J. Phys. Chem. 1996, 100, 15605-15613
15605
Chromophore Reorientation Relative to the Membrane Plane Detected by Time-Resolved Linear Dichroism during the Bacteriorhodopsin Photocycle in Oriented Purple Membrane Qin Song, Greg S. Harms, and Carey K. Johnson*,† Department of Chemistry, UniVersity of Kansas, Lawrence, Kansas 66045 ReceiVed: April 3, 1996; In Final Form: June 27, 1996X
Time-resolved linear dichroism measurements on samples of oriented purple membranes enable reorientations in the plane of the membrane to be distinguished from reorientations with respect to the membrane normal. Our measurements reveal reorientation of the transition dipole of the retinylidene chromophore toward the membrane normal by 14° in the M-state. A smaller reorientation (3°) in the same direction is observed in the L-state and possibly as early as the K-state. The purple membrane was oriented in an 11 T magnetic field and immobilized in a polyacrylamide gel. Time-resolved linear dichroism measurements on purple membrane oriented so that pump and probe beams propagate parallel to the membrane plane are highly sensitive to reorientation with respect to the membrane normal due to the initial orientation of the transition dipole at about 67° with respect to the membrane normal. Measurements where pump and probe beams propagate perpendicular to the membrane plane reveal no significant in-plane reorientation, although the sensitivity of these measurements is not as high. The observed reorientation in the M-state may be related to conformational changes that have been detected in helices C, F, and G.
Introduction The protein bacteriorhodopsin (BR), located in the purple membrane of the halophilic bacterium Halobacterium salinarium, pumps protons across the membrane upon activation by light.1,2 Photoexcitation of the retinal Schiff base chromophore of BR triggers an isomerization from the all-trans to the 13-cis configuration, initiating a sequence of events known as the BR photocycle. Recent work has focused on the relationship of events in the BR photocycle to the structure of the protein, which has been characterized by electron cryomicroscopy.3 After isomerization, the K intermediate is generated in 3 ps and relaxes to form L in about 1 µs. During the L f M transition in about 50 µs the Schiff base proton is transferred to Asp-85,4 located on the extracellular side of the chromophore. Subsequently, in the M f N transition, the Schiff base is reprotonated from an aspartic acid residue, Asp-96, on the cytoplasmic side of the chromophore.5 The all-trans conformation of the chromophore is then restored with the formation of the O intermediate.6 This paper focuses primarily on the K f L f M steps of the photocycle leading to a deprotonated retinal Schiff base in the M intermediate, with the goal of determining the direction of reorientational motion with respect to the membrane plane. The proton-pumping function of BR raises the intriguing question of mechanism. How is proton transport driven from the cytoplasmic to the extracellular aside against a pH gradient? To address this question, it is very important to relate spectroscopically observable photocycle transitions to structural events in the protein. Structural changes in the protein have been observed in the M intermediate by several techniques, including neutron diffraction,7,8 electron diffraction,9 X-ray diffraction,10 and NMR.11 Structural changes in the F and G helices were observed by electron and X-ray diffraction.9,10 The neutron diffraction results show a tilting of the chromophore in the M † X
Email:
[email protected]. Abstract published in AdVance ACS Abstracts, September 1, 1996.
S0022-3654(96)01015-5 CCC: $12.00
intermediate with respect to the membrane normal of 11° ( 6°,8 while 2H-NMR measurements show a tilt in the C-CD3 bond angle at C19 of the retinal polyene chain.11 Reorientations of the chromophore transition dipole over the course of the photocycle have also been characterized by time-resolved linear dichroism measurements. Recently, we have observed chromophore reorientations both in purple-membrane suspensions as well as in nonoriented purple membrane immobilized in polyacrylamide gels.12,13 Reorientations were most pronounced in the M-state, whereas the initial orientation was substantially recovered in the O intermediate, which appears in the later stages of the photocycle. However, in other recently reported experiments no reorientations were detected in the early stages of the photocycle up to the formation of the M intermediate.14 The reason for the disparity in these results has not been resolved. More specific information about reorientations can be obtained in samples with oriented purple membrane.15,16,17 The use of oriented purple membrane has two advantages over an isotropic purple membrane suspension in measuring chromophore orientations during the photocycle. First, since the orientation of the purple membrane is specified with respect to the laboratory frame, reorientations of the chromophore in the purple membrane plane can be distinguished from reorientations out of the plane. Second, the anisotropy change in oriented bacteriorhodopsin is more sensitive to reorientation out of the plane than in an isotropic sample.15 For example, an angle change by more than 7° is required to alter the anisotropy by 0.01 (from 0.40 to 0.39) in an isotropic sample. In contrast, in oriented purple membrane, where the chromophore is oriented initially at about 67° from the membrane normal, a change in orientation of less than 1° results in a change in the anisotropy of 0.01. Isotropic purple membrane suspensions placed in a strong magnetic field have been shown to orient with the membrane plane perpendicular to the magnetic field direction.18 Orientational ordering is a result of the anisotropy of the diamagnetic © 1996 American Chemical Society
15606 J. Phys. Chem., Vol. 100, No. 38, 1996
Figure 1. Axis system describing the orientation of purple membranes and the chromophore relative to the laboratory axes (X, Y, Z). The membrane normal, N, is tilted by an angle β from the axis of orientation Z due to incomplete orientational ordering. µ0 and µi are the transition dipole moments of the ground-state and of photointermediate i, respectively, and ωi is the reorientation angle of the transition dipole in photointermediate i.
susceptibility of the purple membrane. In previous linear dichroism experiments carried out with oriented purple membrane,15 a change in the out-of plane orientation of only 3° was detected in the M-state. In this paper we present a study of the K, L, and M intermediates for two types of oriented samples: one where the pump and probe beams were parallel to the membrane plane and another where they were perpendicular to the membrane plane and parallel to the axis of orientation (see Figure 1). We have adopted an experimental setup with nearly collinear pump and probe beams, permitting measurement of both the out-of-plane and in-plane reorientation in the bacteriorhodopsin photocycle by utilization of results from both alignments of the membrane plane relative to the pump and probe beams. The in-plane and out-of-plane orientations of the K-state, L-state, and M-state have been measured. Utilization of collinear pump and probe beams also avoids artifacts that may arise with perpendicular beams.19 Materials and Methods Preparation of Oriented Purple Membrane. The purple membrane was suspended in a pH 7.0 phosphate buffer (10 mM) and mixed with 7% (w/v) acrylamide, 0.1% (w/v) bis(acrylamide), and 0.03% (w/v) tetramethylethylenediamine. Then, 0.2% (w/v) ammonium persulfate was added to catalyze the polymerization. The liquid was immediately poured into a sample cell and inserted into the 11 T magnetic field at the center of the cryomagnet of a 500 MHZ NMR instrument for at least 1 h. Sample cells with a path length of 5.0 mm were made of plexiglass with windows consisting of microscope cover slides sealed to the cell. The optical density of the sample was approximately 1 at 570 nm. Two kinds of oriented sample were prepared: (1) samples oriented with the purple membrane plane perpendicular to the sample cell window were prepared by placing the sample cell vertically in the magnetic field, (2) samples oriented with the purple membrane plane parallel to the sample cell window were prepared by placing the sample cell horizontally in the magnetic field. Experimental Setup. The time-resolved linear dichroism measurements were carried out in the same manner as timeresolved measurements of transient absorption, linear dichroism, and anisotropy in isotropic purple membrane suspensions and gels.12,13 The sample cell was mounted on a high-resolution rotation stage to allow precise orientation of the sample cell.
Song et al. The mounted sample cell was fixed to a sliding stage, which was translated back and forth horizontally during the experiments in order to prevent repetitive excitation of the same sample region by consecutive excitation pulses. Since samples with a vertical orientation axis (Figure 2, part B) exhibit birefringence, special care was taken to avoid contributions from birefringence to linear dichroism measurements by orienting the polarization of the pump beam precisely along the orientational axis of the sample. This alignment was carried out as follows. First, the analyzing polarizer was adjusted to be parallel ((0.2o) to the pump beam polarization by rotating it to achieve minimum intensity of the rejected pump beam. This defined the 0° or parallel direction. Then, the probe beam polarization was adjusted to be parallel to the pump beam polarization. After that, the sample cell was inserted and rotated to achieve extinction of the rejected probe beam. This resulted in the alignment of the orientational axis of the sample with the polarization of the pump beam. Finally, the polarization of the probe beam was rotated 45° to contain vertically and horizontally polarized components. After the sample cell, the vertical and horizontal polarizations of the probe were separated by the analyzing polarizer and detected as described previously.12,13 The laser system consisted of two synchronized mode-locked Q-switched Nd:YAG lasers.12,13 Pump pulses (100 ps pulse width) were selected from the mode-locked pulse train with a Pockels cell and frequency doubled to 532 nm. Pump pulse energies at 532 nm were less than 0.1 µJ with a 0.1-0.2 mm beam diameter. Probe pulses were generated by a cavitydumped dye laser pumped by the second Nd:YAG laser. The energy of the probe pulses was reduced to less than 0.02 µJ with neutral optical density filters. The pulse repetition rate was 30 Hz. Theoretical Formalism The transient absorption signal from the oriented purple membrane is
∆AIJ(t) ) 〈(eˆ I‚µˆ o(0))2(eˆ J‚µˆ i(t))2〉F(t)
(1)
where I is the pump pulse polarization and J the probe pulse polarization in the laboratory axis system (X, Y, Z in Figure 1), µˆ 0 is the unit vector in the direction of the pump transition dipole, µˆ i the unit vector for the probe transition dipole, F(t) contains the photocycle kinetics, and 〈‚‚‚〉 represents the average over all orientations. The projections of the transition dipole moments µ0 and µi onto the polarization directions I and J depend on the orientation of the molecular axis system (x, y, z) relative to the laboratory frame (X, Y, Z). These two axis systems can be related by the Euler angles Ωs ) (φs, Θs, χs). In order to perform the orientational average, it is convenient to transform the square of the transition dipole moment into a spherical tensor as follows: 2
(eˆ I‚µˆ o(0))2 ) ∑
L
L
(L)* U(II,Lq) D(q,m) (Ωs(0)) Am(L) ∑ ∑ L)0 q)-L m)-L
(2a)
2
(eˆ J‚µˆ i(t))2 ) ∑
L
L
(L)* (Ωs(t)) Am(L) ∑ ∑ U(JJ,Lq) Dq,m L)0 q)-L m)-L
(2b)
where U(II,Lq) is the unitary transformation from spherical
Chromophore Reorientation Detection
J. Phys. Chem., Vol. 100, No. 38, 1996 15607
(L) (Ωs) are the Wigner rotation to Cartesian tensors,20 Dq,m (L) h m(L) are the spherical tensor matrix elements, and Am and A components of the direct square of the pump and probe dipole moments, respectively.21 For simplicity, we choose the transition dipole along the molecular z-axis. Then m ) 0 in eq 2, and the transient absorption signal can be rewritten as
(L)* ∆AIJ(t) ) ∑ ∑ U(II,Lq) U(JJ,L′q′) Dq,0 (Ωs(0)) L,L′ q,q′
(L′)* (Ωs(t)〉 A(L) h (L) Dq′,0 0 A 0 (3)
where the orientational average in eq 1 becomes an average over Wigner rotation functions. In oriented purple membranes, the chromophore orientational distribution over which this average is carried out is intrinsically anisotropic. In a perfectly oriented sample, the retinal chromophore would be oriented somewhere on the surface of a cone with an opening angle θ0, the angle of the chromophore’s polyene chain with respect to the membrane normal (Figure 1). In Figure 1, we define the Z-axis as the orientation axis, along which the membrane normal would be aligned in a perfectly ordered sample. Magnetically oriented purple membrane is not perfectly oriented.18 Consequently, a distribution of angles exists between the purple membrane normal and the Z-axis. For an axially symmetric system, this distribution can be described by two order parameters given by the averages of the second or the fourth Legendre polynomials, 〈P2(cos β)〉 and 〈P4(cos β)〉, respectively, where β is the angle between the membrane normal and the orientation axis defined by the magnetic field. In what follows, these order parameters are written 〈P2〉 and 〈P4〉. Otto and Heyn15,16 have developed a theoretical formalism based on the theory of fluorescence decay in liquid crystals21 to calculate the out-of plane reorientation of the M-state transition dipole for their measurements with mutually perpendicular pump and probe beams. A similar approach is used here to obtain expressions appropriate for our measurements with collinear pump and probe beams. Defining ∆Ωs as the Euler angles relating the laboratory frame to an axis system fixed to the membrane normal, where ∆Ωs ) 0 for a perfectly ordered system, and similarly defining Ωi(t) as the Euler angles relating the molecular axis system for intermediate i to the membrane-fixed axis system (where Ωi(0) ≡ Ω0), we can write the required orientational average as (L)* (L)* (Ωs(0)) Dq′,m (Ωs(t))〉 ) 〈Dq,0 L
L
(L) (L)* (-1)q-m 〈D-q,-m (∆Ωs) Dq′,n (∆Ωs)〉 ∑ ∑ m)-Ln)-L
(4)
These expressions simplify for a uniaxially symmetric distribution, which we assume to be generated by the magnetic field. In particular, for a uniaxial system one has21 (2)* (Ωs(t))〉 ) 〈P2〉P2(cos θi(t)) 〈Dq,m
(5)
(2) (2)* 〈D-q,n (∆Ωs) D-q,n (∆Ωs)〉 ) φqn(0)
(6)
and
where P2(z) is the second Legendre polynomial, 〈PL〉 ) 〈D(L) 0,0(∆Ωs)〉 is the second-rank (L ) 2) or fourth-rank (L ) 4) order parameter describing the degree of orientational order induced by the magnetic field, and φqn(0) are initial values of
Figure 2. Orientation of the purple membrane with respect to the laboratory axis system. The Z-axis is the orientation axis, and the purple membrane sheets are oriented predominantly in the XY plane. A, B, and C show the geometries for measurements with the purple membrane plane oriented parallel to the pump and probe beams, and D shows the geometry for measurements with the purple membrane plane oriented perpendicular to the pump and probe beams. The steady-state anisotropy Rs is measured by geometry A, and the time-resolved anisotropies R1, R2, and R3 are measured by geometries B, C, and D, respectively.
Wigner matrix correlation functions for which explicit expressions are given by Zannoni21 in terms of the second-rank and fourth-rank order parameters 〈P2〉 and 〈P4〉. In the next section, these expressions are used to evaluate the anisotropy parameters measured in our experiments. Definition of Anisotropies. A steady-state anisotropy and three time-resolved anisotropies can be measured with vertically and horizontally oriented purple membrane. While one of the anisotropy parameters defined below is identical to the one measured by Otto and Heyn,15 two additional anisotropy parameters can be defined and measured in the collinear geometry. These anisotropies provide complementary information about the ordering of the purple membranes and the orientation of the transition dipole. The steady-state anisotropy is defined as follows for a vertically oriented orientation axis probed in the absence of the pump beam:
Rs )
AZ - AX AZ + 2AX
(7)
where AZ and AX define the absorbances of the probe beam components parallel to the Z-axis and the X-axis, respectively (Figure 2, part A). Two time-resolved anisotropies can be
15608 J. Phys. Chem., Vol. 100, No. 38, 1996
Song et al.
defined for a vertically oriented orientation axis (Figure 2, parts B and C):
(8)
∆AXX - ∆AXZ ∆AXX + 2∆AXZ
(9)
4
0
2
0
i
i
2
2
4
0
i
i
2
4
(52 + 53〈P 〉) + 2 2 3 sin θ cos θ sin θ cos θ cos φ ( - 〈P 〉) + 5 5 3 2 1 sin θ sin θ cos(2φ )( + 〈P 〉)]/[1 - (P (cos θ ) 4 5 10 4 3 P (cos θ ))〈P 〉 + P (cos θ )P (cos θ )(- 〈P 〉 - 〈P 〉) + 7 7 2 2 3 sin θ cos θ sin θ cos θ cos φ (- 〈P 〉 + 〈P 〉) + 7 7 3 4 1 sin θ sin θ cos(2φ )( 〈P 〉 - 〈P 〉)] (16) 4 7 14
[
R2 ) -〈P2〉P2(cos θi) + P2(cos θ0)P2(cos θi) 0
(10)
2
0
2
i
i
The pump-induced anisotropies for ground-state bleaching are
2 11 36 + 〈P2〉P2(cos θ0) + 〈P4〉P4(cos θ0) 5 7 35 R1 ) 1 + 2〈P2〉P2(cos θ0)
(12)
2 3 - 〈P2〉P2(cos θ0) + 〈P4〉P4(cos θ0) 5 5 R2 ) 4 3 1 - 〈P2〉P2(cos θ0) - 〈P4〉P4(cos θ0) 7 7
(13)
2 4 6 - 〈P 〉P (cos θ0) + 〈P4〉P4(cos θ0) 5 7 2 2 35 R3 ) 10 3 1 - 〈P2〉P2(cos θ0) + 〈P4〉P4(cos θ0) 7 7
(14)
(Equations 11 and 12 were derived previously by Heyn and Otto.15,16) The anisotropies of the photointermediates are functions of 〈P2〉, 〈P4〉, and θ0, and, in addition, depend on the reorientation of the chromophore in the photocycle. The reorientation is described by an in-plane angle change ∆φi ) φi and an outof-plane angle change ∆θi ) θi - θ0. These anisotropies can be derived by methods similar to those used above and are
i
i
i
2
4
4
2
0
0
0
i
2
2
i
i
2
0
2
i
4
2
4
2
0
i
i
2
4
(51 - 72〈P 〉 + 353 〈P 〉) + 1 1 2 6 sin θ cos θ sin θ cos θ cos φ ( - 〈P 〉 - 〈P 〉) + 5 7 35 3 1 2 1 sin θ sin θ cos(2φ )( + 〈P 〉 + 〈P 〉)]/ 2 5 7 70 [1 - (P (cos θ ) + P (cos θ ))〈P 〉 + P (cos θ )P (cos θ ) (74 + 73〈P 〉) + 3 sin θ cos θ sin θ cos θ cos φ (72〈P 〉 - 72 3 4 1 〈P 〉) + (sin θ sin θ cos(2φ ))(- 〈P 〉 + 〈P 〉)] (17) 4 7 14
[
R3 ) 2P2(cos θ0)P2(cos θi) 0
0
i
2
2
i
4
i
i
i
2
i
0
0
0
4
4
2
2
4
2
0
2
2
4
2
i
0
i
2
i
i
2
2
2
(11)
i
4
2
0
Anisotropies in Magnetically Oriented Purple Membrane. Since the orientation of purple membrane by the magnetic field is not perfect, order parameters describing this distribution must be incorporated into the calculation of anisotropies. Similar order parameters arise in descriptions of the orientational distribution in liquid crystals.21 The steady-state anisotropy depends on a second-rank order parameter 〈P2〉, whereas the transient anisotropies depend on both second- and fourth-rank order parameters, 〈P2〉 and 〈P4〉. For a perfectly ordered sample, 〈P2〉 ) 〈P4〉 ) 1. It can readily be shown that, for perfectly oriented purple membrane, R1 is sensitive only to the out-of plane angle change, while R3 is sensitive only to the in-plane angle change. Following the method used by Zannoni,21 the following expressions are obtained for the steady-state and ground-state anisotropies as functions of 〈P2〉, 〈P4〉, and θ0, the angle between the membrane normal and the transition dipole. The steady-state anisotropy is
Rs ) 〈P2〉P2(cos θ0)
i
2
[1 + 2〈P2〉P2(cos θ0)] (15)
An additional time-resolved anisotropy can be measured with a horizontally oriented orientation axis (Figure 2; part D):
∆AXX - ∆AXY R3 ) ∆AXX + 2∆AXY
(51 + 72〈P 〉 + 18 1 1 〈P 〉) + 6 cos θ cos θ sin θ sin θ cos φ ( + 〈P 〉 35 5 7 12 1 2 3 3 〈P 〉) + sin θ sin θ cos(2φ )( - 〈P 〉 + 〈P 〉)/ 35 2 5 7 35
R1 ) [〈P2〉P2(cos θi) + 2P2(cos θ0)P2(cos θi)
∆AZZ - ∆AZX R1 ) AZ + 2AX R2 )
found to be
0
i
i
2
4
(Equation 15 is identical to that presented by Heyn and Otto.15,16) The anisotropy R1 is sensitive to θi, 〈P2〉, and 〈P4〉. On the other hand, R3 is not sensitive to the out-of-plane angle change even with some disordering in membrane orientations. R2 is sensitive to both θ0 and φi. Calculation of 〈P2〉, 〈P4〉, and θ0. The values of 〈P2〉, 〈P4〉, and θ0 that are needed to calculate the reorientation angles ∆θi and φi of the photointermediates in eqs 15-17 can be calculated from the measured anisotropies of the ground state with eqs 11-14. We directly obtain the value of 〈P2〉P2(cos θ0) from Rs. The result of 〈P4〉P4(cos θ0) can then be calculated by inserting the value of 〈P2〉P2(cos θ0) into either R1 or R2. However, in order to determine θ0 from these parameters, it is necessary to find the orientational distribution function over which the value of P2(cos β) and P4(cos β) are averaged to yield the values of 〈P2〉 and 〈P4〉. For purple membranes oriented in a magnetic field, the distribution function can be considered to be Gaussian and depends on a single parameter R:18
f(β) ) exp(R cos2 β)
(18)
where β is the angle between the membrane normal and the orientational axis. The larger the parameter R, the sharper is the distribution. The variables 〈P2〉 and 〈P4〉 are not independent since they both depend on the distribution function and on β:
Chromophore Reorientation Detection
∫0π PL(cos β) f(β) sinβ dβ 〈PL〉 ) ∫0π f(β) sin β dβ
J. Phys. Chem., Vol. 100, No. 38, 1996 15609
(19)
(where L ) 2,4). The distribution function can be expanded: ∞
Rk cos2k β
k)0
k!
f(β) ) ∑
(20)
By substituting eq 20 into eq 19, the values of 〈P2〉, 〈P4〉, and θ0 can be determined from the experimentally measured values of Rs and R1. We tested the convergence of eq 19 and truncated the summation at k ) 50 in the calculation. We also checked the values of 〈P2〉 and 〈P4〉 calculated for a distribution of membrane sizes chosen to approximate the size distribution reported by Lewis et al., based on the expectation that R is proportioned to membrane area.18 The calculated averages were virtually identical to those obtained with a single value of R. Results Steady-State and Ground-State Anisotropies. The anisotropies measured for the ground-state of BR are listed in Table 1. The steady-state anisotropy Rs measured at 570 nm without the pump beam is -0.15 ( 0.01. The negative value of the Rs indicates that θ0 is larger than the magic angle. The groundstate anisotropy RG1 measured at 570 nm with a 390 µs time delay after excitation at 532 nm is 0.20 ( 0.01, in close agreement with Otto and Heyn’s result of 0.19.15 The values 〈P2〉P2(cos θ0) ) -0.15 and 〈P4〉P4(cos θ0) ) -0.0236 were calculated from Rs and RG1 with eqs 11 and 12. The values of 〈P2〉, 〈P4〉, and θ0 were then obtained by finding the values of θ0 and R (eq 18) that simultaneously satisfy the values of 〈P2〉P2(cos θ0) and 〈P4〉P4(cos θ0) with eq 19. The results are 〈P2〉 ) 0.569, 〈P4〉 ) 0.219, θ0 ) 66.6°, and R ) 4.1. The value of θ0 is in close agreement with a number of measurements by other techniques including linear dichroism22,23 and second harmonic generation.24 The corresponding value of RG3 calculated with eq 14 is much less sensitive to 〈P2〉P2(cos θ0) and 〈P4〉P4(cos θ0) and agrees with the experimentally measured value of 0.40 ( 0.01. The value of θ0 determined from Rs and RG1 depends on the assumed distribution function. However, calculations of ∆θi and φi in the photocycle intermediates do not depend strongly on the value of θ0, which is probably determined with higher accuracy by other techniques. The values of 〈P2〉, 〈P4〉, and R are indications of the degree of orientation induced by the 11 T magnet and are, as expected, very similar to Heyn and Otto’s results obtained with BR oriented in a 13 T magnet.15,16 K-State. The anisotropies measured at 640 nm at a 100 ns time delay are dominated by the contribution from the K-state, yielding RK1 ) 0.24 ( 0.03 and RK3 ) 0.39 ( 0.03 (see Table 1). The value of RK1 is slightly larger than the ground-state value, RG1, indicating a possible small out-of-plane reorientation toward the membrane normal in the K-state. The value of RK3, on the other hand, is nearly the same as the ground-state value RG3, indicating the absence of any detectable in-plane reorientation of the K-state. Calculation of the values of ∆θK and φK from RK1 and RK3 with eqs 15 and 17 yields ∆θK ) +7° -2.8° and ∆φK ) 6°-6° , respectively. The time-resolved -2.9°+2.2° scans of the linear dichroism, transient absorption, and anisotropy (RK1) at 640 nm over a time range of 0-2.3 µs show no detectable reorientation during the course of the K-state (data not shown).
L-State. Figure 3 shows the time-resolved linear dichroism, transient absorption, and anisotropy scans at 560 nm over 250 µs for oriented purple membrane. Scans at this wavelength have contributions from both the ground-state and the L-state at early times, but are dominated by the contributions from the BR ground-state after 200 µs. The increase of the anisotropy R1 during the first 100 µs of the scan demonstrates that the value of RL1 for the L intermediate is larger than that of RBR1 of ground-state BR. This is evident from a consideration of the contributions to the measured anisotropy at 560 nm from the L-state and the ground-state BR hole. The value of the anisotropy of the L-state, RL1, was calculated to be 0.24 ( 0.01 with the equation12
R1 ) RBR1 +
∆AL (R - RBR1) ) ∆A L1 ∆A - ∆ABR RBR1 + (RL1 - RBR1) (21) ∆A
where ∆ABR and ∆AL are the transient absorption contributions from ground-state BR and the L-state in Figure 3, and ∆A is the total transient absorption. Since the transient absorption contributions of BR and L have opposite signs at 560 nm, the observation of a lower R1 at early times where both L and BR contribute indicates RL1 > RBR1. The out-of-plane reorientation of the L-state calculated with eq 15 from RL1 is ∆θL ) -0.8° . This result is essentially identical to the out-of-2.7°+0.7° plane reorientation measured in the K-state. The greater precision of the L-state measurement, however, demonstrates clearly a small out-of-plane reorientation of the transition dipole in L with respect to the ground state. Figure 4 shows the time-resolved linear dichroism, transient absorption, and anisotropy at 560 nm over 250 µs for purple membrane oriented in geometry D (Figure 2). R3 remains constant with a value of about 0.4 throughout the course of the L-state decay, indicating that the in-plane orientation of the L-state is the same as that of the ground-state within experimental error, ∆φL ) 0 ( 6°. M-State. The time-resolved linear dichroism, transient absorption, and anisotropy (R1) at 410 nm over 8 ms are dominated by the contribution from the M-state (Figure 5). The anisotropy at 410 nm, R1 ) 0.47 ( 0.03, is much higher than RG1, and is constant during the lifetime of the M-state. The value R3 ) 0.38 ( 0.03 was measured at a fixed time delay of 390 µs and a probe wavelength of 410 nm with the horizontally oriented sample. These results correspond to an out-of-plane -3.2° reorientation of ∆θM ) -20°+2.6° and a in-plane reorientation of ∆φM ) 0° ( 10°. (A -20° change in θ is sufficient in itself to account for the decrease in R3 to 0.375.) However, as pointed out previously,12 the anisotropy measured at 410 nm contains contributions from ground-state BR as well as from M. At the peak M population, the contribution to the transient absorption from BR is about 25% of that of M in magnitude25 but opposite in sign. The anisotropies measured at 410 nm can be corrected for this contribution by the equation12
R1M ) (1 - F)R1 + FR1BR
(22)
where R1 is the measured anisotropy, R1M the M-state contribution, R1BR the ground-state contribution, and F ) -∆ABR/∆AM. (An analogous equation holds for R3M.) In order to calculate the anisotropy of M, the anisotropy of BR at 410 nm must first be known. We assume here that R1BR and R3BR at 410 nm are equal to their values measured at 560 nm. (A possible wavelength dependence of the anisotropy of BR is discussed in ref 12.) With a value of F ) 0.256,25 the out-of-plane
15610 J. Phys. Chem., Vol. 100, No. 38, 1996
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TABLE 1: Anisotropies and Reorientation Angles for Oriented Purple Membrane Samples BR ground statea R1 (Å) R3 (Å) Rs (Å) ∆θ (deg) ∆φ (deg)
0.20 ( 0.01 0.40 ( 0.01 -0.15 ( 0.01 0 0
K-stateb 0.24 ( 0.03 0.39 ( 0.03 -2.8 -2.9+2.2 +7 6-6
L-statec 0.24 ( 0.01 0.40 ( 0.01 -0.8 -2.7+0.7 0(6
410 nmd 0.47 ( 0.03 0.375 ( 0.025 -3.2 -20+2.6 0 ( 10
M-statee 0.40 ( 0.03 0.38 ( 0.03 -3.4 -14.3+2.5 +7 6-6
a Measured with 570 nm probe at 390 µs time delay. b Measured with 640 nm probe at time delay < 2.3 µs. c From fit of eq 21 to 250 µs anisotropy scans at 560 nm. d Measured anisotropy with 410 nm probe at 390 µs time delay (angles uncorrected for ground-state BR contribution). e Corrected for ground-state BR contribution by eq 22 with R BR (410 nm) assumed to be equal to RBR (560 nm).
Figure 3. Time-resolved linear dichroism (top panel), transient absorption (middle panel), and anisotropy (R1, bottom panel) of oriented purple membrane at a probe wavelength of 560 nm. The solid lines in the top and middle panels show single exponential fits to the decays with time constants of 70.9 ( 1.5 µs for the linear dichroism, and 65.3 ( 1.2 µs for the transient absorption. The solid line in the bottom panel is a fit to eq 21 with RBR1 ) 0.209 ( 0.005, and RL1 ) 0.239 ( 0.005. The linear dichroism and transient absorption are plotted in absorbance units. (Uncertainties are the standard errors from the least-squares fits.)
Figure 4. Time-resolved linear dichroism (top panel), transient absorption (middle panel), and anisotropy (R3, bottom panel) of oriented purple membrane at a probe wavelength of 560 nm. The solid lines show single exponential fits to the decays with time constants of 66.9 ( 1.0 µs for the linear dichroism and 67.8 ( 2.3 µs for the transient absorption. The linear dichroism and transient absorption are plotted in absorbance units. (Uncertainties are the standard errors from the least squares fits.)
-3.4° reorientation of M is ∆θM ) -14.3°+2.6° and the in-plane +7° reorientation is ∆φM ) 6°-6°. These values can be compared with the corrected anisotropy at 410 nm for isotropic samples. As reported previously,12 the correction of the measured anisotropy (r ) 0.36) at 410 nm for isotropic BR in polyacrylamide gels yields rM ) 0.37 (with the assumption again that rBR at 410 nm equals its value at 560 nm), which corresponds to a reorientation of 13°. The value of ∆θM is considerably larger than the value (3° ( 1°) found by Otto and Heyn.15,16 We can exclude saturation of the transient absorption as an explanation for our measurement of a larger reorientation angle, since saturation of the sample would cause a decrease in the measured anisotropy, which in this case would result in a smaller calculated
reorientation angle. In contrast, in measurements on nonoriented samples, saturation results in the calculation of a larger reorientation angle. The agreement of the reorientation angle measured in oriented (this work) and nonoriented12 samples argues strongly that our results are measured in the unsaturated regime. Discussion We have studied in-plane and out-of-plane reorientation during the bacteriorhodopsin photocycle with magnetically oriented purple membrane gels. Out-of-plane reorientations have been detected in the photocycle with ∆θK = -3°, ∆θL ) -3°, and ∆θM ) -14°. The out-of-plane reorientation is in each case toward the purple membrane normal. The in-plane
Chromophore Reorientation Detection
Figure 5. Time-resolved linear dichroism (top panel), transient absorption (middle panel), and anisotropy (R1, bottom panel) of oriented purple membrane at a probe wavelength of 410 nm over a time scan of 9.5 ms. The solid lines show double exponential fits to the decays with the rise time fixed at 0.1 ms (representing the unresolved L f M transition) and the decay time fit with 2.3 ( 0.2 ms for the linear dichroism and 2.3 ( 0.5 ms for the transient absorption. The linear dichroism and transient absorption are plotted in absorbance units. (Uncertainties are the standard errors from the least-squares fits.)
orientations of the K, L, and M photointermediates are similar to that of the ground-state. Although no in-plane reorientations larger than the uncertainties were detected, in-plane reorientations of ∆φ of up to 6-13° are not precluded during the photocycle since the anisotropy R3 is intrinsically insensitive to small in-plane reorientation. The chromophore reorientations in oriented gels can be compared to those in the isotropic gels. The in-plane and outof-plane reorientations (∆θ, ∆φ) of the chromophore in oriented gels are correlated to the angle changes (ω) of the chromophore in isotropic gels according to the equation
cos ω ) sin θ0 sin(θ0 + ∆θ) cos(∆φ) + cos θ0 cos(θ0 + ∆θ) (23) The total angle changes (ω) calculated from ∆θ and ∆φ with the above equation are ωK ) 6°, ∆ωL ) 2.7°, and ωM ) 15°, in excellent agreement with the results for isotropic gels.12 Out-of-Plane Motion in the K and L-States. In order to relate the measured reorientations of the transition dipole to the dynamics of the protein, information is required regarding the orientation of the transition dipole with respect to the structure of the chromophore. The transition dipole in all-trans retinal is likely to be roughly parallel to the direction of the conjugated polyene chain.16,26 Isomerization from the all-trans to the 13-
J. Phys. Chem., Vol. 100, No. 38, 1996 15611 cis chromophore involves bending of the polyene around the C13-C14 double bond. This could be accomplished by a tilt of the C5-C13 portion of the chain toward the cytoplasmic side, a tilt of C14-N toward the exterior, or a combination of both. Photovoltaic experiments demonstrate fast positive charge motion toward the cytoplasm, in the direction opposite of the proton pumping,27 favoring a model which involves some degree of tilting toward the cytoplasm. Resonance Raman spectra of the K-state indicate a twisted chromophore conformation caused by rigid, steric constraints in the binding pocket.28 The steric interactions relax in the K f L transition, producing a more planar chromophore with protonated Schiff base.29,30 The similarity of the out-of-plane angle change in the K-state with that in the L-state suggests that the relaxation of the chromophore does not significantly alter its orientation. The angle of tilting of the chromophore in K and L toward the cytoplasm cannot be determined quantitatively at present because an accurate geometry of the transition dipole with respect to the configuration of the chromophore in the all-trans and 13-cis isomers is not known. We discuss here two possible models that can explain the transition-dipole reorientation that we have observed in the K- and L-states. More specific information about the location of the transition dipole in alltrans and 13-cis retinal would allow these models to be refined and might allow one of them to be discarded. In the first model, we suppose that isomerization involves bending of the C5-C13 portion of the chromophore toward the cytoplasmic side. Such a reorientation could account for the observed reorientation of the transition dipole by roughly -3° in the K- and L-states. This model is illustrated in Figure 6. For want of specific information about the direction of the transition dipole, we arbitrarily locate the transition dipole in this model along a line extending from C4 to C14 in both alltrans and 13-cis retinal. The model was constructed on the basis of the structure determination of Henderson and coworkers for ground-state BR,3 which was retrieved from the Brookhaven Protein Data Bank. The chromophore structure in the K f L portion of the photocycle was then modeled by rotating the C14-N segment of the chromophore about the C13C14 double bond by 180° to introduce the 13-cis conformation of the chromophore. The chromophore is also more planar in accordance with experimental results for the L-state29 with a shorter distance between the cyclohexene ring and the Schiff base. The protein structure was adjusted by an energy minimization in CHARMM with respect to protein coordinates. The result shown in Figure 6 is a tilting of the C4-C14 segment up toward the cytoplasmic side and a tilting down of C14-N toward the exterior. The reorientation of the C4-C14 segment by -1.8° is approximately equal to the observed reorientation within experimental uncertainty. Consequently, if the transition dipole is located approximately along the C4-C14 direction, this model can account for the observed out-of-plane reorientation in K and L as a result of isomerization. At the same time, the distance between the Schiff base and the proton acceptor at Asp85 decreases due to the downward tilting of the C14-N segment. This may facilitate proton transfer from the Schiff base to Asp85, which occurs during the L to M transition. The second model recognizes that isomerization may introduce a component of the transition dipole in the 13-cis chromophore that is perpendicular to the C4-C14 vector. This component would in itself cause a reorientation of the transition dipole away from the membrane normal. Without a compensating tilt of the chromophore, this component would be expected to result in a reorientation of the transition dipole that is opposite
15612 J. Phys. Chem., Vol. 100, No. 38, 1996
Song et al.
Figure 6. Model for the chromophore orientation with respect to key residues in the initial (Ground State BR), K and L, and M photointermediates. Part A shows a structural model of ground-state BR in the region of the chromophore based on the structure reported by Henderson and co-workers (ref 3). The view exposes Lys-216, Asp-96, and Asp-85 and shows the distances from the Schiff base to Asp-85 (4.1 Å) and from the Schiff base to Asp-96 (13.5 Å). Part B (K f L) is a view of an energy-minimized structure with a 13-cis chromophore (see text for details). In this structure the distance from the Schiff base to Asp-85 (3.3 Å) is shorter than that in the ground-state, whereas the distance from the Schiff base to Asp-96 (14 Å) is longer. Part C (M-state) is a view of an energy-minimized structure with the 13-cis chromophore tilted by 11° with respect to the structure in part B (see text for details). The distance from the Schiff base to Asp-85 (6.5 Å) is longer than in the K f L structure, and the distance from the Schiff base to Asp-96 (10.1 Å) is shorter. The residues Leu-93, Trp-182, and Phe-219 are exposed to show that they would come into close contact with the chromophore in this structure, which may trigger motion in the C, F, and G helices.
in direction from the detected reorientation. Hence, in this model the observed reorientation of the transition dipole toward the membrane normal requires tilting of the chromophore toward the membrane normal by an angle larger than 3°, the out-ofplane angle change detected in oriented sample. An essentially identical model has been put forth by Heyn and Otto,16 but for the M-state (where they observed a reorientation of only 3°). They propose that the intrinsic reorientation of the transition dipole resulting from isomerization is 8°, so that the total tilt of the chromophore is 11°. The present model would lead to a similar conclusion, but for the K f L portion of the photocycle. M-State Out-of-Plane Motion. In the M-state we have +3,6° . detected an out-of plane reorientation of ∆θM ) 14°-2.5° During the transition of K f L f M, the protein responds to the isomerization and the resulting charge displacements. Timeresolved diffraction studies8-10,31 detected structural changes in the C, F, and G helices in the M-state. The out-of-plane angle changes in the K-state and L-state show that the all-trans to 13-cis isomerization can account for an out-of-plane angle change of the transition dipole of only 3°. Thus, protein or chromophore structural change during the L f M transition must contribute about 11° to the out-of-plane angle change of the transition dipole. Such a reorientation can be accomplished either by motion of the Schiff base end of the chromophore toward the cytoplasmic side, or by motion of the cyclohexene ring toward the extracellular side. A neutron diffraction study of purple membrane regenerated with selectively deuterated retinal demonstrated that the cyclohexene ring stays at the same position during the ground-state to M-state transition.8 Hence the motion of the Schiff base is most likely responsible for the out-of-plane angle change. A model of the M-state in Figure 6 shows an additional rotation of the C4-C14 portion of the chromophore by 11° beyond the orientation in the K f L structure. This model was generated by keeping the position of C5 fixed and rotating the Schiff base up. The purpose of the figure is to illustrate the resulting changes in distances between the Schiff base and the proton donor Asp-96. In this model, the tilting of the chromophore shortens the distance between the Schiff base and Asp96 from 14 Å in K f L to 10 Å in M. This postulated motion of the Schiff base toward the Asp-96 may serve to facilitate reprotonation of the Schiff base. In addition, we note that this
motion would also bring the chromophore into close contact (less than 5 Å) with Phe-219 (4 Å from C20), Leu-93 (3 Å from C20), and Trp-182 (2.5 Å from C19). Consequently, these residues would be expected to undergo changes in position or orientation (or both) to allow the upward tilting of the Schiff base to occur. These residues belong to the G, C, or F helices, each of which has been found to be involved in conformational changes in the M intermediate.7-10,31,32 It may be that structural changes in these helices are correlated with reorientation of the chromophore in the L f M transition. How do these results relate to the findings reported in a neutron diffraction study of the M-state by Hauss et al.,8 where a shortened distance between deuterium-labeled sites was explained by an 11° ( 6° tilt of the chromophore toward the cytoplasm? The issue here is how closely the M-state generated in the neutron diffraction study corresponds to the M-state generated in our measurements on oriented samples. As in the discussion above of the K f L portion of the photocycle, we will discuss two possibilities. In the first we suppose that the M-state generated in the neutron diffraction study corresponds closely to the M-state in the present study. From this viewpoint, the reorientation of 14° measured in the M-state agrees well with the 11° reorientation measured by neutron diffraction. In comparing these results, the affect of pH must be taken into account. The pH in the neutron diffraction study was 9.6, whereas the present study was carried out at pH 7. In measurements on isotropic samples, we have shown that reorientations in the M-state are maximized at a pH of 7-8 and are reduced at higher pH values.33 On the basis of the pH dependence observed in purple membrane suspensions33 and the anisotropy of M in purple membrane immobilized at pH 7 in isotropic polyacrylamide gels,12 we estimate that the anisotropy in polyacrylamide gels at pH 9.6 would be roughly 0.37-0.38 after correction for the ground-state contribution at 410 nm (see eq 22),34 corresponding to a reorientation of 11-13°. Hence, the reorientation measured here agrees well with the neutron diffraction results. We note that this model differs from the picture proposed by Heyn and Otto,16 which explained the 11° tilting of the chromophore in the neutron diffraction experiment with only a 3° reorientation of the transition dipole. The second possibility is that the M-state generated in the
Chromophore Reorientation Detection neutron diffraction study differed significantly from the M-state in the present experiments. In addition to the pH, the conditions under which the M-state was generated differed in several other respects. In the neutron diffraction study, the purple membrane was incorporated into films at 86% relative humidity with 0.1-1 M guanidine hydrochloride, while in the present study the purple membrane was immobilized in gels which consist of roughly 93% water. It is quite possible that the harsher conditions necessary to trap the M-state in the neutron diffraction study restricted conformational and reorientational motions to some degree. In this picture, the 11° tilt observed by neutron diffraction might correspond to a smaller reorientation of the transition dipole than we have observed in oriented purple membranes. Heyn and Otto have shown16 that the 11° tilt could correlate with a reorientation of the transition dipole as small as 3° (see model 2 in the discussion above of the K f L-states). In this case, the reorientation of the chromophore would be larger than the 14° reorientation of the transition dipole. Deciding between these two possibilities will require more detailed knowledge of the location of the transition dipole in the 13-cis chromophore. Significance for Proton Pumping. For purposes of this discussion, we adopt the model depicted in Figure 6, which corresponds to possibility (1) above. That is, we assume that the 14° reorientation of the transition dipole in the M-state corresponds to a tilting of the chromophore by roughly the same angle. In this model, all-trans to 13-cis isomerization results in a decreased distance between the Schiff base and Asp-85, facilitating proton transfer in the L f M transition. The pH dependence of reorientations in the M-state suggests that proton release, which occurs during or shortly after the L f M step, triggers reorientation in the M-state.33 The result is a decreased distance between Asp-96 and the Schiff base, shown in Figure 6. In addition, changes in the environment of the Schiff base as well as conformational changes of the protein may alter the relative proton affinities of Asp-96 and the Schiff base.35 Both changes could serve to induce reprotonation of the Schiff base. What this model fails to explain, however, is how proton translocation occurs at low pH levels (pH < 6),36,37 where reorientational motions are restricted. It may be that at pH < 6, where reorientation is not triggered in the M-state,33 proton transfer from Asp-96 to the Schiff base is facilitated by another mechanism induced by the low pH. Another possibility is that the reorientations observed in M are correlated with an irreversible M1 f M2 transition that has been associated with a reprotonation switch and at pH > 6 occurs roughly simultaneously with proton release.37,38 In this case, however, the correlation of reorientations with the M1 f M2 transition would be disrupted below pH 6, where the M1 f M2 step appears to persist, albeit reversibly with the appearance of an M2 f M1 back reaction.37 Conclusion Anisotropy measurements on oriented samples contain specific information about the direction of reorientational motion in the purple membrane. Due to the defined orientation of the chromophore with respect to the membrane normal, these measurements are particularly sensitive to changes in the angle between the transition dipole and the membrane normal. We have measured changes in this angle of roughly 3° in the K and L photointermediates and 14° in the M photointermediate. There reorientations may be linked to protein conformational changes that have been detected in the C, F, and G helices. Reorientation in the M-state may serve to facilitate reprotonation of the Schiff base.
J. Phys. Chem., Vol. 100, No. 38, 1996 15613 Acknowledgment. We thank Dr. David Vander Velde and Dr. Martha Morton of the University of Kansas NMR Laboratory for the use of the magnet for orienting samples. This work was supported by Grant GM 40071 from the National Institutes of Health and by the National Science Foundation under EPSCoR Grant 9255223. References and Notes (1) Lanyi, J. K. Biochim. Biophys. Acta 1993, 1183, 241. (2) Ebrey, T. G. In Thermodynamics of Membrane Receptors and Channels; Jackson, M. B., Ed.; CRC Press: Boco Raton, FL, 1993; Chapter 10. (3) Henderson, R.; Baldwin, H. M.; Ceska, T. A.; Zemlin, F.; Beckmann, E. J. Mol. Biol. 1990, 213, 899. (4) Braiman, M. S.; Mogi, T.; Marti, T.; Stern, L. J.; Khorana, H. G.; Rothschild, K. J. Biochemistry 1988, 27, 8516. (5) Gerwert, K.; Hess, B.; Soppa, J.; Oesterhelt, D. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 4943. (6) Smith, S. O.; Pardoen, J. A.; Mulder, P. P. J.; Curry, B.; Lugtenburg, J.; Mathies, R. Biochemistry 1983, 22, 6141. (7) Dencher, N. A.; Dresselhaus, D.; Zaccai, G.; Bu¨ldt, G. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 7876. (8) Hauss, T.; Bu¨ldt, G.; Heyn, M. P.; Dencher, N. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11854. (9) Subramaniam, S.; Gerstein, M.; Oesterhelt, D.; Henderson, R. EMBO J. 1993, 12, 1. (10) Nakasako, M.; Kataoka, M.; Amemiya, Y.; Tokunaga, F. FEBS Lett. 1991, 292, 73. (11) Watts, A.; Sternberg, B.; Ulrich, A. S.; Whiteway, C. A.; Seifert, G.; Sami, M.; Fisher, P.; Heyn, M. P.; Wallat, I. Biophys. Chem. 1995, 56, 41. (12) Song, Q.; Harms, G. S.; Wan, C.; Johnson, C. K. Biochemistry 1994, 33, 14026. (13) Wan, C.; Qian, J.; Johnson, C. K. Biophys. J. 1993, 65, 927. (14) Esquerra, R. M.; Che, D.; Shapiro, D. B.; Lewis, J. W.; Bogomolni, R. A.; Fukushima, J.; Kliger, D. S. Biophys. J. 1996, 70, 962. (15) Otto, H.; Heyn, M. P. FEBS Lett. 1991, 293, 111. (16) Heyn, P.; Otto, H. Photochem. Photobiol. 1992, 56, 1105. (17) Otto, H.; Zscherp, C.; Borucki, B.; Heyn, M. P. J. Phys. Chem. 1995, 99, 3847. (18) Lewis, B. A.; Rosenblatt, C.; Griffin, R. G.; Courtemanche, J.; Herzfeld, J. Biophys. J. 1985, 47, 143. (19) Nagle, J. F.; Bhattacharjee, S. M.; Parodi, L. A.; Lozier, R. H. Photochem. Photobiol. 1983, 38, 331. (20) Wan, C.; Johnson, C. K. Chem. Phys. 1994, 179, 513. (21) Zannoni, C. Mol. Phys. 1979, 38, 1813. (22) Cherry, R. J.; Mu¨ller, U. J. Mol. Biol. 1977, 117, 607. (23) Lin, S. W.; Mathies, R. A. Biophys. J. 1989, 56, 653. (24) Huang, J. Y.; Lewis, A. Biophys. J. 1989, 55, 835. (25) Va´ro´, G; Lanyi, J. K. Biochemistry 1991, 30, 5008. (26) Drikos, G.; Ru¨ppel, H. Photochem. Photobiol. 1984, 10, 93. (27) Trissl, H. Photochem. Photobiol. 1990, 51, 793. (28) Braiman, M.; Mathies, R. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 403. (29) Fodor, S. P. A.; Pollard, W. T.; Gebhard, R.; van den Berg, E. M. M.; Lugtenburg, H.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 2156. (30) Mathies, R. A.; Lin, S. W.; Ames, J. B.; Pollard, W. T. Annu. ReV. Biophys. Biophys. Chem. 1991, 20, 491. (31) Koch, M. H. J.; Dencher, N. A.; Oesterhelt, D.; Plo¨hn, H.-J.; Rapp, G.; Bu¨ldt, G. EMBO J. 1991, 10, 521. (32) Hu, J. G.; Sun, B. Q.; Bizounok, M.; Griffin, R. G.; Herzfeld, J. Biophys. J. 1995, 68, A332. (33) Harms, G. S.; Song, Q.; Johnson, C. K. Biophys. J. 1996, 70, 2352. (34) The pH-dependent anisotropies reported in ref 33 were not corrected for the ground-state contribution. (35) Cao, Y.; Va´ro´, G.; Klinger, A. L.; Czajkowsky, D. M.; Braiman, M. S.; Needleman, R.; Lanyi, J. K. Biochemistry 1993, 32, 1981. (36) Liu, S. Y. Biophys. J. 1990, 57, 943-950. (37) Zima´nyi, L.; Va´ro´, G.; Chang, M.; Ni, B.; Needleman, R.; Lanyi, J. K. Biochemistry 1992, 8535. (38) Kataoka, M.; Kamikubo, H.; Tokunaga, F.; Brown, L. S.; Yamazaki, Y.; Maeda, A.; Sheves, M.; Needleman, R.; Lanyi, J. K. J. Mol. Biol. 1994, 243, 621.
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