Complexation of the Signal Transducing Protein HtrI to Sensory

The complexation between the photoreceptor sensory rhodopsin I (SRI) and its signal transducer protein HtrI was examined by assessing titration of the...
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J. Phys. Chem. B 1997, 101, 109-113

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Complexation of the Signal Transducing Protein HtrI to Sensory Rhodopsin I and Its Effect on Thermodynamics of Signaling State Deactivation Bing Yan,†,§ Elena N. Spudich,† Mordechai Sheves,‡ Gali Steinberg,‡ and John L. Spudich*,† Department of Microbiology and Molecular Genetics, UniVersity of Texas Medical School, Houston, Texas 77030, and Department of Organic Chemistry, Weizmann Institute of Science, RehoVot 76100, Israel ReceiVed: June 19, 1996; In Final Form: September 30, 1996X

The complexation between the photoreceptor sensory rhodopsin I (SRI) and its signal transducer protein HtrI was examined by assessing titration of the Schiff base chromophore of SRI with sodium hydroxide and reactivity with hydroxylamine in the presence or absence of HtrI. The apparent pKa of the protonated Schiff base of SRI is 12.2 in the presence and 9.5 in the absence of HtrI. Direct titration of the Schiff base proton was confirmed by titrating an artificial SRI reconstituted with a 14-fluororetinal which reduces the intrinsic pKa of the protonated Schiff base of the HtrI-complexed pigment from >12 to 9.0. The SRI chromophore exhibits high stability to hydroxylamine bleaching in the presence of HtrI; however, removal of HtrI accelerates the bleaching rate 2.4-fold. These results indicate that SRI is physically associated with HtrI in its unactivated (i.e., dark) state. In view of the previously identified association of the SRI signaling state (S373) with HtrI, we conclude that SRI transduces the signal to HtrI through its altered interaction with the prebound transducer protein. The effect of the altered SRI/HtrI interaction on resetting the signaling state of SRI was also examined. At neutral pH the decay of S373 is retarded by 20-fold when HtrI is absent. This effect was found to be due to a raised enthalpic barrier for the transition state during S373 decay. The energy barrier for S373 decay in this pigment can be lowered by providing extramembranous protons (lowering bulk pH). Therefore, lightinduced alteration in SRI/HtrI interaction is important for reducing the energy barrier for S373 decay presumably by providing or assisting a proton supply for retinal Schiff base reprotonation.

Introduction Physical interaction between seven-helix membrane-bound receptors and signal transducing proteins occurs as a key step for transmembrane signal transduction. In principle, ligandinduced structural changes in a receptor may trigger its association with a signal-relay protein or alternatively alter its interactions with a previously bound signal transducer. Sensory rhodopsin I (SRI),1-3 a retinal-containing phototaxis receptor in Halobacterium salinarium membranes, shares the characteristics of other retinylidene proteins in that an apoprotein containing seven-transmembrane R-helices includes a retinal chromophore which binds to the protein via a protonated Schiff base (PSB) linkage. To different degrees, the native apoprotein structure “shields” the Schiff base linkage of the chromophore from attacks of proton-accepting, reducing, and nucleophilic reagents. For example, the pKa of the Schiff base (SB) is raised from 7.2 in solution to 10.6 in octopus rhodopsin4,5 and 13.2 in bacteriorhodopsin (BR).6,7 SRI is transiently activated by the internally bound lightresponding unit, retinal. The absorption of a photon by retinal triggers its all-trans/13-cis isomerization8 which is required for the physiological function of SRI.9 An isomerization-induced conformational change in SRI is believed to activate a signaltransducing protein HtrI (halobacterial transducer for SRI).10 However, the mode of interaction between SRI and HtrI is not known. The photoactivation of SRI leads to the formation of its signaling statesS37311 (photoactivated SRI intermediate †

University of Texas Medical School. Weizmann Institute of Science. § Present address: Sandoz Research Institute, Sandoz Pharmaceuticals Corp., East Hanover, NJ 07936. * Corresponding author. X Abstract published in AdVance ACS Abstracts, December 1, 1996. ‡

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absorbing maximally at 373 nm)spassing through two other intermediates, S610 and S560, within 1 ms.12 The deactivation of SRI is accomplished by the thermal conversion of S373, which contains a deprotonated, 13-cis chromophore, in a first-order process to SR587 (the unphotolyzed species of SRI) in the dark. This process completes three tasks: (1) the reprotonation of the Schiff basesa prerequisite for retinal reisomerization; (2) the reisomerization of retinal from 13-cis to all-trans, and (3) the conformational changes that restore the protein to its unactivated state. In a homologous protein BR, the corresponding recovery process is accomplished by distinct intermediate steps.13 An N intermediate is formed when a proton is transferred from Asp96 to the SB. During the lifetime of N, Asp96 takes a proton from the cytoplasmic medium. Since the proton for this step comes from the medium, the Asp96 reprotonation step is pH dependent. In the D96N mutant of BR, the reprotonation of the Schiff base is directly from the cytoplasmic medium and the decay of M to BR is strongly pH dependent. The decay of S373 is similarly pH dependent in the absence but not in the presence of HtrI14 (see discussion below). The long-lived intermediate S373 has been found to form a complex with HtrI because its decay rate and the pH sensitivity of its decay were altered in HtrI-deficient membranes.14,15 Proton translocation from the Schiff base to the extracellular medium by SRI is blocked by HtrI but allowed when HtrI is not present,16,17 further indicating S373/HtrI association. The physical association of S373 to HtrI is analogous to the association of the Meta II intermediate of rhodopsin to transducin.18 However, the physical separation or association between SRI and HtrI prior to photoactivation is not established by these previous results. In this work, we compare the titration of SRI and reactivity with hydroxylamine in genetically © 1997 American Chemical Society

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engineered bacterial strains in the presence and in the absence of its transducer protein (HtrI). The results unambiguously demonstrate the physical association of SRI and HtrI before activation. This indicates that SRI-induced phototaxis signaling is accomplished by altering the interactions between SRI and HtrI after SRI photoactivation. We further examined the thermodynamics of the deactivation process of S373 in the presence and absence of HtrI in order to characterize the altered SRI/HtrI interactions during signaling. We found that an effect of the stimulus-induced altered interaction between HtrI and S373 is to modify an enthalpic barrier to the transition state by assisting a proton donor in SRI or providing a proton to accelerate S373 decay. Materials and Methods Chemicals, Strains, and Preparations. all-trans-Retinal was purchased from Sigma and all-trans-14-fluoro-, 13-desmethyl-, and 3,7,11-trimethyldodeca-2,4,6,8,10-pentaenal retinal analogues were synthesized as previously reported19-21 and purified by HPLC prior to use. SRI and the artificial SRI pigment were reconstituted in apomembranes of Flx5R, which contains transducer protein HtrI and no bacterioopsin, haloopsin, or detectable sensory rhodopsin-II apoprotein. Reactions with chemical agents were also carried out in apomembranes of Pho81/pTR2∆.16 All reactions were in 4 M NaCl. The preparations of apomembranes were as reported.22 Membrane vesicles were prepared as described.23 Membrane preparations were all in 20 mM Tris/HCl and 4 M NaCl at pH 7.0, unless otherwise noted. Absorption Spectroscopy. Reconstitution was monitored by absorption spectroscopy in cell envelope membrane vesicles from Flx5R. Spectra were recorded on an SLM-Aminco DW2000 spectrophotometer (SLM Instruments Inc., Urbana, Il) at 22 ( 0.5 °C. Vesicle suspensions were degassed before measurements. In reconstitution experiments, 5 µL of ethanol or ethanolic solution of all-trans-retinal or analogues were added to the reference and the sample cuvette, respectively, The path length was 10 mm. Titration of Pigments and Reaction with Hydroxylamine. For pH titrations, the pH of the pigment suspension was measured immediately after each addition of sodium hydroxide solution. The decrease in pigment content was assayed by the absorption of the suspension at the absorption maximum of the pigment or by monitoring flash-induced absorbance changes. Pigment content during the NH2OH bleaching reaction (pH 6.9) was monitored by flash spectroscopy. The actinic flash light was at 550 ( 20 nm with a duration of 250 µs. Absorbance changes were followed at 380 nm. Reactions were carried out at 22 ( 0.5 °C. Flash Spectroscopy. Suspensions of Flx5R membrane vesicles containing reconstituted pigments were used for flash photolysis experiments. The pH of the sample was stabilized by Tris/HCl buffer in 4 M NaCl at pH 7.0. The photochemical reactions of SRI and artificial pigments were measured by a cross-beam kinetic spectrophotometer9 at 23 ( 1 °C in the milliseconds to minutes range. Results and Discussion The pKa of SRI PSB in the Presence and Absence of HtrI. The pKa of the PSB of a retinylidene protein refers to the pH at which the protonated Schiff base linkage of the retinal chromophore deprotonates. The pKa’s of PSB in several retinylidene proteins have been determined, such as 13.2 for BR6,7 and 10.6 for octopus rhodopsin.4,5 The pKa of SRI PSB was not known. Alkalinization (pH 8-10) of the membrane vesicles suspension containing SRI and HtrI causes a blue-shift of the absorption

Figure 1. Sodium hydroxide titration of SRI and the 14-F-SRI analogue. (A) Concentrated sodium hydroxide solution was added gradually to membrane suspensions in 2-5 µL portions into the sample and the reference cuvettes. The pH and absorption spectrum was measured after each addition. The loss of SRI absorbance at 550 nm and the artificial SRI absorbance at 625 nm with increasing pH were plotted as the percentage of pigment remaining (solid circles and triangles, respectively). The decrease of SRI content as observed by flash-induced absorbance changes are also shown (open circles). (B) The titration of SRI in the absence of HtrI (triangles) monitored by absorption. Solid lines in both panels are calculated titration curves for monoacids with the pKa values shown. Dotted line is a polyprotic acid fit indicating reversible denaturation (see text).

maximum of the protein to 552 nm. This shift corresponds to the deprotonation of a protein residue (Asp7624,25) with a pKa of 8.7.26 Further alkalinizing the pigment produces a decrease of the absorbance at 550 nm and a concomitant increase of absorbance at 370 nm, indicating deprotonation of the SB. The titration of SRI in the presence of HtrI (Figure 1A) does not fit with n ) 1 as is evident from the theoretical curve for the dissociation of a monoacid with a pKa of 13.5 (Figure 1A, solid line). At pH higher than 12.0, SRI was bleached abruptly with a steep slope which significantly deviates from the calculated curve. This type of behavior has been analyzed as a cooperative hydroxide ion attack on the protein backbones as well as the retinal SB.27 This transition fit well above pH 12 with a calculated curve for a higher order titration of a protein (dotted line, n ) 3.6, pKn ) 44 and the apparent average pKa is 44/3.6 ) 12.2). To measure the PSB pKa in a pH range below that of the higher order titration, we titrated an artificial SRI derived from a retinal analogue substituted by a fluorine at the C14 position, in the presence of HtrI. This substitution reduces the apparent pKa of the corresponding PSB in solution from ca. 7.2 to 5.0. The 14-fluororetinal bound to SRI apoprotein and formed an artificial pigment with maximal absorption at 627 nm. Presumably this red-shift from the 587 nm absorption maximum of native SRI is due to the electron-withdrawing character of the flourine atom introduced near the PSB, destabilizing the ground state, as discussed for a retinal analogue with trifluoromethyl near the PSB.7 The absorption maximum of 14-F-SRI is shifted

HtrI Complexation to Sensory Rhodopsin I

Figure 2. Reactivities of SRI to hydroxylamine in the presence and absence of HtrI. In the dark, 0.2 M hydroxylamine was added at pH 6.9 and the reduction of SRI content was monitored by flash photolysis for pigment in the presence of HtrI in membranes from strain Flx5R (solid circles) and HtrI-free SRI from strain Pho81 (triangles).

to 375 nm at high pH, indicating Schiff base deprotonation. A PSB pKa of 9.0 was obtained by titrating this pigment and the titration curve fits well with n ) 1 (Figure 1A). Reacidification of the artificial pigment and the native SRI to pH 7.0 restored 90% of the pigment absorbance. In the absence of HtrI the pKa of SRI PSB is lowered to 9.5 (Figure 1B). It is interesting to note that both HtrI-complexed 14-F-SRI and HtrI-free SRI exhibit mono-acid titration behavior, while native SRI/HtrI complex with a higher pKa (pKa > 12.2) does not. Our interpretation is that higher pH affects protein structure by titrating groups other than the Schiff base, and the reduced PSB pKa in the artificial SRI avoids protein denaturation preceding the Schiff base deprotonation. Hence the apparent pKa of 12.2 is only the lower limit value for SRI PSB in the presence of HtrI. HtrI is indicated to be a membrane protein with two transmembrane segments. In principle, in the membranes, it can be free or associated with SRI. However, only its association with SRI can explain its effect on the pKa of SRI PSB in the dark. Reactivities to Hydroxylamine in the Presence and Absence of HtrI. In solution, model PSB compounds readily form oximes in the presence of excess hydroxylamine. However, both bovine rhodopsin28 and BR29 are much more stable to hydroxylamine attack compared to the model PSB. Hydroxylamine bleaching of SRI in the presence of HtrI occurs over a time course of hours under our conditions in the dark and is accelerated 2.4-fold in the absence of HtrI (Figure 2). This result agrees with the SRI titration experiment in suggesting that the accessibility of the SRI chromophore is reduced when HtrI is present. Physical Association of SRI with HtrI. Retinal PSBs in solution can be titrated with a pKa of 7.2, whereas protein structure shields the SB chromophore and raises the pKa of PSBs in retinylidene proteins. The protein-mediated increased PSB pKa in SRI was much less (9.5 versus >12) when HtrI was absent (Figure 1), indicating that HtrI alters SRI structure. The enhanced reactivity of the SRI PSB with hydroxylamine in the absence of HtrI also suggests a more penetrable SRI and an altered SRI structure. These results, which were obtained by conducting the reactions in the dark, establish that SRI and HtrI are complexed before photoactivation. Considering a previous finding14 that the photoactiVated SRI (the S373 form) is associated with HtrI, we conclude that the signal transduction from SRI to HtrI occurs by altering their interactions within a preformed complex.

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Figure 3. Temperature dependence of the signaling state lifetime of SRI and of artificial SRI pigments. Flash-induced absorbance changes at 380 nm were monitored for each pigment at various temperatures. In all cases, the best fit for the decay of the signaling state was a single exponential. The logarithm of the first-order rate constants were plotted against 1000/T for native SRI (triangles), artificial SRI reconstituted with pentaenal analogue (circles), and with 9-demethyl analogue (squares). The apparent ∆Hq for the decay of S373 are each 29 kJ/mol and ∆Sq are -90, -104, and -111 J/(mol‚K) for the native SRI and pentaenal and 9-demethyl SRI analogues, respectively.

Figure 4. Temperature dependence of the signaling state lifetime of SRI expressed in HtrI+ and HtrI- strains. Flash-induced absorbance changes at 380 nm were monitored for SRI pigments at various temperatures. The decay of the signaling state was fit to a single exponential. The logarithm of the first-order rate constants were plotted against 1000/T for the native SRI in the HtrI+ strain (circles) and in the HtrI- strain (squares). The apparent ∆Hq for the decay of S373 are 29 and 74 kJ/mol and ∆Sq are -90 and 43 J/(mol‚K) for SRI in the presence and absence of HtrI, respectively.

Differentiation of the Entropic Effect of Retinal Analogues and the Enthalpic Effect of HtrI on S373 Decay. The absence of HtrI has been shown to retard the decay of S373 at neutral pH, the activated form of SRI.14 Similar retardation of S373 decay was also observed in artificial SRI’s in which the retinal 9-methyl or a major part of the β-ionone ring were deleted.11 Are these similar effects due to the same or different mechanisms? We compared the temperature dependence of S373 decay in artificial SRI pigments missing part of their internal retinal/ protein interactions (Figure 3) and SRI missing complete external interactions with HtrI (Figure 4). Following transitionstate theory as applied to temperature effects on the decay rates of bacteriorhodopsin intermediates,30 activation parameters of

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the S373 decay reaction were evaluated by fitting the rate data to the equation:

ln k ) ln kT/h + ∆Sq/R - ∆Hq/RT where k is the rate constant of the S373 decay reaction, k is the Boltzmann constant, T is the absolute temperature, h is Planck’s constant, R is the gas constant, and ∆Sq and ∆Hq are the entropy and enthalpy changes, respectively, required to reach the transition state. Artificial SRI pigments and wild-type SRI exhibited the same ∆Hq of 29 kJ/mol for reaching the transition state in the thermal decay of S373 to SR587. The ∆Sq were reduced in the order of their reduced decay rates for the native, pentaenal, and 9-demethyl SRI’s (-90, -104, and -111 J/(K‚mol)), respectively (the rate of decay of the pentaenal SRI analogue S373 was reduced by 5-fold and that of 9-desmethyl-SRI by 13-fold compared to the native SRI). To a first approximation, the magnitude of entropy change can be taken as a measure of the conformational change in the protein.31 In these terms, the lack of retinal/protein interactions at the ring and at the 9-methyl position evidently slows down rate-limiting conformational changes during the deactivation and thereby retards the recovery of SRI. Apparently steric interactions between the protein and the retinal 9-methyl group and, to a lesser degree, the β-ionone ring play important roles in conformational changes of SRI for the deactivation of the signaling state. In contrast, the retardation of S373 decay by the absence of HtrI is not due to an entropic effect, since ∆Sq was actually increased from -90.2 to 42.9 J/(K‚mol), which would be favorable for a faster decay of S373 (Figure 4). Therefore, the retarded S373 decay in this case is solely due to the enthalpic factor, i.e., the increase in ∆Hq from 29 to 74 kJ/mol caused by removal of HtrI. ∆Hq, the enthalpy change from the initial state to the transition state, for reactions catalyzed by protein reflects changes in H-bonding, stretching or twisting covalent bonds, and overcoming internal Coulombic effects or dipole moments.32 Hence removal of transducer appears to alter the course of chemical events required for S373 to decay and the reprotonation of the SB. HtrI Facilitates an Internal Proton Migration or Provides a Proton to the SRI Active Site. Extensive analysis of BR has established that the rate of the SB reprotonation step in the photocycle depends on external pH only when the proton comes from the external medium.33-36 The rate of the SB reprotonation step in the SRI photocycle (S373 f SR587) does not depend on pH in the presence of HtrI. Therefore the proton is not from the external medium or an internal proton transfer is ratelimiting, either of which indicates that the proton in the Schiff base reprotonation step comes from a donor in SRI or in the associated transducer protein HtrI. Expression studies demonstrated that in native membranes the absence of HtrI retarded the decay of S373 at neutral pH.14,15 The slow S373 decay in HtrI-free SRI was found to be accelerated by increasing proton concentration in the medium.14 These results further corroborate that the proton for the SB reprotonation is from an internal donor in the presence of HtrI but requires an exterior donor in its absence. Also this is expected from previous reports that the SRI SB exchanges protons with the medium during the photocycle only in the absence of HtrI,16 although this point has recently been disputed37 (however, see discussion in ref 38). We examined the effect of pH on ∆Hq and ∆Sq during S373 decay in the absence of HtrI. Figure 5 shows the temperature dependence of S373 decay at various pH's. A decreasing slope (∆Hq), or enthalpic barrier, correlates well with the increase of proton concentration (value of ∆Hq and ∆Sq are in the legend

Figure 5. Temperature dependence of the signaling state lifetime of SRI expressed in HtrI- strains at various pHs. Flash-induced absorbance changes at 380 nm were monitored for SRI pigment at various pH values and at various temperatures. The decay of the signaling state was fit to a single exponential. The logarithm of the first-order rate constants were plotted against 1000/T. The ∆Hq are 74, 59, 48, 44, and 35 kJ/mol for pH 6.9, 6.4, 5.9, 5.5, and 5.0, respectively. The ∆Sq are 43, 3, -26, -36, and -58 J/(mol‚K) at the corresponding pH values.

for Figure 5). When the proton is from the exterior in the absence of HtrI, the reprotonation of the chromophore relies on the migration of an external proton into the active site. Accordingly, the massive supply of protons at low pH apparently reduces the energy barrier for the transition state in S373 decay (Figure 5). Therefore, HtrI/SRI complexation, among other effects, reduces the enthalpic barrier for the chromophore reprotonation during S373 decay by facilitating proton migration or by providing a proton to the SB. Parallel lines in Arrhenius plots would be expected for a bimolecular pseudo-first-order reaction between the protein and protons in which one of the reactants (protons) is in excess. If there were no effect of protons on the protein other than combining with the SB, the second-order nature of the reaction would produce an apparent pH-dependent activation entropy and constant activation enthalpy. The deviation in slope (activation enthalpy) in Figure 5 indicates a more complex interpretation applies. The observed decrease in slope with increasing proton concentration suggests that protons, in addition to combining with the Schiff base, react with unknown groups in the membrane (e.g., lipid headgroups, protein side chains, bound water) that reduce the barriers between bulk protons and the Schiff base. Concluding Remarks. By examining the accessibility of the retinylidene chromophore in SRI to sodium hydroxide and hydroxylamine in the absence and presence of HtrI, we demonstrated the association of SRI with HtrI in the unactivated (i.e., dark) state of the receptor. Since S373 has been shown also to be complexed with HtrI, SRI is signaling through altering its interaction with the previously bound transducer protein HtrI. The altered interaction between SRI and HtrI after photoactivation has a profound effect on lowering the energy barrier for S373 decay at neutral pH. This specific energy-lowering process by HtrI can be simulated by a nonspecific mass supply of protons. Therefore, the altered SRI/HtrI interaction is important for lowering the enthalpic barrier to the transition state during S373 decay presumably by assisting a proton supply within SRI or providing a proton from HtrI for the SB reprotonation. Acknowledgment. We thank Dr. Wouter Hoff for discussion and for critical reading of the manuscript. This work was

HtrI Complexation to Sensory Rhodopsin I supported by grant GM 27750 (J.L.S.) from the National Institutes of Health. References and Notes (1) Spudich, J. L. J. Bacteriol. 1993, 175, 7755. (2) Oesterhelt, D.; Marwan, W. In Biochemistry of Archaea (Archaebacteria); Kates, M., Ed.; Elsevier Science Publishers: Amsterdam, 1993; pp 173-187. (3) Spudich, J. L.; Zacks, D. N.; Bogomolni, R. A. Isr. J. Chem. 1995, 35, 495. (4) Koutalos, Y.; Ebrey, T. G.; Gilson, H. R.; Honig, B. Biophys. J. 1990, 58, 493. (5) Liang, J.; Steinberg, G.; Livnah, N.; Sheves, M.; Ebrey, T. G.; Tsuda, M. Biophys. J. 1994, 67, 848. (6) Druckmann, S.; Ottolenghi, M.; Pande, A.; Pande, J.; Callender, R. H. Biochemistry 1982, 21, 4953. (7) Sheves, M.; Albeck, A.; Friedman, N.; Ottolenghi, M. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 3262. (8) Tsuda, M.; Nelson, B.; Chang, C. H.; Govindjee, R.; Ebrey, T. G. Biophys. J. 1985, 47, 721. (9) Yan, B.; Takahashi, T.; Johnson, R.; Derguini, F.; Nakanishi, K.; Spudich, J. L. Biophys. J. 1990, 57, 807. (10) Yao, V.; Spudich, J. L. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 11915. (11) Yan, B.; Spudich, J. L. Photochem. Photobiol. 1991, 54, 1023. (12) Bogomolni, R. A.; Spudich, J. L. Biophys. J. 1987, 52, 1071. (13) Lanyi, J. K. Biochim. Biophys. Acta 1993, 1183, 241. (14) Spudich, E. N.; Spudich, J. L. J. Biol. Chem. 1993, 268, 16095. (15) Krah, M.; Marwan, W.; Vermeglio, A.; Oesterhelt, D. EMBO J. 1994, 13, 2150. (16) Olson, K. D.; Spudich, J. L. Biophys. J. 1993, 65, 2578. (17) Bogomolni, R. A.; Stoeckenius, W.; Szundi, I.; Perozo, E.; Olson, K. D.; Spudich, J. L. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 10188. (18) Resek, J. F.; Farrens, D.; Khorana, H. G. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 7643.

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