Transient dissociation of the transducer protein from Anabaena sensory rhodopsin concomitant with formation of the M state produced upon photoactivation
Anabaena sensory rhodopsin (ASR), a microbial rhodopsin in the cyanobacterium sp. PCC7120, has been suggested to regulate cell processes in a light-quality-dependent manner (color-discrimination) through interaction with a water-soluble transducer protein (Tr). However, light-dependent ASR-Tr interaction changes have yet to be demonstrated. We applied the transient grating (TG) method to investigate protein\xe2\x80\x93protein interaction between ASR with Tr. The molecular diffusion component of the TG signal upon photostimulation of ASRAT (ASR with an all-trans retinylidene chromophore) revealed that Tr dissociates from ASR upon formation of the M-intermediate and rebinds to ASR during the decay of M; i.e. light induces transient dissociation of ASR and Tr during the photocycle. Further correlating the dissociation of the ASR-Tr pair with the M-intermediate, no transient dissociation was observed after the photoexcitation of the blue-shifted ASR13C (ASR with 13-cis, 15-syn chromophore), which does not produce M. This distinction between ASRAT and ASR13C, the two isomeric forms in a color-sensitive equilibrium in ASR, provides a potential mechanism for color-sensitive signaling by ASR.
Introduction<!>Sample preparation<!>Measurements<!>Principles<!>TG signal of ASR without Tr<!>Interaction of ASRAT and Tr<!>Interaction of ASR13C and Tr<!>Mechanism of protein\xe2\x80\x93protein interaction of ASR with Tr<!>Conclusion
<p>Anabaena sensory rhodopsin (ASR), a retinal-binding membrane protein in the cyanobacterium Anabaena sp. PCC7120 1, is a member of the microbial rhodopsin family 2, the first-discovered member of which is the light-driven proton pump bacteriorhodopsin (BR). Members of the family have been found in archaea, eubacteria and unicellular eukaryotes such as fungi and green algae, and are recognized by conservation of residues constituting a retinal binding pocket 3–6. The structures of the rhodopsins crystallized so far, including ASR 7, are composed of seven transmembrane helices enclosing within the binding pocket a retinylidene chromophore covalently bound via a protonated Schiff base (C=NH) to a lysine residue. The binding pocket accommodates either an all-trans or 13-cis, 15-syn (C=N cis) chromophore, which photoisomerizes at the C13 double bond inducing a series of conformational changes in the protein moiety which are spectroscopically-distinguished as intermediate states. The common architecture is responsible for diverse functions, such as light-driven ion transport, light-gated passive ion channel activity, and photosensory reception for phototaxis signals transduced through membrane-bound chemotaxis-receptor-like proteins 2–6. Thus far, ASR is the only microbial rhodopsin that associates with a water-soluble protein 1,8,9 believed to be a transducer (Tr). Based on the photochromic properties of ASR, a pigment with photoactive blue-shifted and red-shifted forms in the dark state, it has been suggested that the ASR-Tr pair regulate color-sensitive processes in the cell, e.g. photosynthetic activity, which contains both blue- and red-absorbing pigments 1.</p><p>ASR undergoes chromatic shifts in absorbance to either the red or blue upon illumination with blue or orange light, respectively. These changes have been attributed to the presence of two species of ASR that accommodate either the all-trans (ASRAT) or 13-cis, 15 syn (ASR13C) chromophore with the wavelength of the maximum absorption (λmax) red-shifted for the former compared to the latter (549 nm vs 537 nm, respectively) 1,8 in analogy to the case of BR, which, in the dark-adapted form, exists with two isomeric species in equilibrium. BR with the all-trans chromophore undergoes a cyclic reaction upon photoactivation returning to itself, whereas BR with the 13-cis, 15-syn chromophore is photoconverted to all-trans BR, accumulating exclusively the all-trans species (light-adaptation of BR). In contrast, the two species of ASR are photointerconvertible,10 and thus, orange light (>570 nm) predominantly converts ASRAT into ASR13C establishing a photostationary equilibrium constituting of 20% ASRAT and 80% ASR13C, whereas blue light (460 nm) shifts the equilibrium to one in which ASRAT predominates. In contrast to BR, dark-adaptation increases the population of ASRAT. The fraction of ASRAT in the dark-adapted condition varies with different experimental conditions (67 % 8, 76 % 7, and 96 % 11).</p><p>Time-resolved visible spectroscopy measurements of ASR after photoexcitation have detected a series of conversions between transient intermediate species, analogous to those of all-trans BR 8,12. However, unlike the single cyclic photoreaction of all-trans BR, photostimulation of an ASR population induces two separate photochromic reactions, namely those of ASRAT and ASR13C 8. Notably, the M state produced by Schiff base deprotonation is formed only in the photoreaction process of ASRAT 10. The proton transfer reaction appears to be coupled to cytoplasmic-side structural changes involving the C-terminal extension of the ASR protein, judging from the fact that a C-terminal truncation inverts the Schiff-base proton transfer from an unidentified extracellular-side residue in full-length ASR14 to Asp217 on the cytoplasmic side in the truncated ASR 12,13. In contrast, in the photoreaction of ASR13C, the Schiff base does not deprotonate (i.e. no M-intermediate forms) and instead a red-shifted species forms before converting into ASRAT 10.</p><p>The association of ASR and Tr has been demonstrated by the effect of Tr in slightly accelerating M-decay 1, in slightly shifting the absorption spectrum of ASR13C minus ASRAT 8, and also by direct measurement of their dissociation constant by isothermal titration calorimetry (17 μM) 9. For Tr to function as a transducer molecule, it is presumed to alter its binding to ASR upon photoactivation, although no direct evidence has been presented. To explore the dynamics of the protein–protein interaction, here we apply the pulsed laser induced transient grating (TG) technique, which has been proven to be powerful for detecting dynamics silent to conventional near-UV-visible spectroscopy (spectrally silent dynamics) 15–23. In particular, it has been found that the diffusion coefficient (D) obtained from the TG signals is a very useful property to monitor changing intermolecular interactions with high sensitivity and high time resolution. In the present case of the ASR-Tr system, we observed an increase in D ascribable to release of Tr from the ASR-Tr complex. Since this change was transient during the ASR photocycle, it is not surprising that other techniques without time-resolved capability did not detect this change. The lifetime of the increased D signal coincided with that of the M state during the photoconversion from ASRAT to ASR13C. In contrast, no changes in D were detected during the photoconversion from ASR13C to ASRAT, which produces no M-intermediate. Thus, we suggest that this dissociation is triggered by the conformational change of the cytoplasmic side correlated with the inner protein proton movement during the M-intermediate formation of ASR.</p><!><p>Preparations of the full-length ASR and Tr were similar to those described in the previous reports 1,8. Briefly, the Escherichia coli (BL21(DE3), Invitrogen) transformants containing genes for ASR or Tr with a hexahistidine tag introduced at the C-terminus under the T7 promoter in the plasmid constracts (pET-21d, Novagen) were cultured in 2×YT medium (37 °C) in the presence of ampicillin (50 mg mL−1). Protein expression was induced by adding 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) and 10 μM all-trans retinal. The cell debris was sedimented by low-speed centrifugation (4000×g or 6000 rpm, BECKMAN COULTER, JA-10) and the membrane fraction in the supernatant was then harvested by ultracentrifugation (150000×g or 48000 rpm, Hitachi Koki, S55A). The membranes were solubilized in buffer solution containing 50 mM Tris-HCl (pH 7.5), 300 mM NaCl, and 1 % n-dodecyl β-D-maltoside. After centrifugation of the solubilized membranes, the supernatant was incubated with nickel-nitrilotriacetic acid agarose beads (Novagen), and then washed with 50 mM and 100 mM imidazole in the same buffer and the protein was eluted with 250 mM imidazole. The eluted protein samples were concentrated and dialyzed to remove the excess imidazole. The Tr was expressed and purified using the his-tag by the same method.</p><!><p>The experimental setup for the TG experiment was similar to that reported previously 20,21. A laser pulse from the second harmonic of a Nd-YAG laser (Spectra-Physics, Model GCR-170-10) (10 ns pulse width) was used as an excitation beam. The spot size of the excitation beams at the sample position was ~1 mm-diameter. The TG signal was detected by a photomultiplier tube (Hamamatsu, R1477) and recorded with a digital oscilloscope (Tektronix, TDS-5052). The grating wavenumber (q) was determined from the decay rate of the thermal grating signal of a calorimetric reference sample, Evans Blue, which gives rise to only the thermal grating signal due to the nonradiative transition within the pulse width of the excitation laser. The q was changed by changing the crossing angle of the excitation pulses and the probe beam. The TG signals were averaged over 9–36 pulses to improve the signal to noise ratio. After every shot of the excitation pulse, the sample solution was stirred to avoid significant photoexcitation of the photo-product. Since the excitation volume (~ 1 μL) was much smaller than the sample volume (~200 μL), the sample condition (contributions of the dark- or light- adapted states) should be constant during the TG measurement. All measurements were carried out at 25°C.</p><p>For the measurement of dark-adapted ASR, a sample solution kept overnight in the dark was used. For light-adaptation, the sample was illuminated with a Xe lamp (USHIO, SX-1500XQ) beam passed through an orange glass filter (>570 nm) and IR cut-off filter for 5 min (the light-adapted ASR sample). The concentration of ASR was 40 μM. To prepare the ASR + Tr solution, the same volumes of ASR (pH 7.5, 80 μM) and Tr (pH 7.5, 560 μM) solutions were mixed. Samples were cleared of particulate matter by centrifugation filtration immediately before measurement.</p><!><p>The TG signal is generated from the refractive index change (δn) induced by the photoexcitation of the sample by the interference pattern of the two excitation light beams 22,23. Theoretical descriptions for analyzing the temporal profile of the TG signal are given in section S-1 in the supporting information (SI).</p><!><p>Before investigating the interaction of ASR and Tr, the TG signal of ASR without Tr was measured. Figure 1 depicts the TG signals observed after photoexcitation of ASR in the absence of Tr under dark-adapted (blue curve) and light adapted (low intensity red curve) conditions at q2 = 4.46×1010 m−2. Since most ASR under dark-adapted conditions is ASRAT and the TG signal from ASR13C is negligibly weak as described below, this signal represents the TG signal predominantly from the photoreaction of ASRAT. We will first discuss the dark adapted signal, which consists of an initial rise in the microsecond time range, and decay over a time scale of 100 μs, followed by a large rise-decay feature and a small shoulder signal (~ 100 ms time range) before finally decaying to baseline.</p><p>The total temporal profile of this signal in the time range of μs - s was expressed well by the sum of seven exponential functions.</p><p>
(1)ITG=[a1exp(−k1t)+a2exp(−k2t)+a3exp(−k3t)+a4exp(−k4t)+a5exp(−k5t)+a6exp(−k6t)+a7exp(−k7t)]2 where ki is a decay rate of the TG signal, which indicates the reaction or diffusion kinetics (k1>k2>k3>k4>k5>k6>k7). The pre-exponential factor ai reflects the refractive index change involved in the reaction with rate ki. The first and second terms express the initial rise components in the 1 μs – 10 μs time range. The third term reflects the decaying signal in 100 μs time range. The fourth and fifth terms represent the rise part of the large rise-decay signal in the 1 ms – 10 ms time range, and the sixth term represents the decay part. The last term expresses the small shoulder at the last stage of the signal (>100 ms). Fitting parameters to reproduce the signal are listed in table S1 in the SI. We repeated the measurements on different days several times, and obtained the same values within the experimental error range shown in table S1.</p><p>The assignment of these components was performed using measurements under different grating wavenumbers (q2). The q2-independent rate constants are attributable to the reaction kinetics, whereas q2-dependent rate constants are due to diffusion processes. It was found that k3 and k7 depended on q2 significantly (see Table S2 in the SI). The value of k3 agreed well with the value of Dthq2 (Dth: thermal diffusivity), determined independently from the calorimetric reference sample (Evans Blue) under the same conditions; thus this component was attributed to the thermal grating signal, i.e., a3=δnth, and k3=Dthq2. Since δnth is negative at this temperature, the signs of the pre-exponential factors were determined as a1>0, a2>0, a4>0, a5>0, a6<0, and a7<0. The significant q2-dependence of k7 indicates that this component is the diffusion signal. The q2-dependence of the other time constants was much less, and the values converged to constant values at small q2. This behavior could be explained by a combination of reaction rate and diffusion processes (e.g., eq. S4 in the SI). The time constants at small q2 region (i.e., negligibly small Dq2 compared with ki in eq. S4 of the SI) should represent the kinetics of the reaction. In fact, most of the time constants (k1−1=2 μs, k2−1 = 70 μs, k4−1=1 ms, k5−1 =5 ms, k6−1 =50 ms) in the small q2 region (q2=4.46×1010 m−2 − 1.16×1010 m−2) were similar to the previously reported values determined by flash photolysis 8,12. Therefore, we assigned these kinetic components to the formation and decay processes of the light-induced intermediate species as follows.</p><p>
ASRAT→2μsK→70μsL→5ms→1msM→50msP where P stands for the product species which was produced after the M-intermediate. Therefore, the characteristic large rise-decay component in the 1–100 millisecond time range reflects the formation and decay processes of the M-intermediate, which entail a large absorption shift of the spectrum to shorter wavelength due to the deprotonation of the Schiff base. The rate constant k7 for the last stage of the signal depended on the q2-value and was not observed in the flash photolysis measurement. This last decay phase therefore reflects the diffusion process of ASRAT (diffusion signal).</p><p>It should be noted that the diffusion signal was expressed well by a single exponential function. As reported previously, when the molecular diffusion coefficient (D) of the photoproduct differs from the reactant, the diffusion signals should be expressed by a bi-exponential function 15–17. Therefore, the singularity of the diffusion process indicates that D of the photoproduct of ASRAT (ASR13C) remains unchanged. From the equation k7=Dq2, we determined D of ASR to be D = 2.3(±0.3)×10−11 m2s−1, which corresponds to a globular protein with a molecular mass around 600 kDa, twenty times larger than that of ASR (e.g., 615 kDa for photochlorophyll-protein complex (2.63×10−11 m2s−1) and 612 kDa for hemocyanine (2.80×10−11 m2s−1) 24). The larger than actual value of the molecular weight deduced from the Ds is not surprising for membrane proteins, because the protein molecules are embedded in large detergent micelles. Aggregation of the ASR molecules is unlikely because of the independence of the D-value from the sample concentration (not shown).</p><p>The TG signal measured after the photoexcitation of the light-adapted ASR also shown in Figure 1 (red curve). Compared with the TG signal of the dark-adapted ASR, the TG signal drastically changed both in the time profile and, in particular, the signal intensity (reduced to ~10 % in amplitude). As mentioned in the introduction section, although the dominant ASR species in the light-adapted condition is ASR13C, a small fraction of ASRAT (~20 %) is present 1,8. Therefore, the TG signal due to ASRAT as well as ASR13C is superimposed in the TG signal of the light-adapted sample.</p><p>The TG signal intensity is proportional to the square of the refractive index change (δn2) induced by photoreaction. Since the δn is proportional to the concentration, the contribution of the ASRAT to the TG signal in the light-adapted sample as compared to the dark adapted one is estimated to decrease to [δn(ASRAT)Light/δn(ASRAT)Dark]2=[20%/67%]2~(0.3)2 (using the value 67 % for the population of ASRAT in the dark-adapted condition 8). When the TG signal under the dark-adapted condition is multiplied by (0.3)2 (Figure 1b, blue dotted curve), this signal intensity becomes comparable to that in the light-adapted condition, indicating that the characteristic large rise-decay in the millisecond time range comes from the reaction of ASRAT, which exhibits M-formation and decay. Conversely, ASR13C, which is devoid of M in its photoreaction products as reported previously 10, contributes little to the TG signal. The small amplitude of the TG signal for ASR13C compared to that for ASRAT is ascribable to the weaker absorption coefficient 8 and the smaller quantum yield 25 of ASR13C. However, the imperfect match between the TG signal of the light-adapted samples (Fig. 1b, red curve) and the estimated TG contribution of ASRAT both in signal intensity and time profile suggest the presence of refractive index changes during photoreaction of ASR13C. In particular, the TG signal in the light-adapted samples shows a distinct feature in the last decay phase near 100 ms, which is different from TG of ASRAT.</p><p>The TG signal was analyzed without separating the ASRAT and ASR13C contributions. The total temporal profile of the signal in the light-adapted condition was reproduced by the sum of seven exponential functions similar to that for the dark-adapted ASR.</p><p>
(2)ITG=[b1exp(−k1′t)+b2exp(−k2′t)+δnthexp(−Dthq2t)+b4exp(−k4′t)+b5exp(−k5′t)+b6exp(−k6′t)+b7exp(−k7′t)]2 where k′1>k′2>k′4>k′5>k′6>k′7 (k′2>Dthq2>k′4 at this q2). The pre-exponential factor bi reflects the refractive index change involved in the reaction with rate k′i. Fitting parameters to reproduce the signal are listed in table S1 in the SI. The signal was reproduced well with time constants similar to those obtained in the dark-adapted condition at the same q2. Some small differences were notable, in particular for k5 and k6. These differences may represent a weak contamination of the signal from ASR13C as described above. A significant difference was observed only in the sign of the pre-exponential factor for the last decay phase; the sign was negative in the dark-adapted condition (a7<0), and positive in the light-adapted condition (b7>0) (see Table S1). Since the decay rate was also q2-dependent in the light-adapted condition, this last phase should be a diffusion signal. This diffusion signal reflects diffusion processes of both ASR13C and ASRAT, because both of them exist in the light-adapted condition. The same decay rate of the diffusion signal in dark- and light-adapted conditions (k7 ~ k′7) is consistent with the observation that D of ASR13C is identical to that of ASRAT as described above. On the other hand, the inversion of the sign of the diffusion signal indicates that the refractive index change δn induced by photoreaction of ASRAT is opposite to that from the photoreaction of ASR13C, which is expected because the product for the photoreaction of ASRAT is ASR13C, and vice versa.</p><!><p>The interaction of ASRAT and Tr was studied by measuring the TG signal of ASR in the presence of Tr in dark-adapted samples. For comparison, the TG signals of the dark-adapted ASR in the absence (ITG(ASR)) and presence (ITG(ASR:Tr)) of Tr under the same experimental conditions are depicted in Figure 2a at various q2-values. It should be noted that most of the temporal profile except for the slowest part of the signal (i.e. the diffusion signal) was not changed by the presence of Tr. For example, the orange line in Figure 2a up to 10 ms (representing the M-formation and decay) is not altered by the presence of Tr. This fact indicates that the reaction process as well as the rate did not depend on the presence of Tr. In a previous measurement a slight acceleration of M-decay in the presence of Tr was observed when measured in E. coli cell suspensions 1. The difference may be due to the different environment of ASR in vivo.</p><p>The observed Tr-dependent process is the diffusion signal. For analyzing the Tr-dependent signal, we extracted the difference signal with and without Tr. Since the TG signal intensity is proportional to the square of the refractive index change, taking the difference is not straightforward. We calculated the difference signals by a method described in section S-2 in the SI and plotted them in Figure 2b.</p><p>It is important to note that the difference signals show rise-decay profiles. This profile is a clear indication that there are two diffusing species having different D-values. When the D-value is independent of time, the temporal profile of the species grating signal decays with a molecular diffusion rate for the reactant (DRq2) and the product (DPq2). Hence, the time development of the TG signal in the molecular diffusion part can be expressed by a bi-exponential function 15–17: 
(3)ITG(t)=α{δnPexp(−DPq2t)−δnRexp(−DRq2t)}2 where δnR(>0) and δnP(>0) are the initial refractive index changes due to the reactant and the product, respectively. Using δnth <0, we determined the signs of δn for the rise and decay components as positive and negative, respectively. Hence, the rate constants of the rise and decay components should be DPq2 and DRq2, respectively.</p><p>Interestingly, the intensity of the difference signal exhibited q2-dependence; the intensity is weak in a fast time range (<100 ms, large q2), became stronger in the 100 ms range (q2= 5.66×1011 m−2), and decreased again in the slower time range (>100 ms). The q2-dependence of the diffusion signal intensity indicates that there are reaction dynamics in this time range. Therefore, the signal should be analyzed based on the equation representing time-dependent D. However, before analyzing the signal quantitatively, it may be instructive to describe the qualitative features of this signal, because this is quite a unique observation.</p><p>The origin of the D-change can be examined from the D-value. We first estimated the D-values with bi-exponential analysis of the signal using eq. (3). From the fitting of the difference signal at q2 = 5.66×1011 m−2, where the signal intensity is largest, DP and DR are determined to be 7.5(±0.5)×10−11 m2s−1 and 2.5(±0.5)×10−11 m2s−1, respectively. Since the smaller DR =2.5×10−11 m2s−1 is close to that obtained from the signal in the absence of Tr, we attributed this to D of ASR. On the other hand, the larger DP = 7.5×10−11 m2s−1 was not observed in the absence of Tr. The molecular mass for this diffusion species is expected to be ~ 50 kDa; e.g. comparable to insulin (7.45×10−11 m2s−1; 47.8 kDa) and pituitary growth hormone (7.36×10−11 m2s−1; 49.2 kDa) 24. This estimated molecular mass is similar to that of the Tr tetramer (56 kDa), and Tr is known to form a tetramer in solution 9. Therefore, we attribute the diffusing species of the product to Tr tetramer. Note that in contrast to ASR, Tr is a water-soluble protein and is not contained in a micelle.</p><p>It is important to note that we observed the diffusion of Tr, which does not have a chromophore, upon photoexcitation. We conclude that Tr dissociates from ASR upon photoexcitation of ASR, i.e.,</p><p>
ASR:Tr→hνASR+Tr where ASR:Tr is the complex of ASR and Tr. In this model, three species are involved; ASR:Tr, ASR, and Tr. Since we observed only two diffusion components, the D values for two of the species should be very similar. This consideration appears to be reasonable. From the D-value of ASR, the effective molecular mass of the ASR micelle is very large, ~ 600 kDa as described in the previous section. Upon binding of the Tr tetramer (~50 kDa), the effective molecular mass would change only by ~10 %. According to the Stokes-Einstein equation, D of a molecule is inversely proportional to the radius of the molecule. If we assume that the radius is proportional to the cube root of the molecular mass, the change in the molecular mass of ~10 % corresponds to the change in the D-value of only ~3 %. Hence, the D values of ASR and ASR:Tr should be very similar to each other. Note that ASR contained in a micelle is expected to be able to interact with the water soluble Tr, because the hydrophilic cytoplasmic side of ASR provides an aqueous interaction surface for the water soluble Tr. As stated in the above section, we consider that aggregation of the ASR molecules is unlikely in the micelle. Hence, the ASR:Tr complex should be the 1:1 (transient) complex (where Tr should be a tetramer of the transducer protein).</p><p>In this dissociation model, the intensity of the rise-decay diffusion signal reflects the number of ASR:Tr complexes which undergo the light-induced dissociation of Tr. In the milliseconds time range, the signal intensity increases with time. This time-dependence indicates the dissociation reaction proceeds on this time scale. The signal decreases in the time range of tens of milliseconds. This time dependence indicates the back reaction of the dissociation, i.e. the association reaction. It is notable that the time scales where the intensity changes by dissociation and association were similar to those of the M-formation (~ 1 or 5 ms) and M-decay (~50 ms), respectively. This leads to the suggestion that the dissociation and association events are governed by the M-formation and M-decay of ASR.</p><p>We quantitatively analyzed the diffusion signal based on the following dissociation and association scheme.</p><p>
ASR:Tr→hνASR∗:Tr→kfASR†+Tr→kbASR:Tr where subscripts ASR*:Tr stand for the intermediate after photoexcitation but before dissociation of Tr, and ASR† stands for the intermediate after dissociation. The kf and kb are the rates for the forward conformation change (dissociation) and for the backward conformation change (association), respectively. Based on the above scheme and eq. (S6) in the SI with DI = DR* = DR, the temporal profile of the TG signal can be given by: 
(4)ITG(t)=α[[δnR∗−kfkf−kbδnI−kfkf−kbkf(DR−E)q2+kfδnB+kbkf−kbδnR]exp{−(DRq2+kf)t}+kfkf−kb[δnI+kb(DR−DB)q2+kbδnB−δnR]exp{−(DRq2+kb)t}+[1−kb(DR−DB)q2+kb]kf(DR−DB)q2+kfδnBexp(−DBq2t)]2 where R, R*, I, and B stand for ASR:Tr, ASR*:Tr, ASR†, and Tr, respectively. We analyzed the difference signals at various q2 simultaneously using this equation. During the fitting, we used the D(ASR) value obtained in the previous section as DR. The difference signals were well reproduced by this equation (Figure 2b, black solid curves). Fitting parameters to reproduce the signals are listed in table S3 in the SI. We obtained the rates for the dissociation and association to be 8 (±3) ms and 80 (±40) ms, respectively. The measurements were repeated several times over different days, and the same values within the experimental error range. These time constants were similar to those for the M-formation and M-decay. This result strongly suggests that Tr dissociates from ASR only in the M-intermediate during the photoreaction of ASRAT.</p><p>Under our experimental conditions shown above ([Tr]=280 μM, [ASR]=40 μM), the concentration of the ASR:Tr (Tr as a tetramer) complex is calculated to be 28 μM by using the reported dissociation constant (17 μM). This concentration should be sufficiently large for detection of the Tr dissociation. For further confirmation of the above model, we increased the Tr concentration further, and found that the diffusion signal intensity increased (data not shown), which is consistent with the above Tr dissociation model. The time profile did not change by increasing the Tr concentration, probably because the rate constants are determined by the kinetics of the M state with M- formation and decay as the rate determining steps.</p><!><p>To investigate whether interaction changes occur between ASR13C and Tr during the ASR photoreaction, we compared the effect of Tr on the TG signals of the light-adapted ASR in which ASR13C predominates. Similar to the dark-adapted ASR case described above, Tr-dependent diffusion signals appear in the late-time region (10–100ms) (Fig. 3a). Since light-adapted ASR contains a mixture of ASR13C and ASRAT, the contribution from ASRAT needs to be subtracted. We calculated the ASRAT component based on the amplitude of the 1 ms rise, which is the M-formation-specific TG signal, because M is a photoproduct of ASRAT, but not of ASR13C 10. The detailed procedures are described in the SI and Figure S1. The nearly exact match of the diffusion signal normalized for the M-formation-specific TG signal that defines the contribution of ASRAT shows that the diffusion signal is negligible or absent in ASR13C (Fig. 3b) (in this figure, the calculated difference signal in the dark-adapted condition was reduced by a factor of 0.9 to show that time profiles were also similar).</p><!><p>In this study, we applied the TG technique to investigate the dynamics of protein–protein interaction of ASR with Tr. We observed Tr-dependent transient diffusion coefficient D changes in the sample resulting from the ASRAT, but not ASR13C, isomeric form of the pigment. The appearance of signals with larger D attributable to the release of Tr with an intrinsic time constant of 8 ± 3 ms and the disappearance of the signal at 80 ± 40 ms coincide well with the lifetime of the M-intermediate state of ASR, indicating that Tr is released from the ASR photoreceptor during the lifetime of the M state. The M-intermediate is produced after photoisomerization of the all-trans chromophore in ASRAT to the 13-cis form. M-intermediate formation has been shown to entail a deprotonation of the retinylidene Schiff base, a reaction which is coupled to a conformational change in the ASR protein on its hydrophilic cytoplasmic surface and C-terminal extension 12,14, where Tr is believed to associate. Based on the TG results, we suggest that the cytoplasmic-side conformational change triggers the release of Tr from its ASR binding site. With the reprotonation of the Schiff base that follows (M-decay), ASR13C is formed concomitantly with the recovery of the original conformation in the protein moiety, as suggested from the fact that it rebinds Tr. This transient dissociation of Tr does not take place in the photoreaction of ASR13C converting to ASRAT, where M is not produced. A schematic illustration of the possible reactions is shown in Figure 4. The difference in the manner of interactions with Tr during the photoreactions between ASRAT and ASR13C would allow regulation of the concentration of the free Tr in the cells depending on the environmental light condition; e.g., light intensity and light wavelength, enabling the ASR to function potentially as a photochromic color sensor.</p><!><p>The dynamics of the protein–protein interaction between ASR with Tr were investigated by the time-resolved monitoring of the molecular diffusion process. We observed very different features in the TG signals of ASR in the dark- and light-adapted conditions. When Tr was added to the solution, a significant change was observed in the diffusion part of the TG signal, reflecting the change in the molecular diffusion process of ASR induced by the protein–protein interaction. We conclude that this diffusion change reaction reflected the transient dissociation of the Tr from ASR. We found the time constants for the dissociation and subsequent association reactions were similar to those for M-formation (~ 5 ms) and M-depletion (~ 50 ms) of ASR. Furthermore, this transient dissociation was not observed during the photoreaction of ASR13C, where the M-intermediate is absent. Therefore, we conclude the transient dissociation takes place during M-intermediate formation, when the conformation of the cytoplasmic side is changed after deprotonation of the Schiff base 12,14. This transient dissociation and association should be a key step for ASR to transmit the photo-signal and differences in the interactions between ASRAT and ASR13C with Tr are likely to be an important mechanism for the function as a photochromic color sensor. Since the ASR absorption spectra do not change upon addition of Tr, it is apparent that the dissociation of Tr is a spectrally silent process and cannot be detected by optical spectroscopy. The TG technique was able to overcome this limitation and provided this first direct observation of protein–protein interaction dynamics of ASR with Tr.</p>