “/o for a = 30”. ‘I’lrt~ anomalous dispersiou within the 568 nm absorption band leads to a small diff‘erenct, in refractive index between the apomembrane and thr purple membrane in t,his wavesl(,ngt,ll region. Thr dichroism of this band leads to a ~~avelnngtlI-dependent hirefringf~rrr~~ whictl could in turn, via t,he difference in reflection coefficient, cause an apparelIt wa\~lrngt,h dependence of the dichroic ratio. Such birefringence with the overall shape of thcl anomalous dispersion curve has recently been observed in the related system of orientrltl rod cjuter segments of Rana piypielzs, in which the retinal transition dipole moment is oriented almost parallel to the membrane (Liebman et al., 1974). The maximum birrfrirr. genccl difference across the retinal absorption band was about 0.001. A similar value cat) be estimated for the present system from the absorption spectra using dispersion relations. \I-e conclude therefore that anomalous dispersion effects can be neglected. All measurements uTere made with bacteriorhodopsin in the light-adapted state. itI wlricll the chromophore is in the all-tram form.
3. Results (a) Determination of 8 from transient dichroism measurements with reconstituted vesicles (i) Principles
of the method
ideas of the method as applied to the present system can be understood Excitation of bacteriorhodopsin results in a negative absorbance change at 568 nm (ground-state depletion) due to excited molecules entering the photochemical cycle. By means of a short (2 ps) flash of linearly polarized light an anisotropic distribution of 568 nm transition dipole moments is photoselected from the initial isotropic distribution present in the vesicle suspension. Hence the ground-stabe depletion signal observed immediately after excitation is highly dichroic. The absorbance change decays in a few milliseconds, the lifetime of the 412 nm intermediate. If rotational motion allows complete randomization of the 568 nm transition dipole The
basic
as follows.
612
M. P. HEYN,
R. J. CHERRY
AND
TJ. MijLLER
moment directions within this time, then the anisotropy of the transient absorbance will decay to zero. If, however, rotational motion is restricted, no complete randomization can occur and the anisotropy will attain a constant non-zero value. Intuitively it is clear that the amplitude of the residual anisotropy depends on the kind of motional freedom allowed. It will be shown that this amplitude depends on the angle B (Fig. 1) if we assume that the bacteriorhodopsin molecules incorporated into lipid vesicles are restricted to rotational motion around the normal to the plane of the membrane and are unable to tumble across the membrane. A general theoretical treatment of protein rotational diffusion in membranes which takes into account both the anisotropy of the protein and of the membrane has so far not been presented. However, as pointed out previously (Cherry et al., 1976), it is probably a reasonable approximation to suppose that anisotropic effects are dominated by the anisotropy of the membrane itself. Rotational motion may then be characterized by two diffusion coefficients, D,, for rotation about an axis normal to the membrane and D, for rotation about axes lying in the plane of the membrane (Fig. 1). With this simplification, the description of the rotational motion of the protein becomes formally equivalent to that of a body possessing an axis of symmetry immersed in an isotropic solution. Existing theoretical treatments (Ehrenberg & Rigler, 1972 ; Chuang & Eisenthal, 1972) then give the following expression for the time dependence of the anisotropy parameter r of the flash-induced transient dichroism : r(t)
= i
Ai emEli,
(1)
i=l
where
A, = (6/5) (sin2 0 cos2 0); A, = (3/10) (sin4 0); A, = (l/10) (3 cos2 8 -1)2; E,=(5D,+D,,);E2=(2D,+4D,,);E,=6D,,
0 is the angle between the transition moment for absorption and the normal to the plane of the membrane (Fig. 1). It is assumed that the same transition moment is used for excitation and measurement. Now suppose that the ra,te at which the protein can tumble across the membrane is negligibly small, i.e. D, m 0. Then equation (1) simplifies to give r(t)
= A, eeDllt + A, em4Qt + A,.
(2)
The above equation predicts that r does not fall to zero, but after an initial decay reaches a time-independent value determined by the coe&ient A,. A curve of this general shape has been experimentally observed with band 3 proteins in the human erythrocyte membrane (Cherry et al., 1976), with bacteriorhodopsin in the cell membrane of H. halo&m under conditions where the usual crystalline lattice is not formed (Cherry et al., 1977; Heyn et al., 1977) and with bacteriorhodopsin incorporated into lipid membranes (Cherry et al., 19773). The conclusion that the shape of the curve in these cases is due to restriction of rotation to a single axis is supported by arguments based on membrane structure which also indicate that many and possibly all integral membrane proteins are unable to tumble across the membrane (Bretscher, 1973; Singer, 1974). Now let m be the ratio of the time-independent component A, to r0 (the value of r at zero time). From equation (2) ??h=: A,/?-, = A&i,
$ A, + A,) = (l/4) (3 COS2
e -
1)2,
(3)
RETINAL
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6I3
i.e.
Hence determination of m enables 8 to be calculated. It should be noted that in practice measured values of r are smaller than theoretical predictions because of instrumental effects. These may be taken into account by multiplying the right-hand side of equation (1) or equation (2) by a constant factor. This factor does not appear in equation (3) because it is the ratio of the coefficients which is of importance.
(ii) Time dependence of the anisotropy for bacteriorhodopsin phosphatidylcholine
incorporated
into
vesicles
Figure 3 shows typical plots of r against time obtained with bacteriorhodopsin incorporated into dipalmitoylphosphatidylcholine vesicles. At 45”C, above the phase transition of the lipids, r reaches a time-independent value after an initial rapid decay. As discussed in detail elsewhere (Cherry et al., 1977a; Cherry et al., manuscript in preparation), this fast decay is attributed to the rotat,ional motion of bacteriorhodopsin in the membra.ne. Since the diameter of the vesicles is of the order of 0.4 pm. rotational motion of the whole vesicles would lead to a decay characterized by a rotational correlation time of the order of 10 ms. In order to determine m, we need to measure both r0 and the time-independent value of r. The latter can be read direct’ly from the experimental curve but the determination of r0 is not completely straightforward. We have used two methods to determine rO. In the first method we obt,ain r,, by extrapolating the experimental curve back to zero time. The disadvantage of this n&hod is that at short times the transient absorbance change is a composite signal.
24°C
45oc
0.02
-
I
I
I
0.5
I.0
IL5
I
2-o
1
2.5
Time hs)
FIG. 3. Time dependenceof the enisotropy
r of the 668 nm depletion signal for beoteriorhodopsin incorporated in dipdmitoylphosphatidylcholine vesicles, measured above and below the lipid phase transition (protein to phospholipid ratio, 0.81: 1; 0.1 M-sodium acetate buffer, pH 6.0).
614
M.
P. HEYN,
R. J. CHERRY
AND
U. MijLLER
At room temperature the 568 nm depletion signal is distorted for times up to about 650positive transient (Lozier et al., 1975; Lozier & Nieder50 ps by the overlapping LLA berger, 1977). As pointed out by Rigler & Ehrenberg (1973), r(t) can no longer be assumed to be independent of the signal lifetime when more than one component is involved. Indeed, we have observed variations in r of up to 5% during the first 50 ps of signals obtained from purple membranes (Cherry, unpublished observations), although bacteriorhodopsin is certainly immobilized in this case. Lozier t Niederberger (1977) have also detected a similar effect with purple membranes at 620 nm and 5°C. Because these factors introduce some uncertainty in the extrapolation method, we have also used an alternative method of determining rO, which is based on the observation that bacteriorhodopsin is immobilized when the vesicles are cooled below the phase transition of the lipids (Cherry et al., manuscript in preparation). Circular dichroism, X-ray diffraction and electron microscopy evidence indicate that the hexagonal lattice is reformed under these conditions. Thus below the lipid phase transition r does not decay from its initial value of T,, and is therefore easily measured (Fig. 3). Hence m can be obtained from measurements made with the same sample both above and below the lipid phase transition. When determining m in this way, it is necessary to be careful that the fraction of bacteriorhodopsin molecules excited is the same at the two temperatures, since this fraction is sufficiently high in our experiments for r,, to vary with flash intensity. Within the experimental accuracy, we obtain the same value of m using either of t#hetwo methods outlined above. We also find that m is the same for bacteriorhodopsin
t
0.11,
,
450
500
, 550 Wavelength
,
,
600
650
(nm)
FIG. 4. Linear dichroiem of oriented purple membranes in the 568 nm absorption band. CL,the angle of tilt in Fig. 2, is 30’. In the lower curve (. . .) the state of polarization is horizontal with respect to the plane of Fig. 2, in the upper curve (---) the polarization is vertical.
RETINAL
ORIENTATION
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615
incorporated into either dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine vesicles. The value which we obtain is m = 0*19&0.03. Substituting this value in equation (4) then gives 0 = 78” &- 3” if we take the negative sign in front of the square root. If we take the positive sign we obtain the alternative solution 0 --= 38” & 1.5” which may reasonably be rejected since it is totally incompatible wit,h bot,h the present, and previous static linear dichroism measurements (Blaurock & Stoeckenius, 1971). (b) Determination
of 0 from the linear dichroism
of oriented purple
membranes
A typical linear dichroism spectrum of the light-adapted oriented purple membrane layers, reproduced directly from the recorder paper, is shown in Figure 4. The minima and maxima in absorbance are connected together to give the absorption spectra for the two polarization states. The photochemical cycle is still functional in the driedout layered samples. The cycle is considerably slowed down, however. Light-dark adaptation can also be observed with these oriented samples. The dichroic ratio D, defined as the ratio of the absorbance for horizontally and vertically polarized light, can be calculated for each value of a from the spectra. Figure 5 shons the dependence of D - 1 on cos”a at 568 nm. The experimental points lie on a straight line going through the origin. At normal incidence (a = n/2), no linear dichroism was observed. This lack of dichroism shows, as expected, that the transition dipole moments are randomly oriented around the normal to the plane of the membranes. For such axially symmetric samples an expression for the dependence of D - 1 on cos2a was previously derived for the case of non-interacting chromophores (Cherry et al., 1972). Rearranging their equation and correcting for refraction at the air/layer interface, we obtain the following expression 1 D-1=-(2cot20-l)cos%, n2
(5)
-0.4
D-l
-0.2
FIQ. 5. Dependence tilt a.
of the dichroic
ratio at 568 nm on the square of the cosine of the an.@ of
616
M. P. HEYN,
R. J. CHERRY
AND
U. MifLLER
where 0 is the angle between the 568 nm transition dipole moment and the normal t,o the plane of the membrane (Fig. 1) and n is the refractive index of the oriented purple membrane layer. In the presence of chromophore-chromophore interactions (exciton coupling) equation (5) needs to be modified. We will assume, as was done in all previous discussions of exciton effects in the purple membrane, that exciton coupling occurs amongst bacteriorhodopsin trimers within the hexagonal lattice (Bauer et al., 1976; Kriebel & Albrecht, 1976; Ebrey et al., 1977). This assumption appears to be justified since the lattice is built up of trimers with inter-trimer distances larger than intratrimer distances, and since exciton coupling was also observed when no lattice is present (Cherry et al., 1977a). With exciton coupling between the three chromophores of the trimer, the transition dipole moments belonging to the three new stationary states are linear combinations of the old ones and have directions which differ from those of the uncoupled chromophores. Due to the coupling the threefold degenerate level splits into two levels. One level is twofold degenerate with transition dipole moments in the plane of the membrane. The other level is non-degenerate and its transition dipole moment is perpendicular to the plane of the membrane. Since the two split levels have their transition dipole moments parallel (0 = 90”) and perpendicular (6 = 0’) to the plane of the membrane, one may expect a considerable variation of D - 1 with wavelength. Let X- be the wavelength of the absorption maximum for the twofold degenerate level and let X, be the corresponding quantity for the nondegenerate level. The exciton splitting Ah is then defined by AX = h- - X,. The average wavelength x is defined by 1 = (h, + X-)/2. Assuming Gaussian band shapes for the two split bands with the same bandwidth A, it can be shown (Heyn, unpublished results) that in this case the dichroism at wavelength A is given by 1 D - 1 = _ (2 cot2 (j e (CIA/d) (‘;i-l)/d _ 1) ~082c(, n2 Equation (6) reduces to (5) in the limit Ah/A + 0. The observed linear dependence of D - 1 on cos2 u at a fixed wavelength is thus in accordance with theoretical predictions whether exciton coupling occurs or not. According to equation (6) the slope of the straight line of Figure 5 contains information on the chromophore orientation and on the chromophore-chromophore distances, since it depends on cot2 8 and on the exciton splitting AX. Ah is itself a function of 0, of the chromophore-chromophore distance in the trimer and of a third geometrical variable. In principle both 0 and Ah can be determined if, experimentally, D depends on h. Figure 6 shows, however, that for two representative values of u, D is, within experimental accuracy, independent of h over approximately one bandwidth on either side of 568 nm. One way to reconcile the wavelength independence of D - 1 with a large value for AX is to assume that 0 w 90” so that cota 8 w 0. This case can be excluded however not only on the basis of the large exciton circular dichroism amplitude (Heyn et al., 1975) and the present transient dichroism measurements but also on the basis of the linear dichroism data themselves. With cot2 0 PV0, the slope of Figure 5 is according to equation (6) equal to -l/n2. Since experimentally the slope equals -0.335, the refractive index of the sample would have to assume the unrealistically high value of 1.73. In view of the wavelength independence of the linear dichroism the only other
RETINAL
ORIENTATION
I.0
I
I
I
IN
BACTERIORHODOPSTN I
I
I
I
I
T
I L
61 i
I
~~
II-
0.9
--~---p---p--s--B--~---~--~---~---~--
x. 0
. '4.
04
'\
'\ '-.
T
.
T r
!
. Z-
y
@ *-.
T f
r
-.-.-. -.-.
-.-._
0.7
0.6
I
520
I
I
540
I
L
I
I
580 560 Wovelengthkm)
t
I
600
I
620
FIG. 6. Wavelength dependence of the dichroic ratio L). (0) c( = 30”; (0) CL= 60”. wavelength-independent dichroic ratio is indicated by the horizontal lines (---, a : 30”; ----, 60”). For other values of OLsimilar results are obtained. The predicted wavelength dependence AX -: 20 nm and 0 = 71.2” is shown for G(= 30” (-.-.). For smaller values of o! the deviation the observed wavelength independence is larger than at 30”.
The c( with with
possible interpretation is that Ah/A
