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J. Phys. Chem. 1995,99, 7267-7271
Transient Phase Grating Spectroscopy of Nanosecond Relaxation Dynamics in Bacteriorhodopsin T. Renner, F. W. Deeg,* and C. Brauchle Institut f i r Physikalische Chemie, Universitat Miinchen, Sophienstrasse 1I , 80333 Miinchen, Germany Received: August 9, 1994; In Final Form: March 7, 1995@
The photoinduced reaction cycle of bacteriorhodopsin has been investigated by transient phase grating experiments. This technique with the probe frequency well off any retinal resonance and thus being solely sensitive to refractive index changes of the sample allows the study of energy dissipation and global density changes in the protein. Within the experimental time window between 1 and 20 ns no significant energy release or structural transition of bacteriorhodopsin has been found. This indicates that on this time scale conformational changes of bacteriorhodopsin are synchronized with the well-known dynamics of the retinal chromophore.
1. Introduction Intense experimental efforts in recent years have led to an increased understanding of the pathways involved in energy relaxation and structural transitions of proteins. Special emphasis has been placed on proteins in which a certain function (e.g. electron transfer, proton transfer, ligand dissociation) can be optically triggered, as this allows the access of laser spectroscopic tools with high time resolution and ver~atility.~ One prominent example of this class of light-sensitive proteins is bacteriorhodopsin (BR), the light-driven proton pump in the membrane of halobacteria. The two-dimensional crystal of the retinal protein BR forms the purple membrane domains of the cell membrane of Halobacterium halobium. The 248 amino acids of BR are arranged in seven transmembrane helices which span the 5-nmthick lipid bilayer. The retinal chromophore is embedded in a cage formed by the amino acid side chains on the helices of BR and is covalently bound to the protein moiety via a Schiffbase linkage with the amino acid residue Lys216. The primary events following photoexcitation of BR have been elucidated$-7 and a photocycle as schematically depicted in Figure 1 has been established. Through photoexcitation vibronically excited states on the SIpotential surface are populated and decay within 200 fs to the vibrationally relaxed excited SIstate.8 This is followed by crossing to the SO ground state and the formation of the intermediate J within 500 fs with a quantum efficiency of 11 = 0.64. There is evidence that the isomerization around the 1314 double bond of the retinal is already completed in the J state.I0 With a time constant of 3 ps the intermediate J decays to K, which has been interpreted as the relaxation of the oligomethylene chain of lysine 216. The decay of K with a time constant of ca. 1 ps to the intermediate L has been interpreted as a relaxation of strain in the retinal chain. The L M step with a time constant of 50 ps is associated with the protonation of the amino acid Asp85 from the Schiff base. In the M N transition (ca. 1 ms) the Schiff base is reprotonated from an internal proton donor, Asp96. Afterwards BR returns to its original all-trans configuration through another intermediate, 0, on the time scale of several milliseconds. The transient photophysical and photochemical intermediates in the photocycle of BR have been identified primarily through time-resolved spectroscopy of electronic and vibrational transi-
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Abstract published in Advance ACS Abstracts, May 1, 1995.
tlCH
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fKLl
K15901
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I
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CH = NH
/-
-
- 10 msec / /
1 psec
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Figure 1. Basic photochemical cycle of BR. The parent BR57o and the intermediates K, L, M, N, and 0 are designated by capital letters; the numbers in brackets indicate the maximum of the absorption spectra. The decay times refer to room temperature and are from the literature.
tions of the retiad ckroraophore. mat is, they characterize the retinal m k d e aa8 its inseraCtions with its immediate protein environment. The results of electronic and vibrational spectroscopy obtained so far seem to be in general agreement. However, in order to completely understand the reaction path, specifically in order to comprehend how the energy which is supplied by the absorbed photon is channeled into the cooperative motion of the protein necessary for its function, experimental probes sensitive to chromophore-protein interactions on different length scales and global structuralkonformational dynamics of the protein are needed. Such changes in the tertiary and/or quaternary structure of the protein may not significantly modify absorption spectra of the chromophore probe and therefore remain unnoticed. In a number of studies of heme proteins R. J. D. Miller's group at Rochester has established the transient grating (TG) experiment as a valuable tool in the study of protein dynamics." The main asset of the grating experiment is the fact that it allows the detection of density changes with high sensitivity and picosecond time resolution. In a grating experiment two timecoincident laser pulses of the same wavelength A,, are crossed
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Renner et al.
7268 J. Phys. Chem., Vol. 99, No. 19, I995 inside the sample at an angle 0 and set up an optical interference pattern. This interference pattern generates a coherent spatially periodic response in the material with a fringe spacing or grating constant
A=
4
an An(t,y) = 2 k A ~ - x
a@
X
2 sin (012)
A probe pulse with the wavelength Ap entering the sample at the appropriate Bragg angle is diffracted by the spatially modulated complex index of refraction in the sample. The diffraction efficiency 7 depends on the spatial modulation of absorption Ak as well as the real part of the refractive index An and for small modulation depths has the general formI2
Here y is the rate constant of the slow density change. In the equation above, the decay of the optical grating due to thermal diffusion (characterized by the decay constant b) has also been included. If there are two relaxation steps in the sample which dissipate heat amounts of A a and A( 1 - a) with different time constants yl and y2, the refractive index change is the sum of the contributions of both components, resulting in a diffraction efficiency of the form
([Ak(4I2 + [An(412) (2) where OD@) is the average optical density, p the Bragg angle, and d the thickness of the grating. However, if the probe wavelength is well outside any absorption band, only changes in the real part of the refractive index contribute to the diffracted signal. The diffraction efficiency of such a pure phase grating is given by
where An depends on the density change A@in the material as indicated. Due to their large thermal expansion coefficient, the density change in liquids is in general dominated by thermally induced material expansion. The consequences of this fact for a TG experiment have been elucidated by the Fayer group some time ago.I3 Assuming that the excitation wavelength is resonant with an electronic or vibrational transition in the material, and the excited state is depopulated rapidly through radiationless relaxation channels, leading to a temperature increase in the sample, an optical grating of excited states is followed by a spatially periodic temperature modulation which triggers a density expansion and launches two counterpropagating ultrasonic waves with an acoustic frequency w = 2nlA
(4)
If the thermal energy is deposited in the sample on a time scale much faster than the inverse of the acoustic frequency (which corresponds to ca. 100 ps in the experiments described here), the time dependence of the diffraction efficiency of the grating induced by the ultrasonic waves has the form of a simple damped oscillation,
where c is the acoustic damping constant and the amplitude A is proportional to the dissipated energy. If the energy release and the density change in the sample are not instantaneous, but take place on a time scale comparable to or longer than the inverse acoustic frequency, then the TG signal is altered in a characteristic manner. As exemplified in the Results and Discussion section, such a delayed slow density change leads to an increase of the TG signal with time and/or lifting of the base line. The exact mathematical form of the time-dependent refractive index change in this case (also taking into account acoustical damping) is given byI4
Therefore, it is possible from a TG experiment to evaluate magnitude as well as time constants of energy dissipation mechanisms in a material. For a number of heme proteins it was shown with this technique that within 20 ps after excitation most of the absorbed energy is dissipated throughout the protein and surrounding water layer by efficient vibrational relaxation and energy exchange processes.I5 With short excitation wavelengths a second slow relaxation component (0.8-3 ns) can be seen in the phase grating experiment which is associated with a metastable protein conformation accessible under the original high-excitation conditions. The most interesting result of this kind of experiment, however, was the observation of a nonthermal density wave in the case of carboxy-ligated myoglobin (MbC0).9 It is well-known that the association or dissociation of a ligand at the porphyrin ring is accompanied by a correlated change of the tertiary and quaternary structure of the protein. The protein density wave observed for MbCO now proves that this structural change takes place within less than 30 ps (the experimental time resolution) after dissociation of the CO molecule. Consistent with this interpretation, a similar protein density wave is not found in deoxy-Mb, where no dissociation can take place, nor in oxyhemoglobin, where most of the dissociated 0 2 molecules recombine with the porphyrin within 5 ps. In the following section time-dependent phase grating experiments (Le. the probe frequency is well off resonance) on wild type BR are presented. The basic goal of these experiments is the search for energy dissipation and/or global density changes in the protein on a time scale of 100 ps to 20 ns. Recent photoacoustic and photocalorimetric experiments from Birge et a1.I6 and Rohr et al." have yielded different enthalpies of the intermediate K with respect to BR of 49 and 160 kJ/mol, respectively. As the energy of the exciting photon at hex= 532 nm corresponds to 230 kJ/mol, the amount of energy which is dissipated quasi-instantaneously is ca. 180 kJ/mol in the first case and ca. 70 kJ/mol in the second case. This must result in a strong ultrasonic wave being launched instantaneously compared to the width of the laser pulses and the fastest time scale of the TG experiment considered here. The ensuing relaxation step K L on the microsecond time scale is much too slow to be detected by this experiment. However, there has been an intense discussion in recent years about the appearance of a KL intermediate. First proposed by Shichida et a1.I8 in the discussion of transient absorption measurements, the KL intermediate has also been invoked in transient absorptionI9and resonance Raman20experiments from other groups. However,
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J. Phys. Chem., Vol. 99, No. 19, 1995 7269
Relaxation Dynamics in Bacteriorhodopsin there are also resonance Raman data with no evidence for such an additional intermediatee2' Recently, Atkinson's group has published time-resolved transient absorption and fluorescence data of the K intermediate in the nanosecond time regime.22 They observe no absorption changes or alterations of the fluorescence spectrum; however, they find a strong decay of the fluorescence quantum yield with a time constant of 5.5 ns. This is seen as evidence for a relaxation of the protein around the chromophore. The phase grating results presented in the following section are therefore discussed in the context of a possible second slow relaxation component in the nanosecond time regime. The sensitivity limit of the experiment is given by the ratio of the total energy amount which is dissipated with a slow relaxation rate and the energy amount which is dissipated instantaneously. A value of a = 0.1, which is a relative contribution of the slow relaxation component of 10% (for the relevance of this number see the Results and Discussion section), corresponds to 20 kJ/mol in the first case (AHK-BR = 49 kJ/ mol) and 7.8 kJ/mol in the second case (AHK-BR= 160 kJ/ mol).
2. Experimental Section The experiments were performed with a setup based on a CW-pumped, mode-locked, and Q-switched Nd:YAG laser (based on a Spectron SL 903 resonator). A single pulse from the output pulse train at 1064 nm is selected via a Pockels cell and a Glan-Thomson polarizer. This single pulse is frequencydoubled in a KTP crystal, resulting in a 532-nm pulse with a pulse length of 100 ps and a pulse energy of 12 pJ at a repetition rate of 800 Hz. The pulse is attenuated and split with a beamsplitter into two excitation pulses with maximum energies of 5 pJ each which are focused on the sample with two lenses cf = 500 mm). The remaining 1064-nm pulse with a pulse length of 100 ps is used as the probe beam. After passing a delay line it is also focused on the sample under the appropriate Bragg angle with a f = 250 mm lens. The beam waists are 150 pm for the excitation beam and 100 pm for the probe beam, resulting in maximum excitation powers of less than 280 MW/ cm2. Identical polarizations are used for all beams. One of the excitation beams or the probe beam is chopped, and the signal, which is picked up by a photodiode or a photomultiplier, is fed to a lock-in amplifier. The output of the lock-in amplifier is connected to a PC which allows signal averaging by controlling multiple scans of the optical delay line. Wild type bacteriorhodopsin (Wacker) was treated ultrasonically and dissolved in water. For stabilization 0.01% Na-acid was added. The total volume of 40 mL was pumped through a cuvette with a path length of 1 mm. A flow velocity of well above 12 cm/s was used to guarantee that each set of pulses excites a fresh sample, i.e. BR in a relaxed ground state. An initial optical density OD(568nm,Imm) = 0.8 was chosen for optimum diffraction efficiencies. The sample was spectrally characterized before and after the measurements to test for degradation of the sample, and no alteration of the absorption spectrum was detected. Water was chosen as solvent, as structural relaxation in this solvent (unlike, for example, glycerol) is instantaneous on the time scale considered in this paper. 3. Results and Discussion Figures 2 and 3 show transient phase grating signals of BR dissolved in water for two excitation angles of 9/2 = 2.6" and 9/2 = 10.8", respectively. The experimental data have been
1
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1
1 I
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I
A
1 ;
li
:
4
;
10
12
Time [ns]
Figure 2. Transient phase grating signals and data fits of BR in water for an excitation angle of Ol2 = 2.6'. The excitation wavelength was lex = 532 nm; the probe wavelength was 1, = 1064 nm. The best fits using eq 6 (dashed curve) were achieved with the following parameter values: w = 2.01 ns-', llc = 17 ns, and lly = 100 ps. For the thermal diffusion the literature value llb = 2002 ns was used. The corresponding residuals are shown in part b. However, much better fits are obtained taking into account a second contribution associated with a time-delayed density change. The dotted curve and the residuals in part c correspond to the best fit obtained this way with lly2 = 5.6 ns and a = -0.11.
-s v1
(b)
9
(e)
2 -2
0
2
4
6
8
10
12
Time [ns]
Figure 3. Transient phase grating signals and data fits of BR in water for an excitation angle of fil2 = 10.8'. The best fits using eq 6 (dashed curve) were achieved with the following parameter values: w = 6.23 ns-], llc = 28.8 ns, and lly = 100 ps. For the thermal diffusion the literature value llb = 202 ns was used. The corresponding residuals are shown in part b. However, as for the data presented in Figure 2, a much better fit is obtained taking also into account a second timedelayed contribution. The dotted curve and the residuals in part c correspond to the best fit obtained this way with lly2 = 3.3 ns and a = -0.08.
fitted assuming a density change of the form (6), that is, a single relaxation step with a fast time constant l / y on the order of the laser pulse width. It is evident that these fits describe the experimental data reasonably well. The fitting parameters for the acoustic frequency w and acoustic damping constant c are consistent with the literature values for water. For the slow thermal diffusion which has a negligible effect on the signal form, the literature value has been used. Assuming a signal of the form (7), that is adding a second relaxation step with a different relaxation constant l/y2 and with the same sign as (1 - a), does not improve the quality of the fits. We have performed the same experiments with resonant excitation of Nile Blue in water, which exhibits only one well-known fast relaxation channel.23 We found grating signals (Figure 4) which are indistinguishable from the BR data in Figure 2.
7270 J. Phys. Chem., Vol. 99, No. 19, 1995
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decay with a slow time constant lly2 = 5.5 ns. This corresponds to the value found in the time-dependent fluorescence yield study of Atkinson's group. The time constants for the damping of the acoustic wave (llc) and the decay of the grating due to thermal diffusion (llb)correspond to the values used in the fit described above. The only parameter which has been varied going from part a to part d of Figure 5 is the relative amplitude of the slow dissipation step a with respect to the fast one (1 a). In Figure 5a there is only a fast relaxation step; in Figure 5b there is a 10% (a = 0.1) contribution from the slow step, and this Contribution from the slow decay increases to 30% and 50% in the ensuing signals in Figure 5c,d. The signals have been normalized to the first maximum of the acoustic oscillation. It is evident that with increasing weight of the slow component there is a more and more pronounced slow rise of the whole oscillatory pattern. Comparison of Figure 5a,b and the experimental data in Figure 3 demonstrates that this base-line rise which is associated with a 10% slow component is clearly inconsistent with the experimental data. Closer inspection of the fit results actually allows the conclusion that the relative weight of such a second slow thermal density change with a time constant lly2 must be smaller than 0.05. The error margin of this parameter naturally changes with the decay constant y2 assumed, but the fit results show that the relative contribution of any component with 1 ns 5 l/y2 5 20 ns must be smaller than 0.1.
i a=0.0
- I .
( e )] a = 0 . 3
a=O.5
1
I
1
I
1
Figure 5. Numerical simulation of the transient thermal phase grating signal due to the combined contribution of a fast (yi) and a slow component ( y ~ ) .The parameter a,which varies from part a to d, is the relative amplitude of the slow contribution. The acoustic angular frequency o = 6.23 ns-] was taken from the experimental data; the value for the thermal dissipation ( l l b = 202 ns) and the acoustic damping (I/c = 27.2 ns) of water correspond to the values used in the data fit. The fast component llyl = 100 ps describes the almost instantaneous energy relaxation from BR* to K (3 ps), which is widened by the pulse width. The slow energy relaxation l / y ~= 5.5 ns refers to a possible relaxation process between K and L.
Relaxation Dynamics in Bacteriorhodopsin If during the fitting process the amplitudes of the two contributions are allowed'to have different signs, then actually a small slow contribution with lly2 = 5.6 and 3.3 ns and a relative amplitude a = -0.1 1 and -0.08, respectively, can be found (see Figures 2 and 3). As we know that the initial relaxation step leads to a thermally induced density increase, such a component with a different sign for the change of the refractive index would be indicative of a density decrease. Such a decrease does occur for a global contraction of the protein. This contraction is consistent with a relaxation of the cage around the retinal molecule as postulated by Delaney et a1.22 However, the same analysis for the TG data obtained after excitation of the reference system Nile Blue in water (see Figure 4) yields similar results. That is, the best fit is obtained taking into account two contributions, an instantaneous one with a large amplitude and a delayed one (142 4.0 ns) with a small negative amplitude a = -0.13. As it is well-established that there is no slow relaxation on the nanosecond time scale for the system Nile Blue in water, the amplitude of the slow contribution for this case establishes an experimental margin of error for a of about 10%. Therefore, we have to conclude that the slow nanosecond time scale contribution with negative sign found for the BR protein is compatible with a contraction of the system as postulated by Delaney et al. but that this effect is within the margin of error of our experiment. Going to the zero thermal expansion point of water, where no thermal expansion contributes to the data and the signal can only be due to the contraction of the protein, could help to clarify this issue. Temperature-dependent measurements are therefore planned for the future. Altogether the data indicate that there is no notable global relaxation component of BR in the time range between 1 ns and 20 ns. These results confirm one recent investigation which has focused on the same question. Diller et al. have recorded picosecond time-resolved BR-K difference infrared spectra24 in the spectral range between 1560 and 1700 cm-', which contains vibrations of groups which have a considerable spatial distance to the retinal chromophore. In these difference spectra no change occurs between 100 ps and 14 ns after excitation, demonstrating that only minor alterations of atomic distances can take place in those parts of the protein accessed.
4. Conclusions We have presented transient phase grating investigations of wild type bacteriorhodopsin which demonstrate that within the experimental margin of error there is no evidence for a slow energy dissipation process on a hundreds of picoseconds to tens of nanoseconds time scale. An experimental setup which is
J. Phys. Chem., Vol. 99, No. 19, 1995 7271 able to detect phase gratings on a nano- to millisecond time scale is currently installed in our labs and should clarify if the synchronicity of the BR photocycle is valid for the whole K L M sequence. Such an experiment would close the remaining gap to Fourier-transform infrared experiments by Genvert et al.,25who demonstrated that various parts of the BR protein are indeed very well synchronized on a time scale L 1 ms.
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Acknowledgment. We would like to thank Dr. Maurer and Dr. Miller from Wacker for the generous preparation of the bacteriorhodopsin samples used. References and Notes (1) Miller, R. J. D. Annu. Rev. Phys. Chem. 1991, 42, 581. (2) Fraunfelder, H.; Parak, F.; Young, R. D. A. Rev. Biophys. Biophys. Chem. 1988, 17, 451. (3) Holzwarth, A. Q. Rev. Biophys. 1989, 22, 239. (4) Polland, H. J.; Franz, M. A.; Zinth, W.; Kaiser, W.; Kolling, E.; Oesterhelt, D. Biophys. J. 1986, 49, 651. ( 5 ) Hofrichter, J.; Henry, E. R.; Lozier, R. H. Biophys. J . 1989, 56, 693. (6) Song, L.; El-Sayed, M. A.; Lanyi, J. K. Science 1993, 261, 891. (7) Wu, S.; Brauchle, C.; El-Sayed, M. A. Adv. Mater. 1993, 5, 838. (8) Dobler, J.; Zinth, W.; Kaiser, W. Chem. Phys. Lett. 1988, 144, 215. (9) Govindjee, R.; Balashov, S . P.; Ebrey, T. G. Biophys. J . 1990,58, 597. (10) Atkinson, G. H.; Brack, T. L.; Blanchard, D.; Rumbles, G. Chem. Phys. 1989, 131, 1. (1 1) Genberg, L.; Richard, L.; McLendon, G.; Miller, J. D. Science 1991, 251, 1051. (12) Kogelnik, H. Bell Syst. Tech. J . 1969, 48, 2909. (13) Miller, R. J. D.; Casalegno, R.; Nelson, K. A.; Fayer, M. D. Chem. Phys. 1982, 72, 371. (14) Genberg, L.; Bao, Q.; Gracewski, S.; Miller, R. J. D. Chem. Phys. 1989, 131, 81. (15) Genberg, L.; Heisel. F.; McLendon, G.; Miller, R. J. D. J . Phys. Chem. 1987, 91, 5521. (16) Birge, R. R.; Cooper, T. M.; Lawrence, A. F.; Masthay, M. B.; Zhang, C. F.:Zidovetzki, R. J . Am. Chem. SOC.1991, 113, 4327. (17) Rohr, M.; Girtner, W.; Schweitzer,G.: Holzwmh, A. R.; Braslavsky, S. E. J . Phys. Chem. 1992, 96, 6055. (18) Shichida, Y . ; Matuoka, S.; Hidaka, Y.; Yoshizawa, T. Biochim. Biophys. Acta 1983, 723, 240. (19) Milder, S. J.; Kliger, D. S. Biophys. J . 1988, 53, 465. (20) Doig, S. J.; Reid, P. J.; Mathies, R. A. J . Phys. Chem. 1991, 95, 6372. (21) Brack, T. L.; Atkinson, G. H. J . Phys. Chem. 1991, 95, 2351. (22) Delaney, J. K.; Brack, T. L.; Atkinson, G. H. Biophys. J . 1993.64, 1512. (23) Dutt, G. B.; Doraiswamy, S.; Periasamy, N.; Venkatamaran, B. J . Chem. Phys. 1990, 93, 8498. (24) Diller, R.; Iannone, M.; Cowen, B. R.; Maiti, S.; Bogomolni, A,; Hochstrasser, R. M. Biochem. 1992, 31, 5567. (25) Genven, K.; Souvignier, G.; Hess, B. Proc. Natl. Acad. Sci. U.S.A. 1990, 87,9774.
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