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* Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Fudan University, Shanghai, China; Department of Biology, University of Washington, Seattle, Washington; Department of Physiology and Biophysics, University of California, Irvine, California; and Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai, China
Correspondence: Address reprint requests to Jiandong Ding, E-mail: jdding1{at}fudan.edu.cn.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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8.4, much higher than 5.7 of wide-type BR. The pH-dependence of the rate constant of M-formation shows that the pKa of PRC in the initial state of AR4 is 10.4, whereas it is 9.7 in BR. Thus in AR4, the chromophore photoisomerization and subsequent proton transport from the Schiff base to Asp-85 is coupled to a decrease in the pKa of PRC from 10.4 to 8.4, which is 2 pK units less than in BR (4 units). This weakened coupling accounts for the lack of early proton release at neutral pH and the reversed time sequence of proton release and uptake in AR4. Nevertheless the PRC in AR4 effectively facilitates deprotonation of primary proton acceptor and recovery of initial state at neutral pH. We found also that all pKas of the key amino acid residues in AR4 were elevated compared to those of BR. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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). In contrast to BR, this pump exhibited a reversed order of light-driven proton release and uptake at neutral pH (1). Its amino acid sequence has been determined (2,3) and this pigment was classified as an archaerhodopsin (AR), a name initially given by Mukohata et al. (4–7) for the BR-like pigments isolated from two Australian strains, Halobacteria sp. Aus-1 and Aus-2, and called AR1 and AR2, respectively. Compared with BR-containing strains such as R1M1 which yield the conventional, "standard" BRs, these two strains are from a different genera of Halobacteriacea. The AR-containing strains belong to Halorubrum, whereas those for BRs are from Halobacterium (4,8). A third related pigment found in Halorubrum sodomense was named AR3 (8,9); its sequence has been determined by Ihara et al. (8). The pigment from the strain H. sp. xz515 exhibits 87% sequence similarity to AR1, 97% to AR2, and 84% to AR3, whereas just 59% to BR. It is likely that H. sp. xz515 is also a member of Halorubrum. Hence we name the associated pigment as archaerhodopsin 4 (AR4). The underlying mechanism of its proton pump and the origin of its differences with BR are quite interesting and help elucidate the general features of proton pumps of archaea.
AR4 is localized in the "claret" membrane as a patch in the plasma membrane. SDS-page electrophoresis shows that AR4 is the only protein in the claret membrane. Although data on AR4 is limited, much information on BR is available, which is helpful for a comparative study (10–14) (see also two special journal issues on BR (15,16)). The chromophore of BR, retinal, is covalently bound to Lys-216 of the apoprotein via a protonated Schiff-base. Upon excitation with a photon, the retinal is isomerized from the all-trans to 13-cis configuration, which initiates a photocycle that goes through a series of photointermediates with characteristic absorption maxima (17):
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Detailed spectroscopic studies show that most intermediates include two or more substates, some of which are of great importance for the mechanism (12,18–21). The photocycle is coupled to the unidirectional transfer of a proton. During a photocycle, one proton is released to the extracellular side and another proton is taken up from the cytoplasmic side.
Because of its simplicity and importance as a proton pump, BR is one of the best studied membrane proteins, and it is often used as a model of membrane and energy transducing proteins (e.g., 22–29). Studies of the kinetics of proton translocation across the membrane show that at neutral pH, light-induced proton transfer from the Schiff base to Asp-85 during the L-to-M transition leads to prompt proton release (on a submillisecond timescale) from the proton release complex (PRC or XH) to the extracellular surface (30–32). It is thought that the PRC consists of several amino acid residues, primarily Glu-194 and Glu-204, as well as water molecules (33–37). Most recently, Garczarek et al. identified a protonated water cluster surrounded by six side chains and three backbone groups (Tyr-57, Arg-82, Tyr-83, Glu-204, Glu-194, Ser-193, Pro-77, Tyr-79, and Thr-205) and suggested that this complex might be responsible for the proton release of wild-type (WT) BR (38). After proton release, proton uptake occurs in the N560 
O640 transition (39) or between two N-substates (40), usually in a time of the order of a few milliseconds. Therefore, proton pumping by BR under physiological conditions results in a unidirectional proton release at the extracellular side followed by proton uptake at the cytoplasmic side.
Several polar or charged residues located in the proton channel are highly conserved in all members of the BR and AR families which are proton pumps (8,9). The associated pKas of these groups must be altered during proton transfer, and thus pH titrations are very useful for elucidating proton pumping mechanisms of BR and BR-like proteins. In BR, Asp-85 is the primary acceptor of the Schiff-base proton. Its pKa changes in the photocycle by several pK units (41–43). The pKa of the PRC is quite high (9.7) (42) in the unphotolyzed state and decreases to 5.7 upon the protonation of Asp-85 (39,44). At neutral pH, this would lead to its deprotonation. The protonation of Asp-85 is closely coupled to the dissociation of a proton from PRC at neutral pH, and proton release from the PRC is the result of proton transfer from the Schiff base to Asp-85 upon formation of M. The thermodynamic equilibrium of acid-base transitions between the two interacting groups predicts that the changes in the pKa of Asp-85 are linked to the opposite changes in the pKa of the PRC (42,45–47). This coupling underlines the fast light-induced proton release in BR at pH levels between 6 and 9 (19,42).
In contrast to BR, we found a different sequence of light-induced proton uptake and release at neutral pH in AR4 (1). AR4 is offered by nature as a version of proton pumping machine with all key amino acids residues implicated in proton transport in BR conserved. A series of interesting questions can be raised: What is the cause of the reversed proton pumping behaviors of this protein? It is known that the order of proton release and uptake in BR can be reversed by decreasing the medium pH sufficiently (44). Then if both AR4 and BR operate on similar basic principles (48), could the time order in AR4 be analogously reversed when the medium pH is raised? Is the PRC of AR4 still functional in the photocycle? The answers would be helpful in understanding the mechanism of proton pumping proteins more deeply.
In this work, we compared the main features of the two pigments. The pH-dependence of the photocycle and proton pumping by AR4 were examined. Although fast light-induced proton release was blocked below pH 8.2, it was observed at higher pHs. The pKas of the initial proton acceptor from the Schiff base, of the proton release complex in different states, and of the proton donor to the deprotonated Schiff-base were determined. We found that all pKas of the key amino acid residues in AR4 were elevated compared to those of BR and that the coupling between protonation of Asp-85 and deprotonation of XH in the M-intermediate of AR4 was weakened. The origin of the reversed temporal order of proton release and uptake in AR4 at neutral pH and other differences with BR and other archael proton pumps are also discussed.
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MATERIALS AND METHODS |
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,50). The BR purple membrane was isolated from R1M1, a Halobacterium strain. The culturing of H. sp. xz515 was reported earlier (1). A suspension of AR4 in the "claret" membrane was obtained from the strain H. sp. xz515 by the same procedure as used for purifying the purple membrane from H. salinarum (49,50). A sucrose density step gradient, with concentrations (wt %) of sucrose of 30%, 35%, 40%, and 45%, was employed to purify the claret membrane. After centrifuging the sample (35000 r/min, in the SW41 rotor of a Beckman centrifuge for 12 h), the claret membrane was found in the 40% sucrose layer. It was collected and washed for later use. Besides archaerhodopsin, the claret membrane contained the carotenoid bacterioruberin, which did not separate from AR4 under our purification method; this association had previously been seen for other archaerhodopsins (5,51).
Absorption spectra
Absorption spectra and kinetics of dark adaptation were measured with a Cary-Aviv 14 DS spectrophotometer (Aviv Associates, Lakewood, NJ) as previously described (46). Titration of the purple-to-blue transition was done for dark-adapted samples.
Flash-induced proton pumping behavior
The flash-induced proton release and uptake was detected by the absorption change of pH-sensitive dyes, pyranine and thymol blue, at 456 nm. The signal without a dye was subtracted from that with the dye. The thymol blue as well as pyranine can be separated from AR4 pigment by centrifugation at 25,000 x g for 30 min, which indicates that the dyes are not absorbed on the membranes.
Flash-induced transient absorbance changes
The pH-dependence of proton release and proton uptake was measured with a kinetic spectrophotometer constructed (52–55) similar to that described by Govindjee et al. (56). The actinic flash was provided from a camera photoflash through an optical filter with light around 570 nm transmitted. The measuring light is a relatively weak but constant beam, perpendicular to the direction of the flash.
Other measurements such as the pH-dependence of M-formation, pH-dependence of the formation, and decay of O-intermediate, were obtained with another home-built kinetic spectrophotometer as described by Balashov et al. (57). Actinic illumination at 532 nm was provided by a frequency-doubled Quanta Ray DCR-11 Nd-YAG laser (Spectra Physics, Mountain View, CA). The transient signals were digitized using a LeCroy transient recorder. The instrument was controlled by a computer program written in Lab View. Gels were prepared as described by Liu et al. (58) and were incubated at a given pH for at least 12 h. Light adaptation was carried out by illumination with a 500 W projector (430–550 nm) for 5 min. The measurements of photocycle kinetics were performed for light-adapted samples.
All measurements were performed at room temperature unless otherwise indicated.
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RESULTS |
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). The pyranine signal was eliminated in the presence of a buffer (10 mM tris-HCl, pH 7.1) indicating that it originates from a transient light-induced change of the proton concentration in the medium.
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,44). An interesting question thus arises: will a higher pH alter the time sequence of proton release and uptake in AR4? The pH-dependence of proton release and uptake in AR4 from pH 7.5 to pH 8.2 was then examined using pyranine as shown in Fig. 1 B. This dye has a pKa of 7.2 at the salt concentration that we have used, which is consistent with the data of Govindjee et al. (59). As shown in Fig. 1 B, the temporal order of proton uptake and release was not reversed up to pH 8.2. The different amplitudes of the signal are due to the changes of the sensitivity of pyranine and the different buffering capacity of the sample at different pH levels. The temporal order of proton uptake and release of AR4 was not altered upon increase of salt concentration to 1 M. To examine the pH-dependence of light-induced proton release and uptake at pH higher than 8.2, we used another pH-sensitive dye, thymol blue with a pKa of 8.8. We also measured the kinetics of light-induced proton pumping by BR with thymol blue at alkaline pHs. As expected, the measurements show that proton uptake in BR follows proton release at alkaline pHs. So, thymol blue is suitable for measuring the proton pump at high pHs.
As shown in Fig. 2, the temporal order of release and uptake in AR4 was altered in the pH range between 8.0 and 9.0. It should be noted that an increase of absorption of thymol blue at 456 nm means medium acidification, whereas a decrease represents alkalinization, opposite to pyranine. At pHs above 8.6, the increase of absorption of the dye indicates that proton release is followed by proton uptake. The temporal sequence is thus reversed if the medium pH is sufficiently high. The pKa of proton release complex of AR4 in the M-intermediate was estimated as 8.4 ± 0.2. After 10 mM Tris-HCl buffer (pH 8.8) was added to the system, the signal disappeared, which proves that the signal in Fig. 2 comes from transient proton release and uptake of AR4.
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). Three carotenoids absorption peaks were at 477, 507, and 543 nm. To determine the absorption maximum of AR4, the light-induced absorption changes accompanying transformation of AR4 to the M-intermediate were measured at every 10 nm between 350 and 720 nm. The absorption changes show that the absorption maximum of AR4 is 565 nm (Fig. 3 B), which is very close to that of BR (568 nm), AR1 (568 nm), and AR2 (565 nm) (51). The slight blue shift compared to BR might originate from the Met-145 Phe replacement (60).
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,61). A plot of the absorption changes at 660 nm (which are proportional to the fraction of blue membrane and therefore the fraction of protonated Asp-85) versus pH gives a titration curve for Asp-85, which can be best fitted by the Henderson-Hasselbalch equation with a pKa of 3.6 (n = 0.9) (Fig. 4 B). This result differs from that for BR, which shows a pKa of 2.6 (n = 1.6) for the purple-to-blue transition (42).
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N transition which occurs 4x faster in AR4 than in BR. The formation and decay of the O-intermediate occurs with apparent time constants of 1.5 and 10 ms, respectively. Below we examine the pH-dependence of the kinetics of the formation and decay of the M- and O-intermediates in AR4 which provides information of the coupling with the protonation states of the groups involved in proton release and uptake.
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); thus we can measure the pKa of XH. Such an acceleration is absent in the E204Q (33) and E194C mutants of BR and correlates with the absence of early proton release in these two pigments (34). As shown in Fig. 6 A, the kinetics of formation of the M-intermediate are highly pH dependent. M-formation becomes faster at alkaline pH as it does in WT BR (62). This indicates that although early proton release is absent, the PRC is still present in AR4 and its deprotonation in the pigment's initial state affects the proton affinity of the primary proton acceptor from the Schiff base.
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), is 0.7 pK units higher than the value obtained from pH-dependence of M-rise (9.7 vs. 9.0) (62). Assuming that a similar relationship holds for AR4, the pKa of XH in the ground state or initial state of AR4 is estimated as 9.7 + 0.7 = 10.4. The pKa of the PRC in the initial state of BR can be determined either by direct titration of the blue-to-purple transition which is associated with deprotonation of Asp-85 or from pH-dependence of dark adaptation (42). These two methods could not be used for AR4, because of a very small amount of blue membrane at pHs above 6 and the small absorption changes due to dark adaptation. The presence of carotenoids in the claret membrane and their perturbation upon titration was also a complication. However a third method involving the pH-dependence of M-rise produced a clear result indicating that the PRC has a pKa 10.4 in the initial state.
pH-dependence of O-formation and decay
The pH-dependence of the fraction of O-intermediate and of the rate constants of its formation and decay contain information on the pKa of the groups involved in proton release and uptake (57). As seen in Fig. 7 A, the pH-dependence of the fraction of O-intermediate in AR4 is similar to that in BR, but each of the two pKas are shifted to higher values. The fraction of O decreases with two pKas of 5.8 and 9.2 vs. 3.9 and 7.5 in BR, respectively (57). The pKa of 5.8 is related to the pKa of the XH in O, and the pKa of 9.2 is apparently close to the pKa of Asp-96 in AR4, as suggested by Balashov et al. for analogous pKa of 7.5 in BR (57). The pH dependencies of the apparent rate constants of O-rise and O-decay for AR4 are shown in Fig. 7 B. Both rate constants decrease as the pH decreases below 7 with a pKa of 6.5, which would be the pKa of the proton release group in the O-intermediate (57). The apparent constant of O-decay decreases at high pH with pKa of 8.4–8.8 which we attribute to the pKa of the proton donor during proton uptake (19,57). It should be noted that the rate constants are rather sensitive to measurement conditions such as pH and medium composition, so it is only meaningful to compare data from identical samples.
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BR (39).
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,63,64), O-rise and decay are decided by two proton transfer steps: proton uptake by Asp-96, and proton transfer from Asp-85 to the PRC. But in AR4 at pH 7, fast proton release does not occur and late proton release happens during O-decay, apparently being coupled to the deprotonation of the primary proton acceptor (Asp-85). |
DISCUSSION |
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) and thus should be during or after M-decay. So, proton uptake cannot be very fast. So the origin of the reversed temporal order is the delay of proton release.
Mechanism of proton release in AR4
Now, the question becomes why proton release in AR4 is delayed. The näive interpretation might be disruption of the PRC in AR4, similar to what occurs in the E204Q (33) and E194C (34) mutants of BR. However, if the PRC in AR4 is not a functional unit as in the above BR mutants, then, like E204Q and E194C, we should not observe any pH-dependence of kinetics of M-formation. Moreover, the time constant for the turnover of the photocycle would be in the range of 100 ms, as in the E204Q mutant (57). In contrast to this, we observed a striking pH-dependence of M-rise (Fig. 6) and a relatively fast photocycle (Fig. 5). The possibility of a lack of an intact PRC is thus ruled out.
In the case of BR, the order of proton release and uptake can be reversed by decreasing the medium pH sufficiently (44). When the medium pH is lower than the pKa of PRC in WT BR (<5.7), the PRC is unable to deprotonate. Thus proton release occurs late in the photocycle, during the O640 BR570 transition (39) when the pKa of XH decreases to 4.5 (42). Likewise, the temporal order of proton release and uptake in AR4 should be altered if the pH is raised sufficiently above the hypothetical pKa of the PRC in M of AR4. This phenomenon was found in our experiments where the kinetics of proton pumping were followed up to pH 8.6 (Fig. 2). The pKa of the PRC in the M-intermediate of AR4 is estimated to be 8.4. This result strongly supports the idea that the basic proton pumping principle of AR4 is similar to that of BR because the temporal order of proton release and uptake of AR4 can be reversed by just increasing medium pH.
The pH titration experiments allow us to determine the pKas of several key residues of the proton pump. Table 1 summarizes the pKa changes.
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3.6 for Asp-85 and 10.4 for XH. So the pKa of Asp-85 in AR4 is one unit higher than that in BR (pKa = 2.6); the pKa of XH in the ground state of AR4 is also 0.7 unit higher than in BR (pKa = 9.7). All of the pKas for Asp-85, XH, and Asp-96 are elevated in AR4, which suggests that AR4 is a proton pump whose operating range is significantly shifted to more alkaline pHs.
The pH-dependence of the fraction of the fast component during M-formation (Fig. 6) indicates that the coupling between Asp-85 and XH still exists in AR4 as in BR (19). But, it has been significantly weakened in AR4. The decrease of the pKa of XH after AR4 is illuminated is much less (from pKa 10.4 to 8.4) than that for BR (from pKa 9.7 to 5.7). The higher pKa of the PRC in the photocycle does not allow fast proton release at neutral pH. Such a weakened coupling model is schematically indicated in Fig. 9.
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), Arg-82Gln (65), Arg-134His (66) and Lys-129His (59) created a reversed temporal pattern of proton release and uptake (now proton release lagging proton uptake). Hence we propose that weakened coupling is responsible for the lack of early proton release in AR4 and thus the reversed temporal order of proton uptake and proton release. This weakened coupling in AR4 correlates with the elevated pKa of the proton donor analogous to Asp-96, which might not be an accidental relationship.
Possible residues involved in coupling between Asp-85 and XH in AR4
The coupling in BR can be physically understood from the high-resolution crystal structures of the initial state of BR and the M-intermediate (67,68). In BR, Glu-204, Glu-194, Arg-82 and Asp-85 form a complex hydrogen-bonded network incorporating several bound water molecules. The movement of the positive charge of Arg-82 from its inward position to its outward position upon M-formation, accompanied by changes in hydrogen binding of several water molecules, is what apparently underlies the coupling between the protonated state of Asp-85 and proton release site (69–71). In AR4, this coupling relation between Asp-85 and XH is weakened. Several residues close to Glu-194 and Glu-204, part of the PRC, are changed to residues with smaller side groups in AR4. Ile-191, Ile-198 and Ile-203 are all changed to Vals, whereas Pro-200 and Asn-202 are changed to Glys. Tyr-133 and Leu-207 are replaced with Alas. These changes leave more space around the Glu-194/Glu-204 dyad, which therefore might accommodate more water molecules and alter the H-bonded network in this region. The possible new H-bonds might stabilize the Glu-194/Glu-204 dyad in the M-state and increase the energy barrier for the early light-induced proton release, which is probably from the Glu-194/Glu-204 dyad or a nearby water molecule (38,72). Moreover in BR, Pro-77 and Ser-193 are the residues that surround a protonated water cluster, which is important for proton release (38). The replacement of Pro-77 by Asp and Ser-193 by Thr in AR4 might affect the network in the proton release channel.
The reversed temporal order of proton pumping kinetics in AR4 is not the only archaerhodopsin example. A similar phenomenon was found in AR1 (51). An x-ray crystallographic study of AR shows that the main-chain structures of AR1 and AR2 are similar to that of BR (73,74). The reversion of the order of proton release and uptake in AR1 was attributed to the natural mutation from Lys-129 in BR to His-129 in AR1, for mutation of Lys-129 to His-129 in BR also reversed the normal order (59). This argument however does not work for AR4, which has a lysine at position 129 rather than a histidine.
In contrast to AR4, AR2 exhibits the normal temporal order of light-driven proton pumping kinetics (51). Just eight amino acid residues in AR2 are different from those in AR4 (see Fig. 10), but this seems sufficient to change the proton release/uptake order. Since XH must be located in the half-channel on the extracellular side, the associated three different residues among the eight residues marked in Fig. 10 might be responsible for the change. The most probable candidate is Thr-74, which in AR2 is replaced by an alanine. Threonine is likely to be a part of the hydrogen bond network.
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). Second, the rate constant of O-formation exhibits a strong pH-dependence with pKa 6.5 (Fig. 7). This is similar to WT BR, where an analogous pH-dependence was observed and the O-to-BR transition at low pH was controlled by the protonation state of XH (57). From this we can conclude that the pKa of XH in O is 6.5 in AR4, 2 pK units higher than in WT BR. Last but not least, the overall photocycle turnover (Fig. 5 A) is close to that of WT BR (Fig. 5 B) rather than the E204Q mutant (100 ms), indicating again that the deprotonation of a group analogous to Asp-85 is catalyzed in AR4. Thus the critical role of the PRC in the photocycle is not just in catalyzing early proton release but mainly in catalyzing the deprotonation of primary proton acceptor, facilitating proton transfer in the outward proton channel and recovery of the initial state (shortening turnover of the photocycle). In summary, this work examines the photochemical reactions of AR4 with emphasis of their pH dependencies. By studying the features of AR4 and performing comparisons, we conclude that AR4 is a light-driven proton pump like BR, but with the pKas of the key groups shifted to a more alkaline pH range. The temporal order of proton uptake and release in AR4 can be reversed to that in BR when the pH is increased to 8.6. Although proton release is delayed till the end of the photocycle at neutral pH, it still occurs through the PRC, which is different from the behavior of several mutants of BR in which PRC is disabled. Our analysis indicates that the PRC in AR4 facilitates deprotonation of Asp-85 in the final step of the photocycle. Weakened coupling between Asp-85 and XH is assumed to account for the lack of early proton release and thus the reversed temporal order. Some key residues potentially responsible for this coupling have been suggested.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Submitted on October 22, 2005; accepted for publication January 19, 2006.
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REFERENCES |
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OD456 are normalized to OD412,max, the amount of flash-induced M-intermediate formed in the photocycle of each protein. The downward curve corresponds to acidification of the medium (proton release first), whereas the upward curve corresponds to proton uptake. Samples were measured in 150 mM KCl aqueous solution containing 25% glycerol. (B) pH-dependence of flash-induced absorption changes of pyranine in AR4 suspension. The sample was measured in 100 mM NaCl and 20 mM KCl.
with a pKa of 3.6 (n = 0.9).
assuming an unidirectional N
O back reaction (57
