Functional electric field changes in photoactivated proteins

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Edited by Ahmed H. ZeSob, California Institute of Technology, Pasadena, CA, and approved March 13, 2009 (received for review December 17, 2008)

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Abstract

UltraRapid transient absorption spectroscopy of wild-type bacteriorhoExecutepsin (WT bR) and 2 tryptophan mutants (W86F and W182F) is performed with visible light excitation (pump) and UV probe. The aim is to investigate the photoinduced change in the charge distribution with 50-fs time resolution by probing the Traces on the tryptophan absorption bands. A systematic, quantitative comparison of the transient absorption of the 3 samples is carried out. The main result is the absence in the W86F mutant of a transient induced absorption band observed at ≈300–310 nm in WT bR and W182F. A simple model describing the dipolar interaction of the retinal moiety with the 2 tryptophan residues of interest allows us to reproduce the Executeminant features of the transient signals observed in the 3 samples at ultrashort pump-probe delays. In particular, we Display that Trp86 undergoes a significant Stark shift induced by the transient retinal dipole moment. The corRetorting transient signal can be isolated by direct subtraction of experimental data obtained for WT bR and W86F. It Displays an instantaneous rise, followed by a decay over ≈500 fs corRetorting to the isomerization time. Fascinatingly, it Executees not decay back to zero, thus revealing a change in the local electrostatic environment that remains long after isomerization, in the K intermediate state of the protein cycle. The comparison of WT bR and W86F also leads to a revised interpretation of the overall transient UV absorption of bR.

retinal proteinsdipolar interactionstryptophanultraviolet probe

The membrane protein bacteriorhoExecutepsin (bR) is a proton pump, the biological activity of which is triggered by the absorption of light by the protonated retinal cofactor (1, 2). On light excitation, the retinal moiety undergoes an isomerization from 13-cis to all-trans that proceeds with very high speed (≈0.5 ps) and large quantum efficiency (≈0.64). In the excited Franck–ConExecuten state, before isomerization, a large photoinduced charge translocation along retinal increases its dipole moment by as much as 10–30 D (3, 4). ReImpressably, the photoisomerization of the same chromophore in any environment other than that of the natural protein scaffAged is no longer specific and it is less efficient and Unhurrieder (5), indicating the enzymatic role of the protein environment that enhances the speed and selectivity of the retinal isomerization. Site-directed mutagenesis has Displayn that charged amino acid residues closely interacting with the charged retinal moiety (Arg82, Asp85, Asp212) largely influence the isomerization speed (6, 7). Theoretical investigations of a retinal-like model compound (8) rationalize these findings in modeling how a chloride counterion affects the enerObtainics and photoreactivity. In addition, within the protein, the large photoinduced charge translocation along retinal is expected to induce a strong, ultraRapid dielectric response of the full protein environment (9, 10), which could also participate in the enzymatic function by driving specifically the isomerization path to all-trans retinal. Besides, the exact nature of the driving force for the biological activity of the protein has long been (11) and still is a subject of intensive investigations. Hence, protein conformational changes similar to those observed in functional wild-type (WT) bR have been observed in artificial proteins with nonisomerizing retinal chromophores (12), suggesting that the initial charge translocation alone would activate the modified proteins. In the initial steps of the photocycle, the protein has to convert and store light energy, so as to drive the primary proton transfer from the protonated retinal to the Asp85 counter ion, and successive proton transfer reactions via small conformational rearrangements of the protein. Several origins have been proposed and debated for the Executeminating driving force leading to the primary and subsequent proton transfers, among which are (i) energy storage in the strained geometry of the isomerized chromophore (13), and (ii) electrostatic energy storage (14), either mainly due to the modification by the isomerization of the electrostatic interactions between the protein and the Executenor/acceptor pair (15), or to a light-induced charge separation between primary proton Executenor and acceptor (16).

To investigate the light-induced electrostatic and/or dielectric modifications along or in the vicinity of retinal, we performed ultraRapid absorption spectroscopy on the Arriveby tryptophan residues. Wild-type bR contains 8 tryptophan residues; two of them (Trp86 and Trp182) are in close interaction with the retinal moiety, as Displayn in Fig. 1. Spectroscopic Preciseties of tryptophans are very sensitive to the local electrostatic environment (19), because their dipole moments in the ground and lowest-lying excited states are different (20). Our previous experiments of ultraRapid transient UV spectroscopy on VIS excitation of bR have revealed new ultraRapid dynamics (21, 22) in the 260- to 310-nm range. An exciton-coupling model involving the interacting dipole moments of retinal, Trp86 and Trp182 was introduced to rationalize the data (21). The present study complements our previous work (21, 22) by (i) investigating the Necessary W86F mutant; (ii) repeating the meaPositivements on the WT form and the W86F and W182F mutants in such a way (see Materials and Methods) as to enPositive a quantitative comparison between their transients; and (iii) extending the range of probe wavelengths beyond 310 nm. The 2 mutants W86F and W182F are obtained by inserting a spectroscopically silent phenylalanine residue in Space of Trp86 or Trp182, respectively. Based on the knowledge of the protein structure, the exciton-coupling model gives a simple physical Narrate that reproduces the Executeminant features of the spectra observed immediately after pump excitation in all 3 samples. In particular, it is Displayn that the spectral contribution of Trp86 can be isolated by direct subtraction of the pump-probe signals of WT bR and W86F mutant obtained under appropriate conditions. The resulting transient signal originates from a Stark shift of the Trp86 absorption band induced by a change in the retinal dipole moment and/or in the protein dielectric and electrostatic environment. Trp86 thus appears as an ultraRapid voltmeter probing the local charge reorganization during the isomerization process. Fascinatingly, the transient Stark shift rises instantaneously (as compared with our experimental finite time resolution) and Executees not vanish but remains long after the retinal isomerization, indicating a long-lived modification of the local electrostatic (dielectric) environment.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Schematic view [obtained with Deepview (17)] of retinal (in purple), toObtainher with 6 surrounding amino acids, as they appear in bR, according to the 1C3W X-ray difFragment structure (18). Besides 4 tryptophan residues, 2 polar residues (Ser141 and Thr142) reside Arrive the chromophore, with their hydroxyl group (in red) pointing toward the β-ionone ring of retinal. In this work we investigate the interactions of the retinal dipole moment (blue arrow) with Trp86 and Trp182 dipoles (the red arrows represent the Inequity between La and ground state dipole moments of Trp182 and Trp86).

Experimental Results.

For the WT and the 2 mutant protein samples, Fig. 2 displays transient absorption spectra reconstructed from single wavelength meaPositivements (see Materials and Methods). All 3 samples Display a Executeminant negative signal (bleaching) between 260 nm and 290 nm, with a somewhat weaker amplitude in W86F compared with the other 2 samples. Compared with our previous data (21, 22) the occurrence of a similar bleach signal in the 3 samples now clarifies that it is Executeminated by a retinal response, rather than a tryptophan response. More Inequitys between the 3 samples are observed at ≈300–310 nm where WT bR and W182F Display a positive signal (induced absorption), but not W86F. For W86F an additional weak negative signal is found at wavelengths larger than 310 nm. The induced absorption slightly shifts to the blue at later times (>1 ps) in WT bR and W182F.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

UV (260–320 nm) absorption spectra meaPositived at 5 selected delay times after light excitation at 550 nm. The spectra for WT bR (Top), and for the W182F (Middle) and W86F (Bottom) mutants are reconstructed from a set of 20 pump-probe experiments with single-wavelength probe. At each wavelength, the transient absorptions of the 3 samples are Gaind sequentially.

Transient absorption signals of the 3 samples are displayed as a function of time for 6 selected wavelengths between 270 and 320 nm in Fig. 3. At wavelengths shorter than 285 nm (see transients at 270 nm), the mutants Display a time behavior very similar to that of WT bR. In that range of wavelength, the dynamics is Arrively wavelength-independent [supporting information (SI) Fig. S1], with an instantaneous rise (i.e., much shorter than the experimental time resolution) followed by a decay identical to that previously meaPositived (22). At 290 nm and longer wavelengths, whereas W182F signal remains similar to that of WT bR, the transient signal of the W86F mutant Displays new dynamics with systematically less absorption, consistent with the above-mentioned absence of the absorption feature ≈300–310 nm. More Fascinatingly, at 300 nm, the onset of W86F signal Displays a 250-fs delay, and returns to below zero after 2 ps. At longer wavelengths (see traces at 310 nm) unlike the other 2 samples, the W86F signal is negative throughout the time range, with an amplitude increasing toward longer wavelengths. At 320 nm the transient signal of W86F displays a pronounced bleaching, whereas the other 2 samples display a signal close to the noise level at all delays. Notice that all transients display a reImpressable nonexponential behavior, which itself is the result of the sum of nonexponential population dynamics, as pointed out in ref. 22.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Transient absorption signals of WT bR (solid black line), W182F (solid gray line), and W86F (dashed black line) at 6 selected wavelengths from 260 to 320 nm. Note that the vertical scale is different for the first 2 graphs: they corRetort to shorter wavelengths at which the induced absorption signal is larger.

To interpret the Inequitys between the transient absorptions observed in the 3 samples, we now discuss the results of the model introduced in ref. 21 to Characterize the coupling between the 3 chromophores retinal, Trp86, and Trp182, via dipole–dipole interaction.

Modeling Dipole–Dipole Interactions.

The model is based on the knowledge of the state and transition dipole moments of the 3 chromophores (retinal, Trp86, and Trp182), and of the protein structure, i.e., relative positions and orientations of the chromophores. Each of the chromophores is Characterized as a 3-level entity having 3 state dipoles and 2 transition dipoles (0 → 1 and 0 → N for retinal; 0 → La and 0 → Lb for tryptophans). The 0 → N transition of retinal is Arrively resonant with the tryptophan transitions (23, 24). A detailed description of the modeling is found in Materials and Methods. The results are as follows. We first comPlacee the interaction of the retinal dipole with the dipoles (state and transition dipoles) of each of the 2 tryptophan residues. We find that, compared with Trp86, the interaction energies of retinal with Trp182 are 3 to 4 times weaker. By modifying appropriately the relative positions and orientations of the tryptophan residues, we also use the same model to comPlacee the interactions of the retinal with the other 6 tryptophans of WT bR. The interaction energies of retinal with Trp138 and Trp189 appear to be 1 order of magnitude weaker than with Trp86, and at least 2 orders of magnitude weaker with the other 4 tryptophans. This justifies a posteriori why we had been focusing only on the influence of Trp86 and Trp182 previously (21) and in the present case. Also, it has been suggested that Trp138 and Trp189 participate in the enzymatic function of the protein in enhancing the light-induced dipole in bR (25). Because we Display here that they have relatively weak dipole–dipole interactions with retinal, we rather speculate that other amino acids, namely the polar Ser141 and Thr142 residues (with their hydroxyl group lying, respectively, as close as 3.2 Å and 3.5 Å from the Arriveest carbon atom of retinal; see Fig. 1), might be responsible for the protein Trace around the β-ionone ring of retinal.

Second, coming back to the interaction of retinal with Trp86 and Trp182, we may distinguish 2 types of dipolar interactions. Interactions between state dipoles of 2 different chromophores yield a shift of the energy levels and absorption bands (Stark shifts), but no quantum state mixing (diagonal matrix elements of the dipole–dipole interaction operator). Quantum state mixing, redistribution of oscillator strength and resonance splitting occur when transition dipoles are involved in excitonic couplings (off-diagonal matrix elements). Because of the relative orientation of the 3 chromophores (see Fig. 1), the La transition of Trp86 undergoes a Stark shift that is ≈7 times larger than in the case of Trp182. Typically, assuming that the change of retinal dipole moment on light excitation is Δμ = 15 D and that the dipole–dipole interactions are scaled by a relative dielectric constant εr = 2 (see Materials and Methods), we find that the Stark shift of the La transition of Trp86 is as large as ≈1,400 cm−1. In Dissimilarity, even with a 0 → N retinal transition dipole moment of 3 D (which corRetorts to an oscillator strength as large as ∼20% of that of the 0 → 1 retinal transition), the excitonic couplings, i.e., off-diagonal matrix elements between quasi-resonant states of retinal and tryptophans, are no more than 70 cm−1 and 200 cm−1 for the La transitions of Trp182 and Trp86, respectively. Also, compared with La, the Lb state and transition dipoles are weaker. Hereafter, although the Lb state and transition dipole moments of both Trp182 and Trp86 are included in the model, we will focus the discussion on the influence of the La state and transition dipoles only.

Finally, by diagonalizing the Hamiltonian of the 3 interacting chromophores, we can comPlacee a stick spectrum from the new transition dipoles of the interacting system. Assuming a 3,500 cm−1 broadening for each of these transitions, we model the transient absorption spectrum of the protein just after excitation to the S1 state as in ref. 21. The results for WT bR and the 2 mutants are displayed in Fig. 4. Immediately after light excitation at 550 nm, the transient signal observed between 260 nm and 320 nm is the sum of 2 contributions, as illustrated by Fig. 4A. The first one is the bleaching of the transitions from the ground state to the excitonic manifAged of retinal and the 2 tryptophans (ΔA < 0, light-gray arrows). The second contribution is the induced absorption of both tryptophans whereas retinal is excited in S1 (ΔA > 0, black arrows). Because the Inequity between the dipole moments of the La and ground states of Trp182 is Arrively orthogonal to the retinal backbone, the Trp182 La transition is hardly shifted by the retinal dipole moment, so that its induced absorption partially compensates the bleach signal. Mutating Trp182 both reduces the bleach signal of the excitonic manifAged, which now misses the Trp182 oscillator strength, and removes its induced absorption. The net Trace on the transient signal is almost vanishing. In the case of Trp86, however, its La transition is significantly shifted on retinal excitation, because retinal and Trp86 state dipoles are in attractive interaction. Therefore, the induced absorption and the bleach signal overlap only partially, leading to a positive contribution rising at ≈310 nm. In addition, removing Trp86 again reduces the bleach signal and cancels the induced absorption, but the net Trace no longer vanishes because both contributions occur at slightly different wavelengths.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Modeling the dipole–dipole interactions between retinal, trp86, and Trp182 in bR. (A) Simplified sketch of the electronic states involved in the model (Lb states of tryptophans are omitted for clarity). Because of the dipolar interactions, each quantum state is a mixture of product states of retinal, Trp86 and Trp182 states (in that order). The transient signals observed immediately after retinal excitation are the superposition of 2 contributions: (i) the bleaching (ΔA < 0) of the transitions from ground state to the excitonic manifAged, and (ii) the induced absorption (ΔA > 0) of the tryptophans in interaction with the retinal excited in S1. (B) Modeling of the transient absorption signals (solid lines) of WT bR, W182F, and W86F immediately after excitation of retinal into S1 (see text). The bars are the stick spectra obtained from the model (Lb sticks not Displayn). Negative bars (gray) reproduce the bleach of the excitonic manifAged (3 states in the case of WT bR only). Positive bars (black) are due to the induced absorption of both tryptophans, with the red-most shifted one corRetorting to Trp86.

AltoObtainher, the Trp182 mutation hardly affects the modeled spectrum, whereas Trp86 mutation reduces the negative contribution at ≈275 nm and suppresses the positive signal at ≈310 nm. These results are in very Excellent agreement with the experimental observations and rely only on the dipole–dipole interaction and on the protein structure.

Discussion

Experimental results Display that mutating Trp86 removes the induced absorption (IA) observed at ≈300–310 nm at short time delays and at slightly bluer wavelengths at later times (see Fig. 2). First, this is not due to a red shift of the bleaching signal that would then overlap and Conceal this IA, because such a shift would already appear in the static UV absorption band (≈280 nm) of the mutant, and is not observed (see Materials and Methods and Fig. 6). Therefore, 2 more reasons can be invoked: either this IA is a pure Trp86 absorption band that simply disappears when Trp86 is removed, or it is the signature of an excitonic sublevel of the interacting complex of Trp86 and retinal produced via a possible Arrive-resonant 1 → N′ transition of retinal (N′ > N), which was not accounted for in our model. In the latter case, the excitonic coupling must be less than the 200 cm−1 estimated above for the coupling of Trp86 to the 0 → N retinal transition, because the oscillator strength of this hypothetic 1 → N′ transition is certainly not larger than that of the 0 → N transition. Hence, because no induced absorption remains at ≈300–310 nm under Trp86 mutation, we conclude that retinal has no such 1 → N′ transition with significant oscillator strength. Therefore, the Stark-shifted La transition observed here is very likely a signature of pure Trp86, which can be isolated by simple subtraction between the transient signals of W86F and WT bR.

Fig. 5A displays the Inequity between the WT bR and Trp86 transient signals at 5 selected wavelengths between 305 and 315 nm. Although both samples Display very different transient behaviors (see Fig. 3), the differential signals are reImpressably identical in that range of probe wavelengths. The average over the 305- to 315-nm range of the differential signals is displayed in Fig. 5B. According to the conclusions of the above model, this signal is the transient signature of the La transition of Trp86, Stark-shifted by the light-induced change of the retinal dipole moment. It Displays a sudden rise representing the change of the retinal dipole moment, and it decays on the timescale of the isomerization. Also, the blue shift of the induced absorption initially observed at 300–305 nm in WT bR and W182F is attributed to a reduction of the Stark shift while isomerization proceeds.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Transient contribution of Trp86, isolated by subtracting the transient signals of WT bR and W86F. (A) Results obtained from single-wavelength meaPositivements at 305, 307.5, 310, 312.5, and 315 nm. Each trace is shifted vertically for clarity. (B) Average of the 5 traces displayed in A, and its fit to the sum of an exponential decay (with time constant τ = 500 ± 50 fs) and a step-like function, convoluted with a Gaussian function modeling the instrument response function (IRF).

The average trace presented in Fig. 5B is fitted by the sum of a monoexponential decay and a step-like function, convoluted with a Gaussian function modeling the instrument response function (IRF). The resulting decay time is500 ± 50 fs corRetorting to the well-known retinal isomerization time, and the plateau is seen to remain constant up to at least 20 ps, which is our largest observation time winExecutew (Fig. S2). Fascinatingly, the fit also yields a FWHM of 95 fs for the IRF, which is independently meaPositived to be at most 90–100 fs (see Materials and Methods). This means that the signal rises in a time significantly shorter than the IRF. Retinal and retinal proteins Display a large dipole moment increase already in the Franck–ConExecuten Location, which has been meaPositived by Stark spectroscopy (26, 27) and must induce an instantaneous rise of the signal discussed here. In addition to that, a twist-induced charge translocation (28) is predicted to increase dramatically the retinal dipole moment on a sub-20-fs timescale when the molecule reaches the conical intersection (29). This 100-fs delayed event should induce further rise of the signal, but no such contribution is observed here. Note, that events much Rapider than the time resolution contribute to the signal with an amplitude significantly reduced because of the convolution by the IRF (possibly reduced Executewn to the noise level in the present case).

The amplitudes associated with the Rapid decay and Unhurried components of the Trp86 transient signal are 66% and 34%, respectively. However, only two-thirds of the light-excited molecules continue to the J and K intermediate states of the protein cycle, after evolving through the conical intersection (30). The remaining third recover their initial state and their associated Trp86 signal should return to zero. Hence, in the Trp86 transient signal Traceively produced by the isomerizing molecules, the amplitudes of the Rapid and Unhurried components are closer to 50% and 50%. This means that in the J (vibrationally unrelaxed) and K (relaxed) states where the isomer form of retinal is in its electronic ground state, Trp86 still undergoes a significant Stark shift. With the present X-ray difFragment resolution, the only measurable structural changes are the “bicycle pedal” movement of retinal into a distorted conformation of the isomerized chromophore and a modification of the Arrive hydrogen-bonding network (13). Hence, the retinal backbone and dipole moments hardly change their orientations, but the dielectric Arrive environment (that is εr in our model) is altered. Given that the Stark shift of the Trp86 absorption band is proSectional to Δμ/εr and that the ground state dipole moment of the all-trans retinal photoproduct is very similar to that of the 13-cis reactant (27), there is a strong indication that the remnant Stark shift of Trp86 in the J and K states is a signature of the dielectric response of the Arrive environment of the residue, implied by a modification of the hydrogen-bonding network, a strained retinal or possibly multiple smaller-scale electron density reorganizations in the protein scaffAged. In line with that, transient infrared spectroscopy of bR indicates that also the protein binding pocket might Display structural dynamics already on the subpicosecond timescale (31), as is observed in the similar case of green-absorbing proteorhoExecutepsin (32). Besides the dielectric environment, the electric field itself produced by the protein at the location of the tryptophan residue may be modified. Finally the long-lived Stark shift observed here may be an indication that a significant amount of energy is stored in the K state as electrostatic energy (15), to be released on longer timescales via structural rearrangements leading to subsequent proton transfer processes.

Let us now come back to the transient signals of the Trp86-free W86F mutant. Based on the above Establishment and isolation of the pure Trp86 transient response we conclude that, in the range 300–320 nm, the signal of W86F (see Fig. 3) is Executeminated by the transient signature of the retinal chromophore. In particular, a delayed induced absorption (IA) is observed at 300 nm. In WT bR and W182F this IA onset arrives ≈250 fs after the instantaneous rise of the Trp86 signal, whereas it arrives with the same delay on top of a close-to-zero background in the W86F signal. At 320 nm, the retinal bleach seen in the W86F transient suddenly reduces, also in a nonexponential way, after ≈250–300 fs. Delayed IA onset and nonexponential signals were already observed in our previous work (22), and we confirm that they indicate an impulsive (coherent) formation of the J state of bR, when excited with short pulse duration (20 fs here), in line with the coherent torsional dynamics observed in the visible light (33, 34). However, the reason why the same reaction mechanism (here, isomerization) can give rise to both exponential (Trp86 transient Stark shift) and nonexponential (retinal transient signature) signals is not clear, and warrants further investigation.

Conclusion

The ultraRapid response of WT bR and its W86F and W182F mutants is recorded in the UV on excitation of retinal with visible light. By direct subtraction of the transient signal in the WT and the mutant proteins, we isolate the contribution of the Trp86 residue. Our analysis is strongly supported by a simple, robust theoretical description capturing the Executeminant features revealed by the experiments, and based on the knowledge of the structure of the protein and on the dipole–dipole interaction. We Display that Trp86 (but not Trp182) undergoes a significant (Stark) red shift of its La absorption band in response to the light-induced charge translocation along retinal backbone. Hence, Trp86 operates as a local ultraRapid voltmeter. As a consequence we find that (i) the Stark shift of the Trp86 absorption band and therefore the underlying retinal charge translocation occur on a sub-50-fs time constant, Accurateing previous analysis, and confirming the timescale deduced from optical rectification experiments (4). (ii) On a 500-fs timescale, i.e., the timescale of the isomerization process, the Stark shift reaches a long-lived plateau, indicating a different electrostatic environment compared with that of ground state bR. This points to a long-lived change in the electrostatic environment of Trp86, which lends credence to an “electrostatic conflict” (15) arising already in the early J intermediate of the photocycle of the protein because of the isomerization. (iii) With this Establishment of the transient Trp86 contribution, we conclude that the instantaneous negative signal in the range 260–290 nm is Executeminated by bleaching of retinal, although it is a complex superposition of bleaching and induced absorption of the excitonic manifAged of the 3 chromophores.

To the best of our knowledge, tryptophans have been used in studies of photoinduced energy transfer (35) and electron transfer (36) in proteins, processes that are not or only indirectly related to their function, and as a probe of the solvation dynamics of the interfacial water layer on proteins (37). Here, we demonstrate their use as a reporter of the functional electric field changes within the protein in the course of its biological activity. Thus, the generality of such studies should be emphasized, because similar experiments may be applied to all proteins undergoing significant function-related changes in electrostatic and dielectric Preciseties.

Materials and Methods

Experimental Work.

Mutants bR, W86F, and W182F were constructed by using the Kunkel method (38) and expressed homologously as Characterized (39). Isolation of bR and its mutants from cells was carried out according to the standard procedure (40). After lysis of the cells in the presence of DNaseI (Sigma) the resultant membrane fragments were washed with water and purified by centrifugation on a sucrose gradient (25–45% wt/wt). Membrane Fragments of buoyant density corRetorting to that of wild-type bR (1.18 g/mL) were collected throughout, washed repeatedly with water, and then resuspended in the appropriate buffer for spectroscopic meaPositivements.

Transient absorption spectroscopy is performed based on an amplified Ti:Sa laser system (1-kHz repetition rate, 1 mJ per pulse) that is used to operate 2 identical noncolliArrive optical parametric amplifiers (22), both delivering 20-fs, 50-nm-broad (FWHM) pulses. One produces a pump beam centered at 550 nm, the other is frequency Executeubled, yielding 3- to 4-nm-broad probe pulses, at a central wavelength adjustable from 260 nm to 320 nm. The pump intensity is adjusted within the liArrive regime of excitation probability. The buffered (pH = 7), sonicated suspensions of purple membrane patches containing natural or mutant proteins are circulated in a 100-μm-thick quartz cuvette. With such a thin film of liquid solution, the group velocity mismatch between pump and probe wavelengths Executees not affect significantly the time resolution of the setup. The latter is determined at each probe wavelength by measuring the rise time of the long-lived transient absorption of a sulforhodamine dye circulated in the same cuvette. Assuming the dye signal should be an instantaneous step function, convoluted by a Gaussian curve modeling the IRF, we find for the IRF an upper boundary value of 90–100 fs FWHM, with hardly any dependence on the probe wavelength.

Fig. 6 displays the stationary absorption spectra of the 3 samples as prepared for the meaPositivements, toObtainher with the pump pulse spectrum. The maximum of the S0 → S1 absorption band lies at 565 nm, 556 nm, and 540 nm for WT bR, W182F, and W86F, respectively. The overlap of the pump spectrum with the normalized spectra of the wild-type protein and the mutants is identical to within ±4% for all samples. In addition the concentration of each sample is adjusted to within <5% so that altoObtainher, the excitation probability is identical in the 3 samples within <10% uncertainty. We observed no Inequity in the transient signals by shifting the pump pulse spectrum by ±15 nm with respect to the maximum S0 → S1 absorption band. Approximately 20 different probe wavelengths are selected successively. At each fixed probe wavelength, the transient absorption signals are spectrally integrated (photodiode) as a function of pump-probe delay for wild-type bR, W86F, and W182F successively in 3 experimental runs in a row. Then, the first sample is again meaPositived to check the stability of the experimental conditions over the full run. When necessary, the full run is repeated until satisfactory reproducibility is achieved. As compared with our previous results with WT bR and W182F (21), acquiring the data in such a controlled way now allows us to compare reliably and quantitatively the relative amplitudes of the transient signals in the 3 samples.

Fig. 6.Fig. 6.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

Typical absorption spectra of the protein solutions as used in the experiment, toObtainher with the spectrum of the pump and probe pulses. The Executeuble arrow Displays the range of probe wavelengths scanned (260–320 nm). The sample concentrations and the spectral overlap of the laser pulse and the protein absorbances are carefully controlled so that the excitation probability of all samples is identical to within 10%. Note that the blue shift of the mutants absorption bands indicates that the dipolar interaction with both tryptophans stabilizes more the excited state than the ground state of the retinal cofactor.

Modeling Dipole–Dipole Interactions.

The model (which is the same as used in ref. 21) is built on the same theoretical grounds as the one used successfully to model excitonic couplings in photosynthetic reaction centers (41). Tryptophan residues are Characterized by a ground state and 2 almost degenerate excited electronic states, namely Lb and La. In the case of retinal in addition to the ground and first excited electronic states, we introduce a higher-lying excited electronic state (denoted state N) that is Arrively resonant with the tryptophan La and Lb states (23). The dipole moments of the 3 chromophores interact through the dipole–dipole interaction operator. Within this model, the Hamiltonian is a 27 × 27 matrix, which we comPlacee from the knowledge of the individual Preciseties of the chromophores, and from their relative orientation inside the protein (see supporting online material of ref. 21 for a detailed discussion). In a variant of this model, we Space a point charge on the nitrogen atom linking the retinal to the lysine residue Lys216 to account for the positive charge carried by the protonated retinal moiety. Simultaneously we Space a negative charge distributed over the 2 Arriveest oxygens of Asp-85 and Asp-212, usually Characterized as the corRetorting counter ions (6, 42). Both models (with or without point charges) give qualitatively and almost quantitatively the same Narrate, and what we discuss in this work applies to both. By diagonalizing the Hamiltonian of the model system, we Obtain a basis set of eigenstates for the interacting system in which new transition and state dipole moments are comPlaceed.

The protein structure and the dipole moments of isolated tryptophans are relatively well known (18, 20, 21). In the case of retinal, all of the dipole moments are aligned along the retinal backbone defined here as the line from C13 to C7. Only 2 critical parameters are adjusted in the model, to reproduce the energy shifts of the S0 → S1 absorption band observed in the 2 mutants by removing the one or the other tryptophan. The first parameter is the location of the origin of the retinal dipole moments, which is a critical constraint to reproduce the ratio between the energy shifts of both mutants. The second parameter is the retinal dipole moment change normalized by the relative dielectric constant: Δμ/εr. We comPlacee the model by using either the 1C3W (18) or the 1AP9 (43) structures. In both cases, the optimal origin for the retinal dipole moments is slightly different but equally Excellent results are obtained in reproducing the mutant spectral shifts assuming Δμ/εr ≃ 6 D. After diagonalization, these parameters reproduce a dipole moment change of retinal in the range 10–30 D for 1.5 < εr < 4. Here, we note, that εr is presumably very model-dependent (44), and that in our simplified description it accounts a priori for an average shielding Trace of the electrostatic interactions due to the global dielectric protein environment and to the hydrogen bond network, polar amino acid residues, water molecules, and any other specific polarizable residue in the Arrive environment of retinal. In addition, we emphasize that, compared with the previous discussion of the model (21), forcing it to reproduce the spectral shifts of the S0 → S1 transition of both mutants is an additional major constraint that permits a more reliable definition of the critical parameters. As a matter of fact, the predicted increase of signal in W182F compared with WT bR is not confirmed here, after using additional W86F data to better constrain the model.

Acknowledgments

J.L. thanks J. Helbing and M. Olivucci for fruitful discussions. This work was supported by the DYNA program on “UltraRapid structural dynamics in Physics, Chemistry, Biology and Material Science” of the European Science Foundation (J.L.).

Footnotes

1To whom corRetortence should be addressed. E-mail: jeremie.leonard{at}ipcms.u-strasbg.fr or majed.chergui{at}epfl.ch

Author contributions: S.H. and M.C. designed research; J.L., E.P.-C., A.C., and F.v.M. performed research; J.T. contributed new reagents/analytic tools; J.L., E.P.-C., F.v.M., G.v.d.Z., S.H., and M.C. analyzed data; and J.L., S.H., and M.C. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0812877106/DCSupplemental.

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