Probing Specific Molecular Processes and Intermediates by Time

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Probing Specific Molecular Processes and Intermediates by Time-Resolved Fourier Transform Infrared Spectroscopy: Application to the Bacteriorhodopsin Photocycle Víctor A. Lorenz-Fonfría,†,* Hideki Kandori,‡ and Esteve Padros† †

Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, and Centre d’Estudis en Biofísica, Universitat Autonoma de Barcelona, Barcelona 08193, Spain ‡ Department of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan

bS Supporting Information ABSTRACT: We present a general approach for probing the kinetics of specific molecular processes in proteins by timeresolved Fourier transform infrared (IR) spectroscopy. Using bacteriorhodopsin (bR) as a model we demonstrate that by appropriately monitoring some selected IR bands it is possible obtaining the kinetics of the most important events occurring in the photocycle, namely changes in the chromophore and the protein backbone conformation, and changes in the protonation state of the key residues implicated in the proton transfers. Besides confirming widely accepted views of the bR photocycle, our analysis also sheds light into some disputed issues: the degree of retinal torsion in the L intermediate to respect the ground state; the possibility of a proton transfer from Asp85 to Asp212; the relationship between the protonation/deprotonation of Asp85 and the proton release complex; and the timing of the protein backbone dynamics. By providing a direct way to estimate the kinetics of photocycle intermediates the present approach opens new prospects for a robust quantitative kinetic analysis of the bR photocycle, which could also benefit the study of other proteins involved in photosynthesis, in phototaxis, or in respiratory chains.

’ INTRODUCTION To achieve their function (e.g., proton pump, substrate translocation, etc.) membrane proteins undergo conformational changes in response to external stimuli (e.g., light absorption, substrate binding, etc.). The structural and dynamical characterization of intermediate conformations populated during protein function is arguably a prerequisite for a comprehensive understanding of the molecular mechanism by which they work, often requiring the application of time-resolved techniques.1 The lightdriven proton-pump mechanism of bacteriorhodoposin (bR) is a suitable experimental model where such characterization has been addressed with unparalleled detail.26 However, final progress in understanding its proton-pump mechanism under functional conditions has been largely impeded by the huge analytical difficulties to characterize intermediate states from time-resolved data. Bacteriorhodopsin is a seven transmembrane helix integral membrane protein, containing in its light adapted form an alltrans retinal covalently bound to the protein (opsin) through a protonated Schiff base (SB) with Lys216 (Figure 1a). Upon light absorption retinal photoisomerization from all-trans to a distorted 13-cis 15-anti takes place, triggering a thermal relaxation process known as the bR photocycle (Figure 1a).7 In the photocycle, bR populates a series of distinguishable intermediate states (K, L, M, N, and O) before relaxing back to the initial r 2011 American Chemical Society

ground state (hereafter BR in capital letters), a process taking less than 30 ms at room temperature and neutral pH. The intermediate states are characterized by distinct changes with respect the BR state, namely in the retinal conformation, the SB protonation state, the protein backbone conformation, and/or in the environment and protonation state of amino acid side chains and internal waters.6,810 This photoinduced cyclic process is accompanied by the vectorial transport of one proton from the cytoplasm to the extracellular side per photoisomerized retinal.3 The proton transport is not accomplished in a single step but involves at least five molecular protonation/deprotonation steps,11 which generally tightly correlate with the transition between specific intermediate states, as illustrated in Figure 1a. Intermediary states populated during the bR photocycle were first detected and characterized in the visible (VIS),3 by the distinctive shifts in their retinal absorption wavelength maximum. Later, the same intermediates have been detected and characterized in deeper structural detail by several other techniques, with noteworthy contributions from resonance Raman (RRaman)1214 and infrared difference (IRdiff)911 spectroscopies and more recently from X-ray crystallography.15,16 The structural Received: February 22, 2011 Revised: May 2, 2011 Published: May 26, 2011 7972

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The Journal of Physical Chemistry B

Figure 1. View of the bacteriorhodopsin (bR) photocycle. (a) Backbone and solvent accessible surface of bR in the light-adapted ground state.97 The retinal and the Schiff base (SB), and some amino acid sidechains and internal water molecules are also displayed. Overlaid are the five proton transfer, and places where these occur during the photocycle. (b) The fraction of intermediates after light-induced retinal photoisomerization at 25 °C and pH 7 as estimated by Van Stokkum and Lozier.29 (c) Experimental time-resolved infrared difference spectra of the bR photocycle.

characterization of bR photointermediates has been greatly facilitated by the means of steady-state studies on specific intermediates, performed either by trapping them (e.g., by low temperature) and/ or by artificially enhancing their accumulation (e.g., by site-directed mutagenesis)3,8 hence bypassing the technical and analytical difficulties of time-resolved studies. However, these studies have raised reasonable concerns regarding their functional relevance.17,18 There is also doubts about how purely intermediates have been “trapped”,19,20 and examples where a mixture of intermediates have been considered to represent a single intermediate probably are not uncommon. Last but not least, these steady-state studies have been able to provided only limited insight into the dynamics/thermodynamics of the intermediates transitions. The far more relevant characterization of the functional bR photocycle has been paved with severe challenges. On one side, time-resolution requirements have limited the number of techniques applicable to study the bR photocycle at room temperature, although technical advances continue.2123 On the more fundamental side, final progress has been limited by the complexity of the analysis of time-resolved data. As illustrated in Figure 1b several intermediates (generally an unknown number) appear simultaneously populated at any time, with the collected data (Figure 1c) representing a time-variable weighted mixture of the signal from each unknown intermediate’s spectrum. The habitual goal has been the identification of the number of intermediates, followed by the decomposition of the experimental time-resolved data into the time-evolution and the spectrum for each intermediate.2427 However, in spite some notable analytical efforts,25,2830 such decomposition remains overwhelmingly ambiguous,24,26 as painfully illustrated by the inconsistent estimates for the time-evolution of intermediates reported by different groups31 (see also Figure S1). More robust and computationally simpler alternatives for the analysis of time-resolved spectroscopic data would be desirable.

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A known alternative invokes probing directly the kinetics of the different intermediates,26 by using a time-resolved technique able to probe selectively a molecular process specifically associated to a given intermediate. Among the different spectroscopies, Fourier transform IRdiff spectroscopy has the distinctive property of being reasonably sensitive to changes in the conformation and protonation state of the players in the bR photocycle.9 Moreover, in the time-resolved step-scan collecting mode events from the nanoseconds until the end of the photocycle can be monitored with an adequate signal-to-noise ratio and a wide wavenumber range.3234 However, to attain this potentiality, the appropriate molecular vibrations have to be chosen and selectively probed. This is a nontrivial task given the numerous and considerably overlapped bands that crowd the IRdiff spectra of proteins. Consequently, in spite the considerable number of time-resolved IRdiff works on the bR photocycle published in the last 25 years, the potential information contained in the time-evolution of some selected marker bands has been explored in relatively few cases and to a rather limited extent.17,18,3541 The present work aims to fill this gap, and to inspire other researchers in the bR field and outside it to take full advantage of the rich spectral and kinetic information contained in time-resolved IRdiff spectra of proteins. In the first part of the paper, we briefly discuss some simple but cost-effective approaches to monitor the kinetics of a band with minimal cross-contaminations from the kinetics of a nearby overlapping band. In the second part we characterize the time evolution of a repertory of IR bands with the potential to reveal the kinetics of different molecular processes taking place during the bR photocycle. The suitability of the final choices, i.e., which bands to follow and how to probe which intermediate, has been assessed whenever possible by comparison with previously published analysis of the bR photocycle and/or with parallel experiments using time-resolved VIS spectroscopy (flash photolysis).

’ EXPERIMENTAL METHODS Transient Absorbance Changes in the Visible by Flash Photolysis. We used the data from Peralvarez-Marín et al.41

Briefly, purple membrane suspensions in 1 M KCl pH 6.5 at room temperature (∼25 °C) were monitored from 350 to 700 nm in 5 nm steps, with data collection extending from ∼2 μs to ∼120 ms. Time traces were quasi-logarithmically averaged to a maximum of 20 points/decade. Time-Resolved Step-Scan Infrared Spectroscopy. Sample preparation and data acquisition have been described before.41 An aliquot of purple membrane in 2 mM potassium phosphate and pH 7.0 containing WT bR (∼55 μL) was dried on a 2.5 mm diameter BaF2 window and hydrated in a atmosphere of ∼99% relative humidity. The sample holder was connected to a circulatory thermostatic bath set to 25.0 °C, with the sample temperature at 24.8 °C. The photocycle was initiated 16 times at 632 different mirror retardations by a 532 nm laser at 8.3 Hz repetition rate, and the signal of a MCT detector was sampled at 200 kHz with a 16-bits ADC. The useful spectral range of the obtained time-resolved IR difference spectra extended from 2000 to 800 cm1 at 8 cm1 resolution, with the upper and lower bound limited by an optical filter and the BaF2 window, respectively. The first data point after the laser excitation was set to 2 μs (corresponding to data averaged from 1 to 4 μs, where time 0 corresponds to laser excitation) and from then on in 5 μs steps (6.5 μs, 11.5 μs, etc) until 31.2765 ms. For signal-to-noise 7973

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The Journal of Physical Chemistry B improvement, 12 such data sets were recorded and averaged. Two such experiments using independent samples were collected. We centered the analysis in the data set of higher quality, previously used in Peralvarez-Marín et al.41 However, we should note that the other data set provided similar kinetics (see Figure S2), illustrating the reproducibility of the time-traces provided here. For data reduction the time-resolved data was averaged quasi-logarithmically to a maximum of 20 spectra/decade. The resulting time-resolved IRdiff data is presented in Figure 1c. When probing the kinetics of the intensity at a given wavenumber, the original and deconvoluted time-resolved IRdiff spectra were offset in the 19501850 cm1 region to reduce the baseline oscillations that affect step-scan measurements in the millisecond.32 When measuring the area of the continuum band, a timeincreasing linear drift in the baseline of the IRdiff spectra was corrected assuming it to rise linearly with time as shown in Figure S3. Simulation of Intermediates Kinetics. These were performed as described before.42 Briefly, kinetic matrices were constructed using intrinsic rate constants for the bR photocycle reported in the literature,29,4345 and their eigenvalues and eigenvectors were used to compute the time-evolution of the intermediates predicted by each study. Data Processing and Analysis. The phase-corrected first derivative was performed in the Fourier domain,46,47 using a “Sinc2” filter and a cutoff point of 0.125 cm, corresponding to an instrumental resolution of 8 cm1. Fourier deconvolution was performed using a Lorentzian band of 12 cm1 full width at half height, a narrowing factor of 1.5, and a Bessel filter.48,49 A homemade interactive program with a graphical interface, available by request, was used to extract time-traces of bands from time-resolved spectroscopic data (an screen capture is shown in Figure S4). It allowed following the intensity at any adjustable wavenumber, and the area between any given intervals, with and without an internal baseline included. However, we should clarify that the same time-traces presented here could be obtained manually, albeit with a much longer inversion of time. Another homemade routine was used to automatically compute the peak wavenumber of a band as a function of time, assisted by the buildin Matlab interpolation function “interp1” with the “spline” option. All calculations and programs were run in Matlab v7.

’ RESULTS AND DISCUSSION Improving Band Selectivity in Time-Traces. IRdiff spectra of proteins usually contain numerous positive and negative bands, with considerable band overlap between them. In order to follow the time-evolution of a given band, in possibly hundreds of spectra, we need a computationally fast and robust method to extract its intensity or area. Moreover, this should be done as selectively as possible, i.e., reducing as much as possible contributions from the intensity/area of neighbor bands. The problem of band selectivity in relation to intermediate kinetics is illustrated in Figure 2. In the example portrayed, roughly inspired in the CdO stretching of the Asp85 residue in the photocycle of bR, we constructed synthetic time-resolved spectra made of two Lorentzian bands. One of them is centered at 1762 and the other at 1753 cm1 (Figure 2a, top), each characteristic for a different intermediate and thus with a specific kinetic trace (Figure 2a, bottom). We would like to monitor the kinetics of the 1762 cm1 band selectively, i.e., of one of the intermediates. As we show in Figure 2b, this can not be done

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Figure 2. Methods to improve band selectivity. (a) Two bands (top) and their individual kinetics (bottom). (bf, top) Spectrum of the sum of the two bands (black line), and representation of the percentage of each band that is probed by measuring: (b) the intensity; (b,c) the area with a baseline; (d) the intensity in a phase-corrected first derivative spectrum; and (f) the intensity in a deconvoluted spectrum. (bf, bottom) The resulting probed kinetics in each case (black line), with the individual contributions (dashed color lines).

accurately by simply following the intensity at 1762 cm1 because of substantial contributions (36%) from the kinetic trace of the 1753 cm1 band. In contrast, we could probe the kinetics of the 1762 cm1 band with enhanced selectivity by simply measuring the area around its maximum (including a baseline), with only 1% of contamination (Figure 2c). More modest improvements in selectivity would be obtained if the interval chosen does not efficiently minimize contributions from neighbor bands, for instance by covering a wavenumber region where the neighbor band shows a pronounced curvature, as illustrated in Figure 2d. Another approach to enhance band-selectivity is to preprocess the spectra using a band-narrowing method. One simple way to narrow bands is by the use of derivatives. The second derivative is normally used because it preserved an absorption-like band shape, but it can generate intense side-lobules that can compromise band selectivity (see Figure S5). A generalized approach to derivatives allows applying any derivative order while preserving an absorption-like band shape (i.e., phase-correction).46 Figure 2e (top) shows the phase-corrected first derivative spectra of Figure 2a (top). The intensity at 1762 cm1 provided the kinetics of the 1762 cm1 band with a small (∼5%) contribution from the kinetics of the 1753 cm1 band (Figure 2e). Deconvolution, which attains band-narrowing with a considerably better behaved 7974



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Figure 3. Kinetics of the retinal ground state recovery. Experimental kinetics (red line) from (ad) time-resolved IRdiff spectroscopy and (e) flash photolysis. Scaled kinetics for bR ground state recovery reproduced from van Stokkum and Lozier’s analysis29 (pale blue line). Here and in subsequent figures the insets show three spectral snapshots at some selected time (black line), indicating in red the measured intensity/area.

final band-shape,50 can be used as an alternative to derivation to enhanced band selectivity (Figure 2f). We should finally note that both derivatives and deconvolution go associated with noise enhancement,46,50 and therefore they should be applied with caution. Monitoring Changes in the Retinal Conformation. Retinal Ground State Recovery. The recovery of the retinal back to the BR state conformation, relaxed all-trans retinal, defines the duration of the bR photocycle. Ground state recovery kinetics can be monitored by probing a band characteristic of the retinal in the BR state, absent or shifted in the rest of intermediates. This band should be negative, and display a constant intensity (or area) in all of the intermediates IRdiff spectra. A band with such properties can be used to normalize different samples for the same amount of bR molecules entering the photocycle (spectral normalization).41 The negative band in the retinal fingerprint region at 1254 cm1 seems a priori one of the most suitable bands to monitor BR recovery kinetics (see insets in Figure 3a). This band originates from the coupling of the retinal C12C13 and C14C15 vibrations with the Lys216-SB CεNHþ vibration in the BR state.51 In the RRaman spectrum this band is missing (or reduced in intensity) in all of the intermediates,12,13 possibly because the above commented coupling is only fully preserved in the BR state. The results of RRaman spectroscopy are reproduced in the IR, where a negative band at 1254 cm1 appears in all of the intermediates IRdiff spectra.30,52,53 This band has been used for normalization of IRdiff spectra in the past.41,52 The intensity of the 1254 cm1 band did not provide a reasonable BR recovery kinetics at any wavenumber (not shown). Using the deconvoluted spectra and playing with the integration interval we obtained important improvements (see Figure S6a). Jointly monitoring the area from 1287.5 to 1239 cm1 in the original spectra provided even better results, with a kinetic trace satisfactorily fulfilling the characteristics expected for BR recovery kinetics (Figure 3a, red line). The kinetics remains fairly constant below 1 ms, and when the BR recovery starts the kinetics smoothly decreases to zero in 30 ms,

never taking positive values. Three snapshots of the retinal fingerprint region are shown in the insets of Figure 3a, highlighting in red color the band integrated area. Figure 3a also indicates the time of half decay for the kinetics, 5.1 ms. The same routine of showing spectra snapshots at some selected times and characterizing qualitatively the kinetics in terms of times for half formation/decay will be used in the rest of the paper. In the past we have used with apparent success a similar integration interval as the one used in Figure 3a to follow how the BR recovery is affected by the relative humidity of the sample17 or by the D115A mutation.41 Here, however, we seek also to validate the suitability of the selected interval by an independent method. To do so, we relayed on the BR recovery kinetics estimated by Van Stokkum and Lozier.29 This estimate was chosen over others available at 25 °C and pH 7 because, as shown in Figure S7, it gave the best agreement with the BR recovery kinetics estimated by RRaman spectroscopy at 30 °C,45 arguably the most suitable spectroscopy to get this information.26 Figure 3a shows the estimated kinetics for BR recovery at 25 °C and pH 7 from Van Stokkum and Lozier’s analysis, after appropriate scaling (pale blue line). The agreement with our kinetic trace is remarkably good. The small discrepancies can be accommodated by differences in the sample condition (e.g., humid film in IR vs solution in VIS spectroscopy). Another possible candidate to follow the BR recovery kinetics is the negative band at ∼1200 cm1, also located in the retinal fingerprint region. This band arises from the in-phase retinal CC stretching vibration in BR,51 and it has been commonly used for spectral normalization in the past.9,39 The band arising from this vibration appears downshifted in all the intermediates,12,13 giving a positive band in their corresponding IRdiff spectra (except for the M intermediate, which lacks any positive bands in the retinal fingerprint region as a consequence of the deprotonated SB).9,52 We could not find any integration interval for the raw data giving a BR-like kinetics for this band. However, when the intensity was monitored in the phase-corrected first derivative at 1201.5 cm1 (Figure 3b, red line), the obtained 7975



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Figure 4. Kinetics of several retinal IR bands assigned to different intermediates (see top legend of each plot). Experimental kinetics (red line). Control kinetics (pale blue line) from (a, d, i) flash photolysis and (b, c, f, h) previous kinetic analysis of the photocycle.29,44 In (f) and (h) the insets show the subtraction of two kinetics to obtain the kinetics of interest. FSD stands for Fourier self-deconvolution, and δabs/δv for the phase-corrected first derivative.

kinetics agreed very well with the estimate from Van Stokkum and Lozier’s analysis (Figure 3b, pale blue line). A final candidate to follow the BR recovery is the negative band at ∼1527 cm1 from the retinal out-of-phase CdC vibration,51 present in all intermediates IRdiff spectra.9,52,53 After systematic screening we could not find a wavenumber giving a BR-like kinetics below 1.5 ms (Figure 3c). One possible reason could be the extensive overlap with positive bands from the CdC vibration and/or with amide II bands of some intermediates. Integration using a baseline improved the results (Figure 3d), although not sufficiently. The application of the first derivative or deconvolution did not provide further improvements (not shown). From our results, this band looks not so well suited to monitor the total amount of photoexcited intermediates and to follow the kinetics of BR recovery as the previously discussed ones. Finally, it is worthwhile to give some comments on the BR recovery kinetics directly obtainable from time-resolved VIS spectroscopy. The kinetics of BR recovery has been habitually monitored in the visible by using the negative absorbance transient changes at ∼560570 nm.5456 This negative band is present in all of the intermediates difference spectra but with markedly variable intensities.28,53 Consequently, different intermediates contribute to different extent to this kinetics, being overrepresented for the M intermediate and underrepresented

for the L and N intermediates. As shown in Figure 3e (red line), the flash photolysis kinetics integrated between 550 and 580 nm rises with the time, a feature incompatible with any physically possible BR kinetics. The obtained kinetics shows poor agreement with the estimate from Van Stokkum and Lozier’s analysis below 4 ms (Figure 3e, pale blue line). Retinal in K and O Conformations. The retinal CdC vibration in the K and the O intermediates is red-shifted, with a maximum at ∼1514 and ∼1508 cm1, respectively.12,52,53,57 This opens a window to monitor their kinetics jointly at ∼1505 cm1 (Figure 4a, red line), although with a small contribution from the intense negative band at 1527 cm1 from the CdC of the depleted BR state (see also insets in Figure 4a). We can see than the K intermediate signal decreases from the first available data point, at 2 μs, while the O intermediate builds in the millisecond, with a maximal accumulation at 4 ms. K and O intermediates are the only intermediates with a redshifted retinal absorbance in the visible.58 Their red shift allows monitoring jointly the kinetics of these two intermediates at a sufficient high wavelength (>650 nm), as shown in Figure 4a (pale blue line). In the integrated interval, 655695 nm, contributions from L, M, or N intermediates should not be expected.28,58 The results show that the K intermediate decays from the first data point, as observed in the IR. The O 7976


The Journal of Physical Chemistry B intermediate reaches its maximal accumulation at 3 ms, somehow earlier than that observed in the IR, but with otherwise similar kinetics. This last deviation can be accounted for by the strong pH sensitivity of the O intermediate kinetics. As a matter of fact, at 25 °C the maximal accumulation for the O intermediate at pH 7 (IR) and pH 6.5 (VIS) is predicted to occur at 4.3 ms and at 3.4 ms, respectively.43 Incidentally, the time for the maximal accumulation of the rest of the intermediates is not expected to change from pH 7 to 6.5,43 validating the direct comparison of the kinetics of our time-resolved IR and VIS data for others intermediates. Retinal in L, M, and N Conformations. The retinal CdC stretching of the retinal in the L intermediate gives bands at 1539 and 1551 cm1, as reported by RRaman spectroscopy.12,13 These bands are of relatively small intensity when compared with the intense nearby negative band at 1527 cm1 from the CdC stretching of retinal in BR.13,52,53 Moreover, in this spectral region other positive/negative bands appear at later times in the photocycle, which further complicates monitoring selectively the kinetics of the 1539 and 1551 cm1 bands. In spite of the expected difficulties, we could find a wavenumber in the first derivative (1538.5 cm1) where the kinetics of the retinal CdC stretching in the L intermediate could be probed quite selectively (Figure 4b, red line). Note how the kinetics of L goes practically to zero above 500 μs. This experimental kinetics agrees fairly well with the estimate reported by Ludmann et al.43 for the L intermediate (Figure 4b, pale blue line), although not with the estimates from van Stokkum and Lozier29 or Varo and Lanyi44 (see Figure S1b). Monitoring the kinetics of the retinal in the L conformation using the band at 1551 cm1 resulted to be even more challenging, since it substantially overlaps with a very intense positive band at ∼1554 cm1 that develops in the milliseconds. Nevertheless, the intensity in the deconvoluted spectra at 1547.5 cm1 reported reasonable values for the kinetics of the retinal in the L intermediate conformation (Figure 4c, red line), with possible contribution at later times of the retinal in the N intermediate conformation (see below). For the M intermediate the retinal CdC stretching shifts from the ground state to 1566 cm1.12,13 However, amide II bands from protein conformational changes in transmembrane helices will contribute at ∼1554 cm1.52,59 Also, an unassigned positive band at 1574 cm1 is present during most of the photocycle (see Figure 4b,c insets). Both contributions make the selective measurement of the kinetics of the 1566 cm1 band extremely difficult. Enough selectivity could neither be attained in the first derivative nor in the deconvoluted spectra. In all instances the obtained kinetics were too broad (Figure 4d, red line), not matching well the kinetics of the M intermediate obtained by flash photolysis (Figure 4d, pale blue line). According to RRaman the retinal CdC stretching in the N intermediate gives bands at 1533 and 1549 cm1.13 The former band is hidden in the N-BR IRdiff spectrum by the intense negative band at 1527 cm1 from the retinal CdC stretching in BR,52 as it happens in the N-BR RRaman difference spectrum.13 The second band appears at a wavenumber close to one of the bands arising from the retinal CdC stretching in the L intermediate. Actually, the intensity of the deconvoluted spectra at 1547 cm1 rises back after ∼400 μs, reaching a maximum accumulation at 2 ms (Figure 4c, red line), matching well the kinetics expected for the N intermediate accordingly to van Stokkum and Lozier’s estimate (see the pale blue line).29

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The difficulties of IRdiff spectroscopy to monitor directly the retinal conformation in the L, M, and N intermediates are even higher in the visible. Here, the L and N intermediates spectra overlap strongly with that of the ground state BR,58 with small associated spectral differences.53 As a consequence, their kinetics cannot be selectively probed at any wavelength. In contrast, the large blue shift of the M intermediate clearly defines its kinetics at ∼410 nm.7 Actually, this spectral shift is a consequence of the SB deprotonation, and thus strictly speaking it follows the kinetics of the SB protonation state, not the kinetics of the retinal conformation in the M intermediate. Intermediates with 13-cis and All-trans Retinal. All intermediates with a 13-cis retinal (K, L, M and N) show in their IRdiff spectra a negative band in the retinal fingerprint region at 1166 cm1, whereas the only intermediate containing an alltrans retinal, O, shows instead a positive band at 1172 1168 cm1.9,52,53 This negative band can be potentially used as a marker to follow the kinetics of intermediates with a 13-cis retinal. This requires the use of an integration interval that minimizes the possible contribution of the nearby positive band present in the O intermediate. As when monitoring the BR retinal recovery, the kinetics should display a characteristic behavior: it should keep constant before isomerization to alltrans occurs (∼1 ms), going smoothly to zero, and never taking positive values. This last requirement is to ensure that the positive band from the O intermediate is not being significantly probed, with a net zero contribution to the measured kinetics. The area between 1175 and 1155 cm1 provided a reasonable kinetics for the intermediates with a 13-cis retinal (Figure 4e). It is fairly constant below 1 ms, and then smoothly approaches zero with a half time of 3.5 ms, without taking positive values. In the deconvoluted spectra the negative band at 1166 cm1 appears more resolved, and can be integrated using a narrower interval (11731162 cm1) to give a very similar kinetics for intermediates with a 13-cis retinal (Figure 4g). If the kinetics in Figure 4e accurately represents the total amount of 13-cis retinal intermediates during the photocycle, it turns out that its subtraction from the kinetics in Figure 3a (representing all intermediates) should provide selectively the kinetics of the O intermediate, the only intermediate in the WT photocycle with an all-trans retinal.5,12 The process is shown in Figure 4f (inset), where the kinetic traces in Figure 3a and Figure 4e have been normalized to one, and their first data value corrected. The subtracted time-trace is shown enlarged in Figure 4f (red line), presenting the expected kinetics for the O intermediate accumulation (compare with Figure 4a). This process was repeated for two different kinetic traces (the ones in Figures 3b and 4g), with similar results (Figure 4h). Remarkably, this procedure estimates not only the kinetics of the O intermediate but also its fraction, which was estimated to reach a maximum value of ∼0.16 at 4 ms. Figure 4f,h includes the prediction for the O intermediate accumulation from Van Stokkum and Lozier’s29 (pale blue line) and from Varo and Lanyi’s44 (pale green line) analysis, both showing reasonable agreement with our kinetic mode-free estimate. Changes in Retinal Planarity. When the retinal planarity of an intermediate is lower than in the ground state, we should expect a significant intensity for the retinal hydrogen out-of-plane (HOOP) bending vibrations.9 Only two intermediates are well-documented to display HOOP bands, i.e., to have a retinal more distorted (less planar) than in the BR state: the K and the O intermediates.12 However, the positive HOOP bands observed in some estimates 7977



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Figure 5. Kinetics of the protonated state of the retinal Schiff base. Experimental kinetics (red line). Control kinetics from flash photolysis (pale blue line). (ac) Process to obtain the SB deprotonation/reprotonation kinetics from the intensity of the CC retinal band. (d,e) Attempt to follow the SB deprotonation/reprotonation kinetics from the CdN vibration.

of the IRdiff spectra of the L intermediate at room-temperature have suggested that the physiological L intermediate might display a more distorted retinal that the one trapped at lowtemperatures.17,60 This observation was taken as an example of the potential differences between physiological and cryogenically “trapped” intermediates.61 Figure 4i shows the kinetics of the HOOP band area at ∼980 cm1 (red line). It decreases in intensity steadily with time, following a kinetic trace resembling that of the retinal in the K intermediate conformation (Figure 4a). After reaching zero at ∼500 μs, it raises again up to 3.5 ms as the O intermediate builds up, decaying back to zero as a result of the BR recovery. Note how the initial decay is well-matched by the initial decay of the flash photolysis time-trace at ∼680 nm (Figure 4i, pale blue line), a wavelength where the K intermediate should be probed with a negligible contribution from the L intermediate.58 This observation suggests that contaminations from the K intermediate are responsible for the at least part of the previously reported HOOP intensity in the room-temperature L intermediate.17,60,62,63 It also explains the absence of HOOP bands in the RRaman spectrum of the L intermediate at room-temperature,12,13 fundamentally free from contributions from the red-shifted K and O intermediates by the wavelength of the laser probe. The long life of the K intermediate is most likely caused by its maintained equilibrium with the L intermediate. When this K/L equilibrium is not taken into account during the analysis, features of the K intermediate (e.g., the HOOP bands) will leak into the estimated L intermediate. This will be the case when spectra of the L intermediate are simply obtained from a time slice,17 or when computed from global exponential fitting assuming an irreversible sequential model for the bR photocycle.62,63 This interpretation agrees with the small HOOP bands in a recently estimated room-temperature L-BR IRdiff spectrum where the K-L equilibrium was taken into account.30 Deprotonation and Protonation of the Schiff Base. The deprotonation of the SB in favor of Asp85 and its posterior reprotonation from Asp96 are two of the most important steps in

the proton-pump cycle of bR. The kinetics of the protonation changes of the SB, equivalent to the M intermediate kinetics, can be potentially monitored in the IR using several bands. One of these is the band at 1188 cm1, from the retinal in-phase CC vibration, which displays a negligible intensity in the IR when the SB deprotonates.9 Consequently, the intensity of this band reaches a minimum intensity at 250 μs, moment in which the fraction of deprotonated SB is higher (Figure 5a). The band intensity recovers as the SB reprotonates from Asp96 but before full recovery the intensity decreases to zero (Figure 5a). This last decrease is not associated with SB deprotonation, but it is a consequence of the relaxation of the photocycle to the BR state (see Figure 3a,b). Figure 5b (continuous red line) shows the 1188 cm1 kinetics after conveniently removing the signal attenuation caused by the relaxation to the bR ground state. This was done dividing the kinetics intensity at 1188 cm1 (Figure 5b, dashed violet line) by the kinetics of the total fraction of intermediates (Figure 5b inset). Now, after the SB reprotonation by Asp96 is completed, the band intensity recovers its initial value (see SB-Hþ label and the gray dashed line in Figure 5b). This allows monitoring both the deprotonation and the reprotonation kinetics of the SB from IR spectroscopy, although with the unusual effect of having the effect of the BR recovery in the kinetic trace removed. The “normal” SB protonation kinetics can be restored by offsetting the corrected kinetics in Figure 5b to zero when all the SB is protonated, and undoing the correction for the kinetics of the BR recovery. The result is shown in Figure 5c (red line), which should fairly represent the kinetics of the SB deprotonation/ deprotonation during the photocycle, i.e., the M intermediate kinetics. In order to confirm the reliability of the kinetics presented in Figure 5c we relayed again on flash photolysis (pale blue line in Figure 5c). The agreement between both traces is reasonably good, validating the use of the intensity at 1188 cm1 to follow the SB deprotonation/protonation kinetics. Differences between both kinetics traces do exist, though. The several corrections 7978



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Figure 6. Kinetics of several aspartic carbonyls bands assigned to different molecular processes (see top legend of each plot). Experimental kinetics (red line). In (b) the experimental kinetics was divided by the experimental kinetics in Figure 3b to remove the contribution from BR recovery (dotted blue line). Control kinetics (pale blue line) from (c, d) flash photolysis and (e, f, g, h) previous kinetic analysis of the photocycle.29 In (f) the inset shows the subtraction of two kinetics to obtain the kinetics of interest.

needed to construct the kinetics in Figure 5c (red line) from the intensity at 1188 cm1 (Figure 5a) are likely to be imperfect and to introduce some small errors. We checked if any other IR band could be also used to get a more direct insight into the SB protonation kinetics. When the SB deprotonates it gives a band in the RRaman spectrum at 1620 cm1, from the CdN stretching of the deprotonated SB.12,13 A positive band at ∼1618 cm1 has been reported in the IR when the SB deprotonates in the L-to-M transition.30 Therefore, following its intensity (or area) should allow accessing directly the kinetics of the SB deprotonation/protonation. The major difficulty is that several bands crowd this wavenumber region. For instance, changes in Pro186 have been also assigned to contribute at ∼1616 cm1.64 After deconvolution, two bands become resolved in the IRdiff spectra, at 1626 cm1 and at 1615 cm1 (Figure 5d, see the inset at 400 μs), but only the second band roughly follows the expected time evolution for the SB deprotonation/reprotonation (Figure 5d, red line). Using the first derivative and probing at 1611.5 cm1 improved the results (Figure 5e, red line), but still not accurately enough to match the kinetics of the SB deprotonation/reprotonation (pale blue line). An even more natural target to follow the SB protonation state would be a vibration directly involving the hydrogen of the protonated SB, as for instance the SB NH bending assigned at ∼1396 cm1.9 However, the intensity of this band is weak,

overlapping with many other bands, and we could not find any integration interval giving an M-like kinetics in this region (not shown). Derivation and deconvolution were equally unsuccessful (not shown). Protonation, Deprotonation, and Environment Changes of Aspartic Acid 85. Aspartic acid 85 is probably the most important amino acid in the bR photocycle. Fortunately it is also the one that can be more easily studied by IRdiff spectroscopy. It is deprotonated in BR, but when protonated in the M, N, and O intermediates, it gives a well-resolved positive band at 1762 1753 cm1, from the carbonyl (CdO) stretching of its carboxylic side chain.65 Environment Changes in the Protonated Asp85. Asp85 is protonated during three consecutive intermediates: M, N, and O. In the M intermediate it appears at 17621761 cm1 and shifts to 17551754 cm1 in the N and O intermediates.9,52,53,66 The kinetics of this shift can be nicely visualized plotting the Asp85 CdO maximum wavenumber as a function of time, for instance in the phase-corrected first derivative (Figure 6a). This plot confirms that at least from 30 μs until almost the end of the photocycle Asp85 experiences only two basically different environments. The wavenumber shift of Asp85 starts above 200 μs, showing that the N and O intermediates do not build up before this time. Furthermore, from the time at which Asp85 carboxylic wavenumber maximum is half-shifted we can conclude that at 7979


The Journal of Physical Chemistry B 2 ms the fraction of the M intermediate will be approximately equal to the fraction of the sum of the N and O intermediates. Interestingly, there is not a shift in the Asp85 CdO maximum at the time of proton release complex (PRC) deprotonation, occurring at around 8090 μs.38 This observation contrasts with what might be expected from the pKa coupling between Asp85 and the PRC4,65 but confirms our recent results regarding the nearly complete spectral similarity of the M1 and M2 substates.30 Protonation and Deprotonation of Asp85. The protonation of Asp85 occurs from the SB in the L-to-M transition. Asp85 keeps presumably fully protonated until it deprotonates in favor of the PRC, when the BR state is recovered in the O-to-BR transition.67 Following these two processes requires monitoring the area of Asp85 carbonyl band taking into account its shift (Figure 6b, red line). After correcting the obtained kinetics for the BR recovery, the signal reaches a plateau from 400 μs up to 15 ms (dashed blue line), confirming that M, N, and O intermediates (all with a protonated Asp85) are the only significantly populated intermediates above ∼400 μs in the photocycle. This agrees with the observation that the K and L intermediates are negligibly populated above 400 μs (Figure 4b,i). It also confirms that Asp85 remains fully protonated at least until 15 ms. This observation discourages the idea that some other residue, like Asp212 (see below), could become protonated from Asp85 as a previous step to the ground state recovery in the WT bR at neutral pH. Asp85 and the M Intermediate. Measuring selectively the kinetics of the 17621761 cm1 band should be equivalent to measuring the kinetics of the M intermediate. The result is shown in Figure 6c (red line), where the area between 1772 and 1754 cm1 was considered the best choice to minimize contributions from Asp85 in the N/O intermediates. The kinetics increases as a consequence of Asp85 protonation, and decreases as Asp85 shifts from a M-like to a N/O-like environment. The deprotonation/protonation kinetics of the SB from flash photolysis is reproduced in Figure 6c (pale blue line). The overlap between both is remarkably good, especially in the earlier part corresponding to SB deprotonation and Asp85 protonation kinetics, as it should be from the fact that the proton is transferred from the SB to Asp85.67 The lesser but still good match in the second part of the kinetics suggests that SB reprotonation and the environment changes in Asp85 occur at least with a similar kinetics. The kinetic trace obtained from the phase-corrected first derivative spectra at 1766 cm1 (Figure 4d, red line) is also in good agreement with the M intermediate kinetics observed by flash photolysis (Figure 4c, pale blue line). Monitoring the absorbance changes in the deconvoluted spectra also provided similar results (Figure S6b). Asp85 and the N and O Intermediates. In the N and O intermediates Asp85 is still protonated, but its carbonyl band downshifts to ∼1755 cm1.52,53 This downshift is also observed in the MN intermediate,68 a nonnative intermediate with a deprotonated SB (and presumably an M-like chromophore) but with an N-like protein conformation. Therefore, the protein conformational changes are probably linked to the ∼7 cm1 downshift of Asp85 in the N and O intermediates. We probed the kinetic trace of Asp85 in the N/O environment by monitoring the area between 1761 and 1748 cm1 (Figure 6e, red line). As expected, the intensity of this kinetic trace is close to zero below 200 μs, before N formation starts according to Figure 6a. Its maximum, at 3 ms, agrees with the maximum accumulation expected for the sum of N and O intermediates (pale blue line).29

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We used an alternative strategy to obtain a more accurate kinetic trace for Asp85 in the N/O environment. To the kinetic trace of the protonated Asp85 (Figure 6b), we scaled and subtracted the kinetic trace of Asp85 in the M-like environment (Figure 6c), obtaining as a result the kinetics of Asp85 in the N/ O-like environment (Figure 6f, inset). Additionally, this procedure provides the relative fractions of the M and N þ O intermediates, which equal at 2 ms (see inset), in agreement with the conclusion drawn from Figure 6a. An enlarged view of the obtained N þ O intermediate kinetics is shown in Figure 6f (red line), with the estimate from Van Stokkum and Lozier overlaid (pale blue line).29 Changes in Asp96 and Asp115. The third proton transfer in the bR photocycle is from Asp96 to the SB in the M-to-N transition, and the fourth consists of the reprotonation of Asp96 from the cytosolic medium in the N-to-O transition.67 At high pH deprotonation of Asp96 gives a very clear negative band at 17421741 cm1.52,59 The reason is that the high pH delays Asp96 reprotonation from the cytosolic medium, enhancing the accumulation of the N intermediate.29,43 Also, due to the apparent neutral pKa of Asp96 in the N intermediate,39 Asp96 is completely deprotonated in the N intermediate at pH 9. Therefore, probing selectively the protonation changes of Asp96 at neutral pH is hampered by the relatively low accumulation of the N intermediate, as well as by the incomplete deprotonation of Asp96 in N at this pH. A further difficulty is that in the L intermediate alterations in the vibration frequency of the CdO of Asp96 during the photocycle contribute with an intense negative band at 1742 1741 cm1 and a small positive band at 1748 cm1,69 besides contributing with lesser intensity in the M intermediate at 1742 () and 1736 (þ) cm1.66 In addition, alterations in Asp115 during the photocycle generate a pair of positive-negative bands in all the intermediates IRdiff spectra, with a negative band at 17371733 cm1 and a positive one with a maximum ranging from 1742 to 1730 cm1.52,66,70 The kinetic trace at 1741 cm1, monitored in the deconvoluted spectra, reports first alterations in Asp96 (and Asp115) during the L intermediate (Figure 6g, red line). However, the second phase of the kinetics, with a minimum at 1.6 ms, does not correspond solely to the kinetics of Asp96 deprotonation/ deprotonation. This is evidenced by the fact that it does not exactly follow the kinetics expected for the N intermediate (Figure 6g, pale blue line).29 The most likely reason for this departure is the overlap with changes in Asp115, which contributes with a positive band at this wavenumber in the O intermediate.52,71 Alterations, Deprotonation, and Reprotonation of Asp96. With the aim of canceling the contributions from alterations in Asp115 carbonyl, the area between 1746 and 1727 cm1 was chosen to selectively follow changes in Asp96 (Figure 6h). This wide interval was chosen to encompass all of the positive/negative bands from Asp115, presumably canceling its contribution to the measured area. The initial part of the kinetics corresponds to the alterations in Asp96 hydrogen bonding that occur in the L intermediate. The signal approaches zero at 400 μs, moment in which the L intermediate not longer accumulates (see Figure 4b), suggesting that alterations of Asp96 in the M intermediate are effectively canceled in the chosen integration interval. Then, the negative signal increases again as a result of Asp96 deprotonation, reaching a maximum change at 2.5 ms, in agreement with the expected kinetics for the N intermediate (see pale blue line).29 7980


The Journal of Physical Chemistry B This time is significantly earlier than the time for the maximal accumulation of the O intermediate (4 ms), suggesting that alterations of Asp96 and Asp115 in the O intermediate are successfully canceled in the measured kinetics. In summary, the kinetics in Figure 6h mostly corresponds to the kinetics of the L and the N intermediates as reported by Asp96. Alterations in Asp115. Asp115 and Asp96 are the only two aspartic groups protonated in the BR state.72 However, in contrast to Asp96, Asp115 does not deprotonate during the photocycle. The CdO stretching of Asp115 vibrates at ∼1735 cm1 in the BR state.56,72 Consequently, alterations in its environment give rise to a pair of positive-negative bands in IRdiff spectra, with the negative band centered at ∼1735 cm1: 1737 () and 1729 (þ) cm1 in L,70 1742 (þ) and 1734 () cm1 in M,66 1740 (þ) and 1732 () cm1 in N,66 and 17431738 (þ) and 1732 () cm1 in O.52,71 We monitored alterations in Asp115 by integrating the area between 1735 and 1728 cm1 in the deconvoluted spectra, minimizing in this way contributions from Asp96. The alterations of Asp115 in L contribute positively in this interval and in the rest of the intermediates contribute negatively (Figure 6i). It can be concluded that the alterations in the environment of Asp115 side-chain carbonyl group appear to be especially intense in the L and O intermediates, in agreement with previous observations.52,71,73 Protonation Changes of Asp212. Protonation changes of Asp212 were first proposed for the mutant E194Q,74 where a positive band at 1712 cm1 appears concomitantly to the disappearance of the positive band at 1755 cm1, reporting Asp212 protonation from Asp85.74,75 A positive band at 1712 cm1 was also observed in the E204Q mutant75,76 and in WT at pH 4,52 albeit with a reduced intensity.75 Additionally, it was found that in these mutants the 1712 cm1 band appeared in the presence of chloride but not in the presence of sulfate.76 A possible interpretation is that these conditions provided an enhanced accumulation of a native but otherwise kinetically hidden intermediate that follows the O intermediate (the socalled O0 ). This interpretation would be supported by QM/MM calculations showing that proton transfer from Asp85 to Asp212 is possible in the last stages of the photocycle.77 However, it should be noted that these calculations were done using as a starting point X-ray structures of altered ground states taken to represent the O intermediate structure. Deprotonation of the PRC in the M1-to-M2 transition is prevented in E194Q and E204Q, and in WT at low pH, expectedly affecting the native proton affinity increase of Asp85.4 This might cause Asp85 proton affinity to drop below the proton affinity of Asp212 in the last steps of the photocycle, which could explain the protonation of the later as an induced nonnative feature of the photocycle. In such a scenario, the contribution of a negative charge in the protein interior provided by Cl could be essential to induce Asp212 protonation.76 Therefore, there is a need of confirming protonation changes of Asp212 under conditions where PRC deprotonation in the M1-to-M2 transition is preserved. The kinetic trace at 1712 cm1 did not reveal evidence for protonation changes in Asp212 (not shown). Even after deconvolution, enhancing band intensity, no band build up at 1712 cm1 in any moment of the photocycle (see Figure S6c). This observation agrees nicely with the data in Figure 6b (dashed line), showing that Asp85 remains protonated until the group state recovery. Although not strictly discarding protonation of Asp212, which could be too low to be detectable in our

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experiments at neutral pH, our results argue against such possibility. Protonation and Deprotonation of the Proton Release Complex (PRC). At neutral pH the second proton transfer in the bR photocycle involves the release of a proton from the PRC to the extracellular membrane surface, where incidentally the proton stays for several hundreds of microseconds before diffusing into the extracellular bulk medium.78 Although the proton release is still sometimes cited to occur in the L-to-M transition,20,77 we and others have concluded the PRC deprotonates later, in the transition between two M substates: M1 and M2.30,79 The PRC appears to keep deprotonated until the end of the photocycle, recovering a proton from Asp85 in the fifth and last proton transfer, during the O-BR transition.67 Although the molecular nature of the PRC has been largely disputed, nowadays it is generally accepted to consist of either a protonated water cluster stabilized by several residues,72 or of two glutamates (Glu194 and Glu204) sharing a proton.80 Both can explain the continuum band,80,81 a characteristic negative baseline-like feature in the IRdiff spectra between 2000 and 1770 cm1 (see insets in Figure 7a) that roughly correlates with the proton release kinetics to the extracellular membrane surface.38 Nonetheless, the apparent isotopic shift of the center of gravity of the continuum band in H218O appears to support the former hypothesis.17 Recently, a study combining time-resolved IR spectroscopy, X-ray crystallography and MD simulations of WT and Glu204 and Glu194 to Asp mutants concluded that the protontated water cluster in the ground state could switch to a shared proton between Glu194 and Glu204 briefly before its deprotonation, somehow bringing into agreement both previous hypothesis.82 Probing the area of the continuum band between 2000 and 1800 cm1 should provide the kinetics of the PRC deprotonation/protonation (Figure 7a). Some precautions are needed, because the sample heating caused by the exciting laser will also contribute to the measured kinetics.83 As done before,30 the transient heating was corrected taking into account its exponential relaxation (τ ∼60 μs). The result is shown in Figure 7b (orange line). The violet line in Figure 7b reproduces Asp85 CdO kinetics (taken from Figure 6c) after appropriate scaling. Two things are visually clear: (i) Deprotonation of the PRC, as reported by the continuum band, occurs significantly later than Asp85 protonation and (ii) both Asp85 deprotonation and PRC reprotonation follow the same kinetics, as far as we can tell with the present data quality. Subtracting both kinetic traces should provide the kinetics of an intermediate with a protonated PRC and Asp85. This intermediate should correspond to the M1 substate if, as we affirm,30 PRC deprotonation takes place in the M1-to-M2 transition. Although the resulting kinetic trace is very noisy (Figure 7b, inset), the general trend expected for the M1 substate is clear, achieving a maximal accumulation at ∼70 μs. This is in almost perfect agreement with a previous estimate implied in doublepulse experiments,84 and slightly faster than a previous estimate of 120 μs obtained under reduced hydration conditions,30 known to delay the M1 to M2 transition.85 Protein Structural Changes. Protein structural changes occurring during the bR photocycle have been confirmed by several techniques,23,54,86 consisting mostly in changes in the tilt of some transmembrane helices and in their accompanying cytoplasmic loops. The timing of these structural changes is still disputed though, with some studies reporting them to occur 7981



Figure 7. Kinetics of the proton release complex (PRC). (a) Experimental kinetics of the continuum band. (b) Corrected kinetics of the continuum band, corresponding to the kinetics of the PRC deprotonation and reprotonation (orange line) and the scaled kinetics of Asp85 protonation state (violet line), taken from Figure 6b. The inset shows their subtraction (gray line), assigned to the kinetics of the M1 intermediate, and its smoothed version after a maximum entropy multiexponential fitting (red line).42.

mainly upon formation of the M intermediate or in-between M substates,21,87 whereas others locate most of them later, upon formation of the N intermediate.30,54,86 Some of the bands arising in the IRdiff spectra have been assigned to amide vibrations, and thus to report backbone conformational changes during the photocycle.6,9 The negative band at ∼1670 cm1 is customarily used as a reporter of structural changes in the transmembrane helices of bR.6 This assignment is supported by its wavenumber, its very high dichroism,88 its H/D exchange resistance,89 and its partially reduced intensity upon site-directed [1-13C] backbone labeling of Try185 in WT90 and Cys46 in the T46C mutant.91 Also, the fact that this band is not observed by RRaman spectroscopy discards contributions to this band from the retinal and the SB.12,13 To monitor the kinetics of the band at ∼1670 cm1, we have to take into account the presence of two neighbor negative bands at ∼1673 and ∼1659 cm1 (see Figure 8a insets). We monitored the intensity at 1671 cm1 in the deconvoluted spectra (Figure 8a, green line), although it becomes apparent that the nearby band at 1673 cm1 is also partially probed in this way (see inset at 240 μs). Trying to minimize its contribution we monitored instead the intensity at 1667 cm1, just at the valley

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between the ∼1673 and ∼1659 cm1 bands (see inset at 240 μs), with apparently good results (Figure 8a, red line). The intensity at 1669 cm1 in the phase-corrected first derivative provided a similar kinetic trace (Figure S6d). The resulting kinetic trace fairly reproduces the experimental kinetics expected for the sum of the N and O intermediates (Figure 8a, pale blue line),29 and it is also similar to that of Asp85 carbonyl in the N/O environment (Figure 6e,f). Both observations suggest that the negative band at ∼1670 cm1 is present in both the N and O intermediates and absent in the rest of intermediates. As an alternative strategy to monitor the 1670 cm1 band we also followed in the original spectra the area around its maximum (Figure 8b, red line), matching well the expected kinetics for the N þ O intermediates (pale blue line).29 Therefore, we can safely conclude that the structural changes responsible for the 1670 cm1 band, presumably the tilt change of some transmembrane helix/ helices,30 fully occur upon formation of the N intermediate and are basically maintained in the O intermediate. Another characteristic band in the amide I region is the negative band at 1694 cm1. This negative band shows H/D exchange resistance,89 reduced intensity upon [1-13C] labeling of Cys46 in the T46C mutant91 and insensitivity to 13N and C15-deuteration of the retinal,59 and it is not observed by RRaman,12,13 justifying its assignation to an amide I vibration. Given its wavenumber and moderate dichroism,88 this band is unlikely to arise from changes in helices, and thus might report changes in turns, possibly located in some of the cytoplasmic loops. When the area of this band is probed, the resulting kinetics arises soon, reaching a maximum at 1.8 ms (Figure 8c, red line), earlier than for the band at 1670 cm1 (compare with Figure 8a,b). This kinetics can be well-reproduced by summing (pale blue line) the scaled experimental kinetics of the M intermediate (light green line) and the N þ O intermediates (light violet line), both taken from the inset in Figure 6f. On one hand, the good fit implies that contributions from structural changes between M substates must be negligible. On the other hand, the factor used to scale the M kinetics to respect the N þ O kinetics implies that the structural changes reported by the 1694 cm1 band are 2.5 times larger in the N and O intermediates than in the M intermediate; that is, 70% of the protein structural change would occur in the M-to-N transition, and the rest upon formation of the M intermediate. Interestingly, these percentages are in rough agreement with our previous estimate of protein structural changes from the analysis of time-resolved X-ray scattering data.30 Amide II bands are known to be less secondary structuresensitive than amide I are,92 and thus they can provide a more global view about the timing of the main structural changes in the bR photocycle. The positive band at ∼1556 cm1 originates from the amide II vibrations of the protein, but with overlapping contributions from vibrations of the retinal.59 To enhance its selectivity, we monitored the intensity of the positive band at 1556 cm1 in the deconvoluted spectra (Figure 8d, red line). Its kinetics resembles much that in Figure 8c, with a maximum intensity at 1.8 ms. But the negative intensity at earlier times points to a negative contribution from K and L intermediates. We fitted the kinetics to the experimental kinetics of the M and the N þ O intermediates (Figure 8d, pale blue line), but only above 200 μs to minimize contributions from earlier intermediates. The good fit confirms the notion that significant structural changes do occur upon formation of the M intermediate (30%). However, it also confirms that most of the structural changes occur upon 7982



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Figure 8. Protein structural changes in the bR photocycle. Experimental kinetics (red line). Control kinetics (pale blue line). In (a, b) the control kinetics is from a previous analysis of the photocycle.29 In (c,d) the control kinetics comes from the weighted addition of the experimental kinetics of the M (from Figure 6c) and N þ O (from Figure 6f) intermediates, shown in dotted color lines.

formation of the N intermediate (70%) and are maintained in the O intermediate. Practical Considerations. The wavenumbers provided here to probe specific molecular processes should be of general applicability to study the bR photocycle by time-resolved IRdiff spectroscopy. In spite of that, notice that any instrumental change that alters the shape of IRdiff spectra might require some tuning/readjustments of the wavenumbers used here. Changes in the spectral shape might occur, for instance, when the timeresolved data is collected by attenuated total reflection instead of by transmittance. The shape of IRdiff spectra will also change depending on the instrumental resolution of the interferogram and the apodization function used for the Fourier transform, and some readjustments will be forcedly required for experiments at resolutions different from 8 cm1 and the triangle apodization, the conditions used here. The tilt of the sample normal to respect the IR beam will alter the intensity of dichroic bands, and so the shape of the IRdiff spectra. When this angle is different from ∼0° some of the wavenumber values provided here might need some tuning. Finally, it is also evident that at different pH values, or in mutants, some bands might be shifted or altered in shape, requiring corresponding adjustments in the probed wavenumber values. A less obvious problem to reproduce our results might be encountered by the fact that the Fourier transform of an interferogram with standard zero-filling will provide only one data point every 4 cm1 for spectra acquired at 8 cm1 resolution. Such data spacing will be too sparse to select suitable wavenumber values. One data point at least every 2 cm1 is recommended, while additional interpolation to one data point every 0.51 cm1 might be required for some kinetics requiring a finer wavenumber tuning. To save computation time, and storage resources, such additional interpolation can better be done after quasi-logarithmic averaging of the time-resolved IRdiff spectra. Based on our experience we recommend to use either Fourier or piecewise cubic spline interpolation.

’ CONCLUSIONS We have provided a thoroughly discussion on how the different molecular processes taking place in the bR photocycle can be selectively probed by time-resolved IRdiff spectroscopy. Furthermore, our analysis sheds light into some still disputed or unclear aspects of the bR photocycle. For instance we could conclude the following: (i) Previously described differences in the retinal HOOP bands between the estimated roomtemperature and the trapped low-temperature L intermediate, interpreted as differences in the retinal torsion, are probably exaggerated by contributions of the K intermediate in the estimated spectrum of the L intermediate at room-temperature (Figure 4i). (ii) Asp85 remains fully protonated until the end of the photocycle (Figure 6b). This observation discourages the proposal of a proton transfer from Asp85 to Asp212 before the recovery of the bR ground state. (iii) The kinetics of Asp85 protonation precedes the appearance of the negative continuum band, confirming that PRC deprotonation follows Asp85 protonation (Figure 7). (iv) The main protein structural changes in the photocycle occur upon formation of the N intermediate (in the M2-to-N transition). Structural changes of lower amplitude also occur upon formation of the M intermediate (in the L-to-M1 transition), whereas there is not evidence for structural changes in between M substates (Figure 8). We have also related the obtained kinetic traces to specific intermediates. In this way, we have shown that we can successfully estimate the kinetics of the L intermediate (Figure 4b), M intermediate (Figures 5c and 6c,d), O intermediate (Figure 4f,h), K þ O intermediates (Figure 4a,i), L þ N intermediates (Figures 4c and 6h), N þ O intermediates (Figures 6e,f and 8a,b), M þ N þ O intermediates (Figure 6b), M2 þ N þ O intermediates (Figure 7b), K þ L þ M þ N intermediates (Figure 4e,g), and K þ L þ M þ N þ O intermediates (Figure 3a,b). These spectral estimations of intermediate kinetics 7983


The Journal of Physical Chemistry B can open new perspectives for solving the bR photocycle, as well as for the robust characterization on how external conditions (e.g., pH or temperature) or site specific mutations affect different molecular steps/intermediates of the photocycle. We should finally comment that the method presented here demands considerable interaction from the user. Furthermore it feeds on previous spectral assignments, and so it might give the best fruits on well-characterized systems such as the bacteriorhodopsin photocycle. Nevertheless, we foresee that timeresolved IR studies of many proteins involved in photosynthesis, phototaxis, respiratory chains, or enzymatic reactions73,93,94 could largely benefit from similar studies as the one presented here. These benefits might also extend to time-resolved IR studies of the folding and miss-folding of proteins and peptides.95,96

’ ASSOCIATED CONTENT Supporting Information. Figure S1 Comparison of the estimated intermediates fractions of the K, L, M, N, and O intermediates at 25° and pH 7 by different authors. Figure S2 Comparison of some selected time-traces for two independent time-resolved IR data. Figure S3 Procedure to correct the time-trace of the continuum band for the baseline drift. Figure S4 Screen capture and brief description of the program used to extract time-traces from time-resolved data. Figure S5 Second derivative and band selectivity. Figure S6 Additional experimental kinetic traces. Figure S7 Comparison of relaxation kinetics of the bR photocycle at 30° and pH 7 estimated by different authors. This material is available free of charge via the Internet at http://pubs.acs.org.

bS

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT We are grateful to Alex Peralvarez-Marín for critically reading the manuscript and for their valuable comments. This work was supported by a Marie Curie Reintegration Grant PIRG03-6A-2008-231063 to V.A.L.F., by a Ministerio de Ciencia e Innovacion Grant BFU2009-08758/BMC to E.P., and in part by Grants 22247024 and 20108014 from Japanese Ministry of Education, Culture, Sports, Science, and Technology to H.K. ’ REFERENCES (1) Henzler-Wildman, K.; Kern, D. Nature 2007, 450, 964. (2) Lanyi, J. K. J. Phys. Chem. B 2000, 104, 11441. (3) Stoeckenius, W. Protein Sci. 1999, 8, 447. (4) Balashov, S. P. Biochim. Biophys. Acta 2000, 1460, 75. (5) Lanyi, J. K. Annu. Rev. Physiol. 2004, 66, 665. (6) Haupts, U.; Tittor, J.; Oesterhelt, D. Annu. Rev. Biophys. Biomol. Struct. 1999, 28, 367. (7) Lanyi, J. K.; Varo, G. Isr. J. Chem. 1995, 35, 365. (8) Lanyi, J. K. J. Phys. Chem. B 2000, 104, 11442. (9) Maeda, A. Isr. J. Chem. 1995, 35, 387. (10) Kandori, H. Biochim. Biophys. Acta 2000, 1460, 177. (11) Heberle, J. Biochim. Biophys. Acta 2000, 1458, 135. (12) Smith, S. O.; Lugtenburg, J.; Mathies, R. A. J. Membr. Biol. 1985, 85, 95.

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