Protein Response to Chromophore Isomerization in - American

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Chapter 16

Protein Response to Chromophore Isomerization in Microbial Rhodopsins Revealed by Picosecond Time-Resolved Ultraviolet Resonance Raman Spectroscopy: A Review Misao Mizuno and Yasuhisa Mizutani* Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan *E-mail: [email protected].

Proteins function by changing their structures, and external stimuli facilitate the sequential change of specific structural sites. To understand the mechanism of protein functions, it is essential to clarify how external stimuli can bring about these site-specific structural changes. Time-resolved ultraviolet resonance Raman spectroscopy probes the structural dynamics of specific sites in protein structure by selectively enhancing the vibrational Raman bands assignable to aromatic amino acid side chains as well as polypeptide bonds. We have applied picosecond time-resolved ultraviolet resonance Raman spectroscopy to observation of protein response to chromophore isomerization in microbial rhodopsins.

Structural changes of proteins regulate their functions. Studies on protein dynamics is therefore important for elucidating mechanisms how protein functions. In photoreactive proteins, functionally-important structural changes of protein are initiated by light absorption to the chromophore. The local photoinduced structural change of the chromophore triggers a series of changes in the higher order structure, thereby facilitating function. Clarification of the

© 2015 American Chemical Society In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


propagation of structural changes induced by the local structural changes can help to understand the functional mechanism of protein. Microbial rhodopsins are typical photoreactive proteins. Figure 1a shows crystallographic structure of bacteriorhodopsin, which is the best studied microbial rhodopsin. Microbial rhodopsins consist of seven transmembrane α-helices. A retinal chromophore is covalently bound to the Lys residue through a protonated Schiff base linkage. All-trans configuration of the retinal chromophore is thermodynamically most stable. Absorption of a photon by the chromophore gives rise to isomerization from all-trans to 13-cis configuration as shown in Figure 1b. The photoisomerization of the chromophore takes place within subpicoseconds (1–4), which induces sequential changes in protein structure important for their functions.

Figure 1. (a) Crystallographic structure of bacteriorhodopsin (PDB ID = 1C3W). The red molecule represents the unphotolyzed retinal chromophore with the all-trans configuration. (b) Isomerization of the retinal chromophore from all-trans to 13-cis configurations. (see color insert)

Elucidation of the structural dynamics of the protein moiety associated with the chromophore isomerization is of great interest in understanding of protein mechanisms. To discuss protein response to chromophore isomerization, we have measured picosecond time-resolved ultraviolet resonance Raman (UVRR) spectra of four types of microbial rhodopsins, bacteriorhodopsin (BR) (5), sensory rhodopsin II (SRII) (6), sensory rhodopsin I (SRI) (7), and Anabaena sensory rhodopsin (ASR) (8). In this review, we summarize our recent studies on the protein response to chromophore isomerization of the microbial rhodopsins. 330 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Time-Resolved UVRR Spectroscopy UVRR spectroscopy is a versatile technique for studying protein structures because it enables us to observe of Raman bands of aromatic amino acid residues and polypeptide backbones with high selectivity (9–11). Several vibrational bands of aromatic residues can be utilized as structural markers of proteins; hence, time-resolved UVRR spectroscopy can provide site-specific information about protein dynamics. Although time-resolved UVRR spectroscopy has been successfully applied to protein dynamics in the nanosecond to second region (12, 13), application to protein dynamics in the picosecond region was limited (14). The most crucial factor was a lack of a light source that fulfills the requirements for small timing jitters, appropriate repetition rates, and the wavelength tunability of pulses applicable to time-resolved UVRR spectroscopy. Time-resolved UVRR measurements require pump and probe pulses. The wavelength of the pump pulse needs to fall within the electronic absorption band of the cofactor (retinal, flavins, heme, etc.) in proteins to photoexcite it. At the same time, the wavelength of the probe pulse has to be close to that of an electronic transition of the specific part of interest in the protein for resonance enhancement of Raman bands. Furthermore, the spectral width of the probe pulse has to be narrow enough to record well-resolved vibrational bands. To obtain the time-resolved UVRR spectra of a wide variety of proteins with high S/N ratios within a reasonable measuring time, it is necessary to generate independently tunable pump and probe pulses with a high repetition rate. We constructed an apparatus consisting of two widely tunable light sources for time-resolved UVRR spectroscopy using a 1-kHz picosecond Ti:sapphire laser/regenerative amplifier system (15).

Time-Resolved UVRR Apparatus and Measurements Figure 2 is schematic of the time-resolved UVRR measurement apparatus. A Ti:sapphire oscillator (Tsunami pumped by Millennia-Vs, Spectra-Physics) and amplifier (Spitfire pumped by Evolution-15, Spectra-Physics) system operating at 1 kHz provided 778-820 nm pulses, each with an energy of about 0.8 mJ, and duration of 2.5 ps in a nearly TEM00 mode under operation at 1 kHz. The whole laser system was covered with the plastic sheet, which was equipped with dust cleaners containing high-efficiency particulate air (HEPA) filters to keep the laser system free of dust. In the pump arm, a pump pulse of 530-600 nm was generated with a home-built optical parametric generator (OPG) and amplifier (OPA), which were pumped with the second harmonic of the output of the amplified laser. To generate a pump pulse with shorter wavelength (439-494 nm), stimulated Raman scattering in compressed methane or hydrogen gas was excited by the second harmonic of the laser output. Also, the second harmonic of the laser output (389410 nm) was directly applicable to a pump pulse. The tunability of the pump pulse is shown in the right panel of Figure 2c. In the probe arm, the second harmonic of the laser output was focused into a Raman shifter filled with methane or hydrogen gas to generate first Stokes stimulated scattering in 439-494 nm. For example, a UV probe pulse at 225 nm was generated with a BBO crystal as the second harmonic of the 450-nm output. In this way, a UV pulse was generated in the 331 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


wavelength range of 220-247 nm. Sum frequency generation between the second harmonic and the stimulated Raman scattering was generated to produce the UV probe pulse in 206-218 nm. The tunability of the probe pulse is also shown in the right panel of Figure 2b. Light components other than the probe pulse were eliminated spatially with a Pellin-Broca prism and spectrally with dichroic mirrors.

Figure 2. Picosecond time-resolved UVRR spectrometer. (a)Schematic optical setup of the spectrometer. (b) Optical configuration in the probe arm. (c) Optical configuration in the pump arm. L=lens, BS=beam splitter, ND=neutral density filter, HWP=half wave plate, SH=mechanical shutter, LBO=lithium triborate, BBO=β-barium borate, DM=dichroic mirror, PB=Pellin-Broca prism. (see color insert) 332 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


After the pump and probe beams were made coaxial using a dichroic mirror, they were focused with a spherical lens onto a flowing thin-film of the sample solution. Focused spot sizes were 150 μm (fwhm) for the probe beam, and 250 μm (fwhm) for the pump beam. At the sample point, energies of the probe and pump pulses were attenuated to 0.5 and 5 μJ, respectively, using Cr-coated quartz ND filters. The two beams were configured for 135° backscattering illumination and collection. The spectral features of photoproducts in the pump-probe spectra were also shown to be invariant to a two-fold change of the probe power. The pump power was selected so that no saturation effect or spectral changes occurred by a doubling of the pump power. Cross correlation trace of the pump and probe pulses measured by difference frequency generation with a thin BBO crystal indicated a width of 3.0-3.7 ps. Intensities of pump and probe pulses were monitored with photodiodes (S2387-1010R, Hamamatsu Photonics) and found to be stable within ±10%. Raman scattered light was collected by an F/2 quartz doublet achromat and focused by an F/4 quartz doublet achromat onto the entrance slit of a Czerny-Turner configured Littrow prism prefilter (16) coupled to a 50-cm single spectrograph (500M, SPEX). The spectrograph was equipped with a 1200 grooves/mm, 500 nm blazed grating operating in second order or a 2400 grooves/mm, 250 nm blazed grating operating in first order. Dispersed light in the spectrograph was detected with a liquid nitrogen-cooled CCD detector (SPEC-10:400B/LN, Roper Scientific) with Unichrome UV-enhancing coating. Raman shifts were calibrated with cyclohexane to an accuracy of ±4 cm-1. The time-resolved UVRR data acquisitions were carried out as follows. The sequence of the delay times in the time-resolved measurements were determined to be random in each scan. At each delay time, Raman signals were collected for three 20-second exposures with both the pump and probe beams present in the sample. This was followed by equivalent exposures for pump-only, probe-only, and dark measurements. This method enabled us to avoid the errors caused by a slow drift of laser power and to obtain quantitatively reproducible spectra from one day to the next, which is possible because of the excellent long-term stability of this laser system. The pump-only spectrum was directly subtracted from the pump-and-probe spectrum, yielding the “probe-with-photolysis” spectrum. The dark spectrum was directly subtracted from the probe-only spectrum, yielding the “probe-without-photolysis” spectrum, namely the spectrum of the photolyzed state. The probe-without-photolysis spectrum was subtracted from the probe-withphotolysis spectrum to yield the photoproduct spectrum. The scattering intensity for the change in the optical absorption of the sample at each time point was corrected by normalizing the data to the intensity changes of the OH stretching band of water (~3400 cm-1) of the sample solution. After normalizing the band intensities in all the spectra, the probe-without-photolysis spectrum was subtracted from the probe-with-photolysis spectrum to generate the difference spectra.

333 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Bacteriorhodopsin (BR) BR functions as a light-driven proton pump found in the purple membrane of halobacteria. So far, changes in the protein structure during a photocyclic reaction have been examined by time-resolved spectroscopy at room temperature (1, 2, 17–35) as well as cryo-spectroscopy (36–45) and crystallography (46, 47). In the photocycle, a series of intermediates, J, K, KL, L, M, N, and O are observed, each of which is characterized by a distinct absorption band. Structural information on the retinal chromophore in the photointermediate was studied based on time-resolved visible resonance Raman spectroscopy (48). The chromophore isomerizes around the C13=C14 bond from all-trans to 13-cis configuration upon the photoexcitation, to produce the J intermediate. The highly twisted chromophore with a 13-cis configuration is formed upon the J formation. In the K intermediate, the chromophore relaxes to a more planar 13-cis configuration. In the K-to-KL and KL-to L transition, the chromophore undergoes conformational changes although it keeps its configuration in 13-cis form. The Schiff base of the chromophore is deprotonated in the M intermediate and reprotonated in the N intermediate. The configuration of the chromophore returns to all-trans form in the O intermediate. For the time-resolved UVRR study, structural changes at Trp182 and Trp189 in helix F in the late intermediates, L, M, and N, were discussed based on microsecond time-resolved data (13). Nanosecond time-resolved experiments have been performed for M (49) and KL intermediates (50) by Mathies and co-workers. To study ultrafast protein response to the isomerization, we measured picosecond time-resolved UVRR spectra of BR (5). The UVRR spectrum of BR in the unphotolyzed state probed at 225 nm is shown in the top trace in Figure 3. This spectrum contains all the Raman bands of eight Trp and eleven Tyr residues in BR. Vibrational bands of Trp and Tyr side chains are noted as W and Y, respectively. The mode assignments made by Harada and Takeuchi (10) are shown in green and blue characters. The 1615-cm-1 band is attributed to the overlap of the W1 (Trp) and Y8a (Tyr) bands. The bands at 1555, 1357, 1013, and 763 cm-1 are assigned to the vibrational modes of Trp, W3, W7, W16, and W18, respectively. The other spectra in Figure 3 represent time-resolved UVRR difference spectra, obtained by subtracting the unphotolyzed BR spectrum from the spectrum measured at each delay time from -5 to 1000 ps. Upon photoexcitation, negative UVRR bands were clearly observed for Trp within the instrument response time. The negative bands indicate the depletion of the Raman intensity due to the change in protein structure during the photoreaction. The negative bands decayed from 10 to 50 ps, indicating that the band intensity of Trp recovers due to structural changes subsequent to the instantaneous change. In the region from 100 to 1000 ps, the difference spectra did not change. Figure 4 shows the temporal intensity changes of five remarkable bands observed in Figure 3. Data analysis revealed that the intensity of all the bands instantaneously decreases within the instrumental response time and recovers with a time constant, τrecovery, of approximately 30 ps. The retinal chromophore isomerizes from the all-trans to 13-cis form within ~0.5 ps upon the formation of the primary photoproduct of the J intermediate (1, 2). The initial UVRR 334 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


intensity depletion can be attributed to the protein response to the chromophore isomerization. The subsequent intensity recovery is likely to reflect the protein response to the structural change of the chromophore. It was revealed, for the first time, that the dynamics of the protein structure in the BR photocycle takes place with a time constant of 30 ps in this study (5).

Figure 3. Picosecond time-resolved UVRR spectra of BR. Probe and pump wavelengths are 225 and 565 nm, respectively. The top trace is the probe-only spectrum divided by a factor of 50, representing the UVRR spectrum of BR in the unphotolyzed state with an all-trans configuration. The spectrum of the buffer has been subtracted. The other spectra are time-resolved difference spectra generated by subtracting the probe-only spectrum from the pump-probe spectrum at each delay time. The accumulation time for obtaining each spectrum was 120 min. (Reproduced with permission from reference (5). Copyright 2009 American Chemical Society.) (see color insert) 335 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Figure 4. Temporal intensity changes of the bands in the range from -10 to 100 ps. (a) 1615 cm-1, the overlap of W1 and Y8a; (b) 1555 cm-1, W3; (c) 1357 cm-1, W7; (d) 1013 cm-1, W16; (e) 763 cm-1, W18. Markers indicate the intensity changes measured at each delay time relative to the intensity in the probe only spectrum. Solid lines are the best-fit with a function of [A1 × exp(−t/τrecovery) + A2] convoluted with the instrument response function. The obtained parameter, τrecovery, for each trace is indicated in the figure. (Reproduced with permission from reference (5). Copyright 2009 American Chemical Society.) (see color insert) We also measured picosecond time-resolved UVRR spectra probed at 238 nm (Figure 5). The different spectral patterns were observed at 5 and 100 ps, implying that the different intermediates were detected. It was found that the intensity of the band at 1620 cm-1, attributable to the W1 (Trp) and Y8a (Tyr) bands, decreased within the instrument response time, and recovered with a time constant of 30 ps. The W18 band at 765 cm-1 exhibited an intensity loss within 30 ps (5). These intensity changes suggests that the 30-ps process detected under the 238-nm probe condition results from the protein response to the chromophore relaxation as observed in the spectra probed at 225 nm. The two time-resolved UVRR difference spectra shown in Figure 5 reflect the structure before and after the 30-ps process in the protein dynamics, respectively. We found that the temporal behaviors of the observed spectral changes in each Raman band of both Trp and Tyr were not uniform. The W16 and W18 bands were not observed in the 5-ps difference spectrum while negative bands emerged in the 100-ps spectrum due to the intensity decrease of these bands. On the contrary, the spectral pattern of the W3 band showed the sigmoidal form at 5 and 100 ps. If the observed spectral change arose from the structural change of single Trp residue, the temporal 336 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


behaviors of the spectral changes for these bands would be identical. Therefore, the non-uniform temporal behaviors of the Trp bands indicate that the spectral changes in Figure 5 are attributable to at least two residues. The same is true for the Tyr bands. The Y8a band showed the intensity bleach at 5 ps and recovered at 100 ps, whereas the Y7a band exhibited the sigmoidal form at both 5 and 100 ps. This implies that the observed spectral changes of the Tyr bands arise from at least two residues.

Figure 5. Picosecond time-resolved UVRR spectra of BR (probe laser, 238 nm; pump laser, 565 nm). The top trace is the probe-only spectrum divided by a factor of 50, representing the UVRR spectrum of BR in the light-adapted state. The spectrum of the buffer has been subtracted. The others are time-resolved UVRR difference spectra at (a) 5 ps and (b) 100 ps. The accumulation time for obtaining each spectrum was 295 minutes. (Reproduced with permission from reference (5). Copyright 2009 American Chemical Society.) (see color insert)

The present UVRR results provide structural information on the primary protein response associated with the photoreaction of the chromophore in BR. The observed process can be attributed to structural rearrangement of protein moiety in the vicinity of the retinal chromophore. Based on the spectral changes of the structural marker bands in the UVRR difference spectra, we discuss the primary protein response to the chromophore isomerization. In the picosecond region, photoexcited BR sequentially relaxes to the J, K, and KL intermediates. All the Trp bands probed at 225 nm (Figure 3) as well as the Y8a band in the 238-nm spectra (Figure 5) bleached within the instrumental response time. The initial intensity bleach is likely to arise from the J-intermediate formation which occurs within 0.5 ps (17, 18, 20). Because the J intermediate is supposed to convert to the K intermediate within 3 ps (17, 18), the negative bands in the UVRR difference spectrum at 5 ps are assignable to bands of the K intermediate. The subsequent K-KL transition is less well defined. Kaminaka and Mathies reported that the KL intermediate was observed in the 10-ns UVRR difference spectrum probed at 240 nm (50). The features of our UVRR difference 337 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


spectrum at 100 ps measured by using the 238-nm probe pulse (Figure 5b) are very similar to those of their 10-ns spectrum, indicating that the KL intermediate was observed in the 100-ps UVRR spectrum. Thus, the observed spectral changes suggest that the protein response with a time constant of 30 ps is associated with the formation of the KL intermediate from the K intermediate. In the time-resolved 225-nm UVRR difference spectra (Figure 3), the intensities of the Trp bands bleached in response to the photoreaction of retinal. The resonance enhancement of the Raman bands depends on the electronic transition to which the frequency of the probe pulse is resonant. The environmental changes around the Trp residue, such as changes in the hydrophobicity and the hydrogen-bond strength, give rise to the energy shift of the electronic transitions. Also the change in the dipole moment of retinal responding to the photoreaction would cause the changes in the transition dipole moment of Trp due to excitonic coupling (51). These changes affect the resonance enhancement and, thus, result in changes of the band intensities. The negative W7 band in the time-resolved difference spectra in Figure 3 showed an asymmetric form compared to the UVRR band in the unphotolyzed state. The peak of this negative band was located around 1360 cm-1, whereas the shoulder was detected at about 1340 cm-1. The W7 mode is known to show a doublet, of which intensity ratio (I1360 / I1340) is a marker of hydrophobicity around Trp. When the Trp residue is located in a hydrophobic environment, the intensity ratio becomes larger (52). Since the spectral width of our laser was as wide as 20 cm-1, the Trp doublet could not be resolved. The enhancement of the shoulder band at 1340 cm-1 relative to the band at 1360 cm-1 in the difference spectra may indicate that the intensity ratio I1360 / I1340 in the early photointermediate is smaller compared to that in the unphotolyzed state. Thus, it is likely that the hydrophobicity of a Trp residue is reduced in the early picoseconds. Under the 238-nm probe condition (Figure 5), the W3 mode exhibited a sigmoidal form arising from the frequency shift in the difference spectra at both 5 and 100 ps. The W3 frequency is correlated with the torsion angle, χ2,1, which is defined as the dihedral angle of the C2-C3-Cβ-Cα linkage of the indole side chain (53, 54). The observed downshift implies a decrease in the torsion angle χ2,1 of the indole ring in the K and KL intermediates. Negative bands appeared at the positions of the W16 and W18 bands at 100 ps in Figure 5b. These negative bands indicate that the intensities of the W16 and W18 bands in the KL intermediate are smaller compared to that in the unphotolyzed state. The Raman intensities of the W16 and W18 modes are enhanced in resonance with the Ba and Bb states (55, 56). The absorption band of the Bb transition is blue-shifted when the hydrophobicity around the Trp residue decreases (57, 58). Because the probe wavelength, 238 nm, is located at the red side of the maximum of the Trp Raman excitation profile, which is located at 224 nm (59), the blue-shift of the absorption band results in decrease of the resonance enhancement. Therefore, the intensity loss of the W16 and W18 modes is associated with the reduction of the hydrophobicity in the KL state. For Tyr bands observed in Figure 5, two apparent marker bands were detected. A derivative-like feature was observed in both the 5- and 100-ps UVRR difference spectra at the position of the Y7a mode. This is caused by the lower frequency shift 338 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


of the Y7a band in the K and KL intermediates. The Y7a frequency is an indicator of the degree of the proton-donating state in the hydrogen bond on the phenolic OH group (60). The lower frequency shift suggests that the Tyr has more proton-donor character in the K and KL states. A large negative band of the Y8a mode instantaneously appeared upon photoexcitation, decaying within 30 ps. This indicated that the Y8a band intensity bleached in the K intermediate and subsequently recovered in the KL intermediate. The Raman intensity of the Y8a mode is resonantly enhanced by the Franck-Condon A-term mechanism via the La absorption (peak wavelength 222 nm). The maximum of its Raman excitation profile is around 225 nm (61). Thus, under the present probe condition, the intensity of the Y8a band decreases when the Raman excitation profile exhibits blue shift. It has been reported that the La absorption band systematically blue-shifts when the Tyr residue is in a more protic environment (57). The present results show an increase of the hydrogen-bond strength in the J and K intermediates and the subsequent decrease in the KL intermediate. In picosecond time-resolved UVRR spectra of BR at room temperature, we observed spectral changes of both the Trp and Tyr bands, which reflect the primary protein response to the photoreaction of the retinal chromophore. The time constant of the primary process in the protein moiety was determined to be 30 ps, for the first time. The time constant of 30 ps suggests that this change is associated with the transition from the K intermediate to the KL intermediate, which has been less defined.

Sensory Rhodopsin II (SRII) SRII serves as a negative phototaxis receptor found in halobacteria. It forms the signaling complex with its cognate transducer protein, HtrII, in the cell membrane. SRII is activated by a blue light around 500 nm and regulates the kinase phosphorylation. The structure of SRII and its complex with HtrII have been studied by X-ray crystallography (62, 63) and various other spectroscopic methods (64–66). Structural differences between the unphotolyzed state and the cryogenically trapped photointermediates have been deduced from FTIR studies (67–70) and X-ray diffraction on protein crystals (71). The primary chromophore reaction dynamics of SRII are quite similar to those of BR (3, 72). For instance, the formation time constants of the J and K states are approximately 0.5 and 3 ps, respectively (3). However, little is known about the primary protein dynamics in SRII. We investigated the primary protein response to retinal isomerization of SRII in the early intermediates on the basis of picosecond time-resolved UVRR spectra (6). The time-resolved UVRR spectra clarified the structural changes that occur around the Trp and Tyr residues located in the vicinity of the retinal chromophore. Figure 6a shows picosecond time-resolved UVRR spectra of SRII probed at 225 nm. Similarly to the BR spectra, the negative bands were clearly observed at the positions of the Trp bands after 450-nm pump pulse irradiation. Based on the temporal intensity changes in Trp bands as shown in Figure 6b, it was found that 339 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


the Trp band intensities instantaneously bleached within the instrumental response time and recovered with a time constant of ~30 ps. The initial UVRR intensity depletion can be attributed to the protein’s response to chromophore isomerization associated with the formation of the J intermediate. The subsequent intensity recovery reflects protein response to the relaxation of the chromophore in SRII in the transition from the K to the “post-K” (the KL state analogous to the case of BR) with a time constant of 30 ps, which is comparable to the time constant reported for the BR protein response.

Figure 6. (a) Picosecond time-resolved UVRR spectra of SRII (probe laser, 225 nm; pump laser, 450nm). The top trace is the probe-only spectrum divided by a factor of 40. The other spectra are time-resolved difference spectra. The accumulation time for obtaining each spectrum was 80 min. (b) Temporal intensity change of the W3 band. Markers indicate the intensity changes measured at each delay time relative to the intensity in the probe only spectrum. Solid line is the best-fit with a function of [A1 × δ(t) + A2 × exp(−t/τrecovery) + A3] convoluted with the instrument response function. (Adapted with permission from reference (6). Copyright 2011 American Chemical Society.) (see color insert) 340 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Figure 7. (a) Picosecond time-resolved UVRR difference spectra of WT-SRII (red) and the Y174F mutant (blue) in the 1400−1800 cm-1 region. Probe and pump wavelengths were 238 nm and 450 nm, respectively. The accumulation times for obtaining WT and Y174F mutant spectra were 79 and 76 min, respectively. (b) Details of the crystallographic structure of the SRII−HtrII complex in the unphotolyzed state (PDB ID=1H2S) in the vicinity of the retinal chromophore, Tyr174 and Thr204. Black dashed lines indicate hydrogen bonds. The helices of HtrII are in contact with the transmembrane helices F and G of SRII, in which Tyr174 and Thr204 are involved, respectively. SRII Tyr199 is hydrogen-bonded to HtrII Asn74. The HtrII helix involving Asn74 is displayed by a yellow-colored helix. (Reproduced with permission from reference (6). Copyright 2011 American Chemical Society.) (see color insert)

Another important result on picosecond protein response of SRII is that we succeeded to detecting structural and/or environmental changes of Tyr174, of which functional importance was pointed out previously (68, 73), taking the advantage that Raman band intensities of Tyr residues in proteins are greatly enhanced in UV excitation. Figure 7a shows a comparison of the picosecond time-resolved UVRR spectra of WT SRII and the Y174F mutant probed at 238 341 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


nm. Red and blue traces show the spectra of WT SRII and the W174F mutant, respectively. The spectral features of all the Trp bands observed in the difference spectra of the Y174F mutant were consistently close to those of WT SRII (data not shown), indicating that the mutation does not significantly perturb the Trp residues. However, Figure 7a shows that the negative bands of the Y8a mode in the Tyr174 spectra were much weaker than the corresponding bands in the WT spectra. This is strong evidence that Tyr174 is responsible for the intensity change of the Y8a band and that the structure and/or environment of Tyr174 changes in SRII following chromophore photoisomerization. Figure 7b shows the X-ray crystallographic structure of the SRII-HtrII complex around Tyr174 and Thr204, which are located close to the retinal chromophore and form a hydrogen bond with each other (74). One of the HtrII helices is displayed by a yellow-colored helix. The all-trans-retinal molecule binds to Lys205 via a protonated Schiff base linkage (magenta-colored side chain). The HtrII helices are in contact with the SRII transmembrane helices F and G (62), in which Tyr174 and Thr204, respectively, are located. Assay measurements of SRII mutants showed that Tyr174 and Thr204 are key residues in the SRII signal transduction pathway (73). Sudo et al. found that phototaxis function was lost in Thr204 or Tyr174 mutants and claimed that these residues are functionally important (68). They also demonstrated the presence of steric constraint between the C14H group and Thr204 (75). Furthermore, the extent of the steric constraint correlated with the physiological phototaxis response (68). They therefore proposed the model that the light signal is transmitted to HtrII from the energized interhelical hydrogen bond between Thr204 and Tyr174. The energized hydrogen bond is located in both the retinal chromophore pocket and in helices F and G that form the membrane-embedded interaction surface, and is transmitted to the signal-bearing second transmembrane helix (TM2) of HtrII (76). Thus, while the roles of Thr204 in structural changes and in negative phototaxis are clearly understood, the role of Tyr174 in these processes is not. Our data clearly show that the structure and/or environment of Tyr174 does change in accordance with chromophore photoisomerization. If the steric constraint between the C14H group and Thr204 is present, its effect would be exerted on Tyr174 simultaneously with photoisomerization. In fact, the contribution of Tyr174 to the Y8a band intensity was observed immediately upon photoisomerization. Thus, the present data strongly support the model described above.

Sensory Rhodopsin I (SRI) SRI was discovered in the halobacterium, regulating both negative and positive phototaxis. Kitajima-Ihara and co-workers characterized a new SRI-like protein from the eubacterium, Salinibacter ruber, and named this photosensor protein SrSRI (77). SrSRI showed remarkable stability compared to that of halobacterium, Halobacterium salinarum, SRI (HsSRI), which has been less studied due to its thermal instability. In SrSRI, it is suggested that the Cl− ion 342 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


binding site is located around the β-ionone ring of the retinal chromophore (78). Photochemical properties and structural changes of SrSRI are very similar to those of HsSRI (77). To analyze the protein response in the SRI, we employed picosecond time-resolved UVRR spectroscopy (7).

Figure 8. (a) Picosecond time-resolved UVRR spectra of SRI (probe laser, 225 nm; pump laser, 549nm). Red and blue traces are the spectra in the presence of 1 M NaCl (with Cl−) and 333 mM Na2SO4 (without Cl−), respectively. The top trace is the probe-only spectrum divided by a factor of 40. The other spectra are time-resolved difference spectra. The accumulation times were 100 and 70 min for obtaining the spectra with and without Cl−, respectively. (b) Temporal intensity change of the W3 band. Filled and open circles indicate the intensity changes measured at each delay time relative to the intensity in the probe only spectrum with and without Cl−ion, respectively. Solid lines are the best-fit with a function of [A1 × exp(−t/τrecovery) + A2] convoluted with the instrument response function. (Adapted with permission from reference (7). Copyright 2014 American Chemical Society.) (see color insert)

Figure 8a shows picosecond time-resolved UVRR spectra of SRI with and without Cl− ion probed at 225 nm. After photoexcitation, negative bands due to Trp residue were clearly observed within instrumental response. The negative bands represent depletion of the Raman intensity resulting from the change in protein structure upon photoisomerization. The negative bands decayed from 10 to 343 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


50 ps. This indicates that the Trp band intensity recovers due to further structural changes following the intensity depletion associated with photoisomerization. After 100 ps, the difference spectra did not change. The temporal intensity change of W3 band is shown in Figure 8b. The band intensity instantaneously bleached with in the instrumental response and recovered within 100 ps. The retinal chromophore isomerizes from the all-trans to 13-cis configuration in 0.64 ps (7). Thus, initial intensity depletion is attributed to the protein response to the chromophore isomerization. The time constants of band recovery of SRI with and without Cl− ion were estimated to be 17 ± 3 ps and 12± 2 ps, respectively. Both were similar to each other, indicating the similar structural changes of the Trp residue(s) following the K formation in the chromophore structure in both conditions.

Anabaena Sensory Rhodopsin (ASR) ASR is found in a freshwater cyanobacterium and exhibits a unique photoreaction different from other microbial rhodopsins. In the ground state, ASR has two stable configurations of retinal, all-trans, 15-anti (ASRAT) and 13-cis, 15-syn (ASR13C) (79), which exhibit photoinduced interconversion (80). Thus, the photoreaction of ASR is not cyclic but photochromic. The retinal chromophore is predominantly of the all-trans configuration in the dark-adapted state (DA-ASR) and contains a large fraction of the 13-cis configuration in the light-adapted state (LA-ASR) (80–82). In the photochromic reaction of ASR, ASRAT is photoconverted to the primary photointermediate, K-ASRAT, with the 13-cis, 15-anti configuration, and ASR13C is converted to the K-ASR13C intermediate with the all-trans, 15-syn configuration. Taking an advantage of the photochromic character of ASR, we can compare the protein responses upon the all-trans→13-cis and 13-cis→all-trans isomerization. Spectroscopic studies indicated that the photoreaction of both ASRAT and ASR13C includes several distinct intermediates (4, 80, 82–87). FTIR spectroscopy revealed that the distortion of the chromophore in K-ASRAT is localized in the Schiff base region, while that in K-ASR13C is distributed widely along the polyene chain. In addition, although the hydrogen-bond strength between the Schiff base and the water molecule in ASRAT is similar to that in ASR13C, the hydrogen bond is broken in K-ASRAT but not in K-ASR13C (83, 84). Femtosecond absorption spectroscopy demonstrated that the gross appearance of transient absorption of DA-ASR and LA-ASR is similar whereas the time constants of both intersystem crossing and buildup of K-ASR13C are much faster than those of K-ASRAT (4). We obtained the picosecond time-resolved UVRR spectra of DA- and LA-ASR to compare the primary protein dynamics beginning with ASRAT and ASR13C (8). Figure 9 shows picosecond time-resolved UVRR spectra of ASR probed at 225 nm. The initial states of photoreaction for green and pink traces in Figure 9 are the DA and LA states, respectively. The top traces are the probe-only spectra of DA-and LA-ASR, representing the UVRR spectra of each initial state. No difference was observed between UVRR bands for DA- and LA-ASR, suggesting that no significant difference exists in the structures of the Trp and Tyr residues 344 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


in DA- and LA-ASR. Figure 9 also shows time-resolved difference spectra. The difference spectra were obtained by subtracting the probe-only spectrum of the initial state from the pump-probe spectrum measured at each delay time from -10 to 1000 ps. In the time-resolved difference spectra, negative bands were clearly observed for Trp vibrational modes. The negative bands represent the intensity bleach of the Trp Raman bands due to the change in protein structure upon photoexcitation of the retinal chromophore. It should be noted that the frequency of the negative W3 band in the 10-ps difference spectrum of LA-ASR was higher than that of DA-ASR, suggesting that the Trp residue in LA-ASR giving rise to spectral changes upon retinal isomerization had a higher frequency component of the inhomogeneously broadened UVRR bands of the W3 mode than that of DA-ASR.

Figure 9. Picosecond time-resolved UVRR spectra of ASR (probe laser, 225 nm; pump laser, 549nm). Green and pink traces are the spectra of DA- and LA-ASR, respectively. The top trace is the probe-only spectrum divided by a factor of 40. The other spectra are time-resolved difference spectra. The accumulation times for obtaining the spectra of DA- and LA-ASR were 48 and 100 min, respectively. (Adapted with permission from reference (8). Copyright 2013 Elsevier B.V.) (see color insert)

The negative bands of Trp appeared within the instrument response. The negative bands decayed from 10 to 50 ps. Based on the temporal intensity changes of the Trp bands (Figure 10), the time constant of the intensity recovery was calculated to be 34 ± 7 ps for DA-ASR and 31 ± 4 ps for LA-ASR. Accordingly, the time constant of the change in protein structure during the photoisomerization of LA-ASR was almost the same as that of DA-ASR. In the unphotolyzed state of both DA- and LA-ASR, no difference was observed between the UVRR spectra. Under the present experimental condition, HPLC analyses revealed that LA-ASR contains 62.5% ASR13C, while DA-ASR is composed of 98.6% ASRAT. The UVRR spectrum of DA-ASR reflects the protein 345 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


structure of ASRAT. Although the UVRR spectrum of LA-ASR is a mixture of the spectral contributions of ASR13C and ASRAT, the spectral contribution of ASR13C can be examined by looking at the differences in the LA- and DA-ASR spectra.

Figure 10. Temporal intensity change of W3 band of (a) DA-ASR and (b) LA-ASR. Markers indicate the intensity changes measured at each delay time relative to the intensity in the probe only spectrum. Solid line is the best-fit with a function of [A1 × exp(−t/τrecovery) + A2] convoluted with the instrument response function. In panel (b), the black solid and gray dashed curves represent the temporal change of ASR13C and ASRAT, which was calculated based on the isomer composition in the unphotolyzed state of LA-ASR. (Adapted with permission from reference (8). Copyright 2013 Elsevier B.V.) (see color insert)

In the primary intermediate states appearing in the picosecond temporal frame, the intensity change of the Trp bands was observed both in the DAand LA-ASR spectra (Figure 9). Because DA-ASR solely consists of ASRAT, the observed temporal intensity changes of Trp bands in the time-resolved spectra starting in DA-ASR (Figure 10a) is originated from the photoreaction of ASRAT. Therefore, for the intermediate starting in ASRAT, the intensity of Trp bands bleached within the instrumental response time and recovered with a time constant of 30 ps. On the other hand, it was not straightforward to determine the spectral contribution of ASR13C in the time-resolved spectra starting in LA-ASR (Figure 10b), because the absorption coefficients at the pump pulse wavelength (80) as well as the quantum yields of photoisomerization (88) are different in ASRAT and ASR13C. The black solid and gray dashed curves Figure 10b shows estimated spectral contributions of ASRAT and ASR13C in the temporal intensity changes in the time-resolved UVRR spectra of LA-ASR, based on the isomer composition. Wand and co-workers investigated the ultrafast relaxation 346 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


process in photoreactions of the chromophore both in ASRAT and ASR13C by time-resolved absorption spectroscopy (4). They succeeded in isolating the spectral contributions of ASRAT and ASR13C in the LA-ASR spectra. Both in the photoreaction of ASRAT and ASR13C, the photoisomerization of retinal occurs within sub-picoseconds. Accordingly, the initial intensity bleach observed in the present time-resolved UVRR spectra is attributable to the protein structural change due to retinal photoisomerization occurring within a picosecond. The intensity of the Trp bands recovered with a time constant of 30 ps both for ASRAT and ASR13C. The photoisomerized ground-state intermediate is formed within sub-picoseconds (4). It does not convert back to the original state because the barrier height for the torsion around the C13=C14 bond is too high to pass over it in thermal reaction. The observed intensity recovery can be explained not by a recovery of the original structure, but by a transition from the K intermediate to the subsequent intermediate showing different band intensities. So far, the 30-ps process has not been reported in the photoreaction of ASR. This process is attributed to further rearrangements of the protein moiety around retinal. The rate of the structural rearrangement in K-ASRAT is similar to that in K-ASR13C. The robustness of the relaxation rate implies that the mode of structural change of retinal does not significantly affect the protein response of surrounding residues.

Comparison of Primary Protein Responses in Microbial Rhodopsins We carried out real-time observation of the primary protein response to the chromophore isomerization in microbial rhodopsins based on the spectral changes in Trp bands. During the picosecond region, it is expected that residues in the vicinity of retinal change their structures. Figure 11 shows the crystallographic structure in the vicinity of the chromophore of BR. Three Trp residues are located in the retinal biding pocket. Trp86 and Trp182 sandwich the polyene chain of retinal. Trp189 are positioned near the β-ionone ring. Many amino acid residues are conserved among microbial rhodopsins. The three Trp residues are also conserved for BR, SRII, SRI, and ASR. Owing to the selective Raman enhancement, we can utilize the Raman bands of Trp as good probes to discuss the structural change around the chromophore in microbial rhodopsins. The temporal changes of the Trp band intensities among the four microbial rhodopsins described above were compared. It was commonly found that the intensities of Trp band in these rhodopsins bleached upon photoisomerization of retinal and recovered with tens of picoseconds. The intensity recovery of the Trp band was attributed to the rearrangement of the protein moiety following the structural relaxation of the chromophore. The comparison shows that the rates of rearrangements of the protein moiety are insensitive to the functions, ion binding, and direction of the isomerization among these rhodopsins, suggesting that the primary structural response of the protein moiety to the chromophore isomerization is very similar in the microbial rhodopsins. 347 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Figure 11. Crystallographic structure in the vicinity of the retinal chromophore of BR (PDB ID=1C3W). Three Trp residues (Trp86, Trp182 and Trp 189 in BR) are conserved at the same positions among BR, SRII, SRI, and ASR. (see color insert)

The similar primary structural response of the protein moiety to the chromophore isomerization observed for the four microbial rhodopsins. Two pathways can be proposed for the propagation of structural changes from the chromophore to the protein moiety. One is a pathway through hydrogen bonding network including the protonated Schiff base. The orientation of the hydrogen bond of the protonated Schiff base changes upon the photoisomerization and can quickly perturb the protein moiety. The other is through van der Waals contacts between the retinal and the surrounding amino acid residues. In the time-resolved visible resonance Raman spectra, an intense Raman band due to the hydrogen-out-of-plane (HOOP) wagging mode of the retinal was observed in the J intermediate of BR (1). The HOOP band gains its intensity when the polyene chain is distorted (89). In fact, no intense HOOP band is observed for polyenes in solution because the polyene quickly adopts the stable planar form following the photoisomerization in solution. The distorted polyene chain with measurable lifetime in the proteins would be due to highly packed structure around the retinal. The propagation of structural changes through the van der Waals contacts is possible in such highly packed environment around the chromophore. It is interesting to compare the primary protein response of the microbial rhodopsins with that of visual pigment rhodopsin. Time-resolved UVRR spectra of visual pigment rhodopsin have been reported by Kim and co-workers. Visual pigment rhodopsin undergoes the 11-cis to all-trans isomerization (14). They reported that the intensity of negative Trp band gains up to 20 ps using probe wavelength of 233 nm. This temporal behavior is similar to that of BR probed at 238-nm excitation. The similarity of the protein response to the chromophore isomerization suggests that the protein response is insensitive to the retinal configuration and that a time constant of tens of picoseconds is common as the protein response to the retinal isomerization. 348 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Concluding Remarks It is essential to clarify how external stimuli can bring about site-specific structural changes to understand the mechanism of protein functions. For that purpose, measurement technique with high time resolution and site-selectivity is necessary. Time-resolved UVRR spectroscopy is able to probe the structural dynamics of specific sites in protein structure by selectively enhancing the vibrational Raman bands assignable to aromatic amino acid side chains as well as polypeptide bonds with picosecond time resolution. We here demonstrated high potential ability of time-resolved UVRR spectroscopy in observation of primary protein response to chromophore isomerization in microbial rhodopsins.

Acknowledgments We are grateful to Professors Hideki Kandori (Nagoya Institute of Technology) and Yuki Sudo (Okayama University) for kindly supplying the protein samples and stimulating discussions. We thank Mr. Seisuke Inada (Osaka University) who carried out time-resolved UVRR measurements of ASR. We also thank Dr. Mikihiro Shibata and Mr. Junya Yamada (Nagoya Institute of Technology) for hard work for preparation of a large amount of BR samples. The works presented in this review were supported by a Grant-in-Aid for Scientific Research in the Priority Area “Molecular Science for Supra Functional Systems” (No. 19056013) to Y.M. from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a Grant-in-Aid for Scientific Research on Innovative Areas “Soft Molecular Systems” (No. 25104006) to Y.M. from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a Grant-in-Aid for Scientific Research (B) (No. 20350007) to Y.M. from the Japan Society for the Promotion of Science, and a Grant-in-Aid for Young Scientists (B) (No. 23750015) to M.M. from the Japan Society for the Promotion of Science.

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