COMMUNICATION:
The Retinal Specific Protein RGS-r Competes with the gamma  Subunit of cGMP Phosphodiesterase for the alpha  Subunit of Transducin and Facilitates Signal Termination*

(Received for publication, January 14, 1997, and in revised form, February 10, 1997)

Thomas Wieland Dagger §, Ching-Kang Chen § and Melvin I. Simon

From the Division of Biology, 147-75, California Institute of Technology, Pasadena, California 91125

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

In vertebrate photoreceptor cells, transducin mediates signaling between rhodopsin and cGMP phosphodiesterase by transiently binding its gamma  subunit (PDEgamma ). For the termination of signaling GTP hydrolysis by the transducin alpha  subunit (TDalpha ) GTPase is required. This reaction can be accelerated by GTPase-activating proteins (GAPs), e.g. PDEgamma . Recently we identified a second retinal GAP that interacts with TDalpha , RGS-r. Here we compare the GAP function of RGS-r and PDEgamma . Both proteins stimulated single turnover GTPase of TDalpha ; however, RGS-r was more effective than PDEgamma . When added together, PDEgamma competitively inhibited the RGS-r-stimulated GTPase. In addition, the interaction of TDalpha in its GTP-bound form (TDalpha GTPgamma S), the transition state (TDalpha GDP*AMF) and the GDP-bound form (TDalpha GDP) with RGS-r and PDE, respectively, was measured by surface plasmon resonance. PDEgamma displayed highest affinity for TDalpha GTPgamma S, weaker affinity for TDalpha GDP*AMF, and weakest affinity for TDalpha GDP. RGS-r exhibited only a comparable high affinity for TDalpha GDP*AMF. We conclude that the observed competition between RGS-r and PDEgamma for TDalpha occurs when the hydrolysis of GTP is initiated. By competing with PDEgamma and removing it from TDalpha as well as increasing Pi release, RGS-r apparently facilitates signal termination and TDalpha recycling.


INTRODUCTION

Transducin (TD),1 the heterotrimeric guanine nucleotide-binding protein (G protein) in vertebrate photoreceptor cells mediates the signal between rhodopsin and the effector enzyme cGMP phosphodiesterase (PDE). Photon absorption activates rhodopsin which then catalyzes GDP/GTP-exchange with the alpha  subunit of TD. The GTP-bound transducin alpha  subunit (TDalpha GTP) activates PDE by binding to its inhibitory gamma  subunit (PDEgamma ). Activated PDE lowers cGMP levels, thus closing the cGMP-gated cation channels and hyperpolarizing the photoreceptor cell membrane (1-3).

Electrophysiological recordings of isolated photoreceptors revealed that the entire cellular response to light occurs in less than a second (4), suggesting that both the activation and inactivation of TDalpha occur on this time scale. In vitro studies have demonstrated that TDalpha activation occurs in 100 ms (5). The subsequent interaction between TDalpha and PDE occurs in less than 5 ms (6). The deactivation of PDE in intact photoreceptor is also a rapid process (<2 s). To achieve this, GTP hydrolysis by the intrinsic GTPase of TDalpha has to occur fast. However, this reaction is a relatively slow process (>10 s) in vitro, and therefore it has been postulated that a GTPase-activating protein (GAP) exists to account for the rapid shutoff of TDalpha in vivo. Several studies have shown that PDEgamma itself has GAP activity (7-11). The significance of this GAP activity of PDEgamma is still a matter of debate (12-15). In addition, a thus far, unpurified membrane-bound component with GAP activity for TDalpha has been described (14, 15). We have identified (16) a retinal specific member of the RGS protein family (for review, see Refs. 17 and 18) termed RGS-r. Like other RGS domains (19-22), RGS-r exhibits GAP activity and, specifically, recognizes a conformation of TDalpha that exists during the transition state in the hydrolysis of GTP by transducin (16). In this report, we compared the GAP activities of RGS-r and PDEgamma to get insight into their physiological relevance.


EXPERIMENTAL PROCEDURES

Cloning and Purification of Proteins

His6-RGS-r was expressed in the Escherichia coli strain BL21(DE3) and purified as described (16).

The cDNA for bovine PDEgamma was subcloned into the NdeI and BamHI sites of pET15B (Novagen). The amino-terminal His6-tagged PDEgamma was expressed in the the E. coli strain BL21(DE3) and purified from bacterial cytosol as described for RGS-r.

Bleached bovine ROS membranes were prepared from bovine retinae as described (23). Transducin was eluted from the membranes by hypotonic elution in the presence of 100 µM GTP or guanosine 5'-(gamma -thio)triphosphate (GTPgamma S), and the subunits were separated by affinity chromatography on blue Sepharose (Bio-Rad) (24). Urea-treated ROS membranes were prepared from the eluted membranes as described (25).

GTPase Measurements

Transducin single turnover GTPase was determined by a method described before (8) except that all measurements were conducted at 0 °C in a buffer containing 50 mM Tris-HCl, pH 7.5, and 2 mM MgCl2. Briefly, the reaction was started by mixing 20 µl of ROS membranes depleted of PDE (12 µM final rhodopsin concentration) with 20 µl of [gamma -32P]GTP (0.2 µCi, 0.25 µM final concentration) and incubated for the indicated periods of time. Where indicated, RGS-r or PDEgamma was added before mixing. The reaction was stopped by addition of perchloric acid and inorganic 32P was measured by Cerenkov radiation in a liquid scintillation counter. The GTPase rate constant was determined by the exponential fit of the time course of inorganic 32P release.

The multiple turnover GTPase of TD was determined in a reconstituted system as described (16) in a reaction mixture (100 µl) containing 50 mM triethanolamine-HCl, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 0.1 mg/ml bovine serum albumine, 0.2 µM TDalpha GDP, 0.2 µM TDbeta gamma , 10 µg of urea-treated ROS membranes, and the indicated concentrations of RGS-r and/or PDEgamma . The reaction mixture was thermoequilibrated for 5 min at 30 °C, and reaction was initiated by addition of 10 µM [gamma -32P]GTP (0.2 µCi). After incubation for 10 min at 30 °C, termination of reaction and determination of inorganic 32P were performed as described.

Analysis of the Transducin-RGS-r and the Transducin-PDEgamma Interaction by Surface Plasmon Resonance (SPR, BIAcore)

His6-RGS-r or His6-PDEgamma were tethered to a NTA-sensor chip (Pharmacia Biosensor) after priming the chip with Ni2+. In detail, the chip was equilibrated at a continuous flow rate of 5 µl/min with a buffer (BIA buffer) containing 20 mM Hepes, pH 7.5, 100 mM NaCl, 2 mM MgCl2, 10 mM imidazole, and 0.01 mg/ml bovine serum albumine. First, the chip was washed with 10 µl of BIA buffer containing 50 mM EDTA. The chip was then primed with 10 µl of 0.1 M NiCl2 and again equilibrated with BIA buffer. 20 and 10 µl of 0.01 mg/ml RGS-r and PDEgamma were applied, respectively, creating a stable surface of about 300-400 RU. For kinetic studies 35 µl of varying concentrations of TDalpha GDP, TDalpha GDP*AMF, or TDalpha GTPgamma S were injected at a flow rate of 5 µl/min. Each injection was followed by a buffer flow for several minutes to monitor the dissociation of the complex. The data were analyzed after subtraction of the background signal (blank injections) with the BIAevaluation software (Pharmacia Biosensor). The values of the dissociation constants (kd) were determined by exponential fitting of the SPR signal decay after replacement of the analyte solution by BIA buffer. The values obtained and the applied concentration of the analyte were used to calculate the association constants (ka) by exponential fitting of the SPR signal increase after application of the analyte.


RESULTS

Expression and Purification of Functional PDEgamma

To compare the two putative retinal specific GAPs: the RGS-r domain (16) and PDEgamma (8-11), we expressed and purified PDEgamma in a manner similar to that described for RGS-r (16). The ability of the recombinant PDEgamma (10 µM) to interact with TDalpha GDP, TDalpha GDP*AMF, or TDalpha GTPgamma S was studied by the column trap assay used previously (16). TDalpha GDP bound PDEgamma only weakly; however, activation of TDalpha GDP by AMF significantly increased the binding to PDEgamma (data not shown). The binding of TDalpha GDP*AMF to PDEgamma was clearly less than the binding observed with the metabolically stable GTP analog GTPgamma S bound TDalpha (TDalpha GTPgamma S). Using similar conditions, RGS-r trapped only TDalpha GDP*AMF but not TDalpha GDP or TDalpha GTPgamma S. No binding of either form of TDalpha to the matrix was observed in the absence of a trapping His-tagged protein (16). The data therefore indicate that the recombinant PDEgamma was fully functional with regard to the generally accepted scheme of effector-G protein interaction, in which the interaction of these proteins is dependent on the G protein activation state.

Comparison of the GAP Activities of PDEgamma and RGS-r

We next compared the ability of both these proteins, RGS-r and PDEgamma , to enhance GTP hydrolysis by TDalpha in a single turnover assay with bleached ROS membranes (8-11). In this assay the concentration of TD (approx 1 µM, estimated by measurement of the rhodopsin concentration) is several times higher than the concentration of [gamma -32P]GTP (0.25 µM), and as [gamma -32P]GTP is quickly bound to excess TD·rhodopsin complex subsequent, formation of 32Pi reflects a single synchronized turnover of the TDalpha -GTPase. As with other RGS proteins (19-22), RGS-r was very effective in stimulating GTP hydrolysis by TDalpha at room temperature (maximal hydrolysis of added GTP occurred within seconds, data not shown). Therefore, the temperature at which the reaction was performed was lowered to 0 °C. Under these conditions we were able to obtain reliable measurements of single turnover GTPase activity. Maximally 75% of the added GTP was hydrolyzed (Fig. 1). A maximal stimulating concentration (10) of PDEgamma (2 µM) was compared for its ability to stimulate the GTPase with 200 nM RGS-r (Fig. 1A). The calculated rate constant for the basal GTPase reaction was 7 ± 0.3 × 10-3 s-1. In the presence of PDEgamma and RGS-r this rate increased to 14 ± 0.4 × 10-3 s-1 and 36 ± 1.8 × 10-3 s-1, indicating a 2- and 5-fold acceleration of GTP hydrolysis, respectively. The RGS-r concentration used was submaximal (Fig. 1B; EC50 and EC100 for mRGSr were about 0.1 and 1 µM, respectively). The maximal increase in stimulation of the rate of GTP hydrolysis was about 10-fold. When added together the GAP proteins did not increase GTP hydrolysis in a synergistic manner. In contrast, addition of PDEgamma inhibited the RGS-r-stimulated GTPase about 50% (rate constant: 22 ± 0.5 × 10-3 s-1). These data were further confirmed when the influence of both recombinant proteins on the rhodopsin-catalyzed multiple turnover GTPase of TD (200 nM) was studied in a reconstituted system described previously (16). RGS-r stimulated Pi release maximally about 6-fold (Fig. 2). Half-maximal and maximal stimulation of GTP hydrolysis occurred at 40 and 200 nM, respectively. In contrast, no stimulation of Pi release by PDEgamma was observed in the multiple turnover assay even when tested in concentrations up to 2 µM. Furthermore, at concentrations of >1 µM, PDEgamma inhibited the rhodopsin-catalyzed steady-state GTPase (data not shown). As suggested before (15), these data can be best explained by a complex of TDalpha GDP with PDEgamma that is refractory to reactivation.


Fig. 1. Increase in the single turnover GTPase of TD by RGS-r and PDEgamma . ROS membranes containing about 12 µM rhodopsin and 1 µM transducin were incubated with 0.25 µM [gamma -32P]GTP in the absence (open circle , control) and presence of the indicated concentrations of RGS-r (A: bullet , black-diamond , 200 nM; B: black-triangle, 6.25 nM; black-down-triangle , 12.5 nM; black-diamond , 25 nM; bullet , 50 nM; square , 100 nM; triangle , 200 nM; down-triangle, 1000 nM) and PDEgamma (A: black-down-triangle ,black-diamond , 2 µM) for the indicated periods of time at 0 °C. The reaction was stopped by addition of perchloric acid, and the amount of 32Pi released was determined. Mean ± S.D. of triplicate determinations are shown (A, B). The data were analyzed by exponential fitting, and the rate constants for GTP hydrolysis were calculated. In C, the rate constants obtained from the data presented in B were plotted against increasing concentrations of RGS-r.
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Fig. 2. Competitive inhibition of the RGS-r stimulated multiple turnover GTPase of TD by PDEgamma . The rhodopsin-catalyzed multiple turnover GTPase of TD was measured in a reconstituted system consisting of 10 µg of urea-treated ROS membranes and 200 nM TDalpha beta gamma . The reaction was initiated by addition of 10 µM [gamma -32P]GTP and conducted for 10 min at 30 °C. The release of 32Pi was determined as described under "Experimental Procedures." A, GTPase activity was measured in the absence and presence of RGS-r (open circle , 100 nM; bullet , 200 nM; square , 500 nM) at increasing concentrations (20-2000 nM) of PDEgamma . B, GTPase activity was measured in the absence (diamond ) and presence (black-diamond ) of 200 nM PDEgamma at increasing concentrations (20-500 nM) of RGS-r. GTPase activity in the absence and presence of a maximally stimulating concentration of RGS-r was 3.5 and 20.5 pmol Pi/min in A and 10.8 and 63.4 pmol Pi/min in B, respectively. Mean ± S.D. of RGS-r-stimulated GTPase determined in triplicates are shown.
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When the inhibitory effect of PDEgamma on RGS-r-stimulated GTPase was titrated (Fig. 2A), we found that the extent of inhibition was dependent on the RGS-r concentration used to stimulate TDalpha GTPase (Fig. 2A). As more RGS-r was present, more PDEgamma was required to obtain the equivalent degree of inhibition. Conversely, when the effect of increasing concentrations of RGS-r was studied in the absence and presence of 200 nM PDEgamma (Fig. 2B), the addition of PDEgamma caused a shift to the right in the concentration dependence for RGS-r and the half-maximal stimulation by RGS-r occurred at 40 and 200 nM in the absence and presence of PDEgamma , respectively. The maximal stimulation achieved by RGS-r was virtually not altered by PDEgamma . This suggest that RGS-r is more effective than PDEgamma in stimulating both single and multiple cycles of transducin activity and that the two proteins compete for transducin at some stage in the activation cycle. This interpretation was further supported by the finding that the extent of inhibition of the RGS-r-stimulated GTP hydrolysis was dependent on the concentration of TD used in the reconstitution (data not shown).

Analysis of the Interaction of TDalpha with PDEgamma and RGS-r by SPR

We studied the direct interaction of TDalpha GTPgamma S, TDalpha GDP*AMF, and TDalpha GDP with RGS-r and PDEgamma , respectively, using SPR (BIAcore). Either RGS-r or PDEgamma was tethered to a Ni2+-NTA sensor chip via their His6 NH2 terminus as described under "Experimental Procedures." Surfaces of 300-400 RU were created with either protein and TDalpha GTPgamma S, TDalpha GDP*AMF, or TDalpha GDP was used as analytes at various concentrations. Examples of the measurements of the interaction of RGS-r with TDalpha GDP*AMF and TDalpha GDP or of PDEgamma with TDalpha GDP*AMF are shown in Fig. 3. A summary of the results is presented in Table I. Binding of TDalpha GDP*AMF to RGS-r was relatively fast and apparently reached saturation at about 500 nM. The maximal increase in SPR was about 350 RU on a 300-RU surface of RGS-r, which approximates the value expected for one to one binding of a 39-kDa protein (TDalpha ) to 30-kDa protein (RGS-r). The dissociation constant (KD) calculated from these measurements was 45 nM. As observed before by the column trap assay, TDalpha GDP exhibited a relatively low affinity (KD approx  2 µM), which was due to a drastic reduction in the association rate. Saturation was not observed under the conditions used. Binding of TDalpha GTPgamma S to RGS-r was also very weak, and the maximal increase in SPR observed was in the same range as observed with identical concentrations of TDalpha GDP (data not shown), consistent with data most recently reported for the RGS4-Goalpha interaction (20), these data taken together indicate that the affinity of TDalpha GTPgamma S for RGS-r is low. However, exact calculations of kinetic constants for the TDalpha GTPgamma S-RGS-r interaction were not possible as the binding data recorded were multiphasic and could not be readily analyzed. PDEgamma displayed the highest affinity for TDalpha in its GTPgamma S-liganded form. The KD value obtained from our measurements (33 nM) was virtually identical to that reported (10), with association (ka) and dissociation rate (kd) constants within the same order of magnitude. Consistent with the data obtained by the column trap assay, the affinity of PDEgamma for TDalpha GDP*AMF was lower than for TDalpha GTPgamma S. The difference in affinity was mainly due to an about 4.3-fold increase in the dissociation rate compared with TDalpha GTPgamma S. Interestingly, even TDalpha GDP displayed relatively high affinity for PDEgamma (300 nM), which is almost an order of magnitude higher than the affinity of RGS-r for TDalpha GDP. In summary, the data suggest that RGS-r has the ability to compete with PDEgamma for TDalpha when the protein is in the transition state stabilized by AMF.


Fig. 3. Binding of TDalpha to RGS-r or PDEgamma on the SPR-sensor chip. His6-RGS-r (A, B) or His6-PDEgamma (C) were attached to a Ni2+-NTA-SPR chip as described under "Experimental Procedures." Binding of TDalpha GDP*AMF (A, 200 and 500 nM; C, 100 and 200 nM) or TDalpha GDP (B, 500 and 1000 nM) was monitored after injection of 35 µl (flow rate, 5 µl/min) of the respective analyte for the indicated periods of time at 25 °C.
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Table I.

Kinetic constants for the interaction of TDalpha with RGS-r and PDEgamma

His6-mRGSr or His6-PDEgamma were attached to a Ni2+-NTA-SPR chip as described under "Experimental Procedures." TDalpha GDP, TDalpha GDP*AMF, and TDalpha GTPgamma S in various concentrations were used as analytes to study the interaction between TDalpha and PDEgamma or RGS-r in real time. Kinetic constants were obtained by fitting the data to appropriate mathematical functions using the BIAevaluation software (Pharmacia Biosensor). Constants obtained from single measurements differed not more than 2-fold from the values displayed (ND, not detected).


RGS-r PDEgamma

TDalpha GDP ka (M-1 × s-1) 868 6590
kd (s-1) 0.0018 0.0020
kD (nM) 2074 303
TDalpha GDP*AMF ka (M-1 × s-1) 62160 36600
kd (s-1) 0.0028 0.0057
kD (nM) 45 156
TDalpha GTPgamma S ka (M-1 × s-1) ND 39500
kd (s-1) ND 0.0013
kD (nM) ND 33


DISCUSSION

Evidence for Competition between RGS-r and PDEgamma for TDalpha in the Transition State of GTP Hydrolysis

When RGS-r and PDEgamma were compared for their GAP activity, it was evident that RGS-r was more effective than PDEgamma . At maximally stimulating concentrations, the increase in GTPase reaction velocity mediated by RGS-r was about 5-fold higher than that achieved by PDEgamma . In fact PDEgamma inhibited the RGS-r-stimulated GTPase in the single turnover GTPase as well as in the multiple turnover GTPase. The inhibition by PDEgamma was dependent on the concentration of RGS-r used to stimulate TDalpha GTPase and was fully reversible when the concentration of RGS-r was several times higher than the concentration of PDEgamma . The extent of inhibition was also dependent on the concentration of TD. Thus, it is reasonable to suggest that PDEgamma and RGS-r compete for TDalpha . This interpretation is further supported by data obtained by measurement of the protein-protein interaction using SPR. Consistent with recently reported data (16, 20, 21), TDalpha exhibited its highest affinity for RGS-r in the transition state mimicked by AMF. The affinty of TDalpha GDP*AMF for RGS-r was about 4-fold higher than for PDEgamma , while in the two other nucleotide stabilized conformations of TDalpha , TDalpha GTPgamma S and TDalpha GDP, the affinity of TDalpha for PDEgamma was much higher than for RGS-r. Therefore, the data suggest that RGS-r competes with PDEgamma for TDalpha only at the stage after GTP hydrolysis is initiated.

Function of RGS-r in the Termination of TDalpha and Effector Activation

Interestingly, in contrast to RGS-r, PDEgamma was not able to increase the rate of Pi release from TDalpha in the multiple turnover GTPase assay, which mainly monitors the recycling of TDalpha . At higher concentrations, PDEgamma (>1 µM) inhibited the rhodopsin, and TDbeta gamma catalyzed recycling of TDalpha itself, an effect that has been described previously by others (26, 27). This effect has been attributed to the formation of a complex of TDalpha GDP with PDEgamma that is refractory to reactivation (15) and is therefore sequestered from the further interaction with TDbeta gamma and rhodopsin (26). As already outlined above, RGS-r was able to overcome the inhibition by PDEgamma and thus enhance again the recycling of TDalpha . The SPR measurements revealed that RGS-r in contrast to PDEgamma displayed very low affinity for TDalpha GDP, suggesting that after release of Pi, TDalpha GDP will easily dissociate from RGS-r and thus is capable of rebinding to TDbeta gamma and rhodopsin. Our data are consistent with the following reaction cycle (Fig. 4). After TDalpha is loaded with GTP by rhodopsin, it dissociates from the receptor and TDbeta gamma . The free TDalpha GTP then binds specifically to PDEgamma and releases the inhibitory effect of this subunit from PDEalpha beta (6, 28-30). When TDalpha reaches the state where GTP hydrolysis is initiated, RGS-r effectively competes with PDEgamma and thus releases TDalpha from PDEgamma , which then again inhibits the enzymatic activity of PDEalpha beta . The GAP activity of RGS-r, i.e. its ability to stabilize a form of TDalpha that facilitates GTP hydrolysis and Pi release, rapidly converts it into its GDP-bound form, which dissociates easily from RGS-r and is available for rebinding to TDbeta gamma . It is known that TDalpha activated by AMF is capable of stimulating the effector PDE in reconstituted systems (3), suggesting that PDE is active as long as the gamma -phosphate of GTP is not released from TDalpha . By competing with PDEgamma in the stage when GTP hydrolysis is initiated and subsequently catalyzing the complete hydrolysis, i.e. the release of gamma -phosphate, RGS-r can apparently play a major role in PDE deactivation and recycling of TD. Studies are currently in progress to address the relationship between RGS-r and the membrane-bound GAP described by Angleson and Wensel (14, 15).


Fig. 4. Proposed activation-deactivation cycle of transducin in the presence of RGS-r and PDEgamma . R*, activated rhodopsin; TDalpha beta gamma , transducin and its subunits; TDalpha GDP-P, transducin alpha  subunit in the transition state; PDEgamma , gamma  subunit of cGMP phosphodiesterase; RGS-r, retinal specific RGS protein; TDalpha star PDEgamma , complex of TDalpha with PDEgamma ; TDalpha star RGS-r, complex of TDalpha with RGS-r; Pi, inorganic phosphate; solid arrows, reactions likely to occur in the presence of RGS-r; dotted arrows, reactions less likely to occur in the presence of RGS-r; kD, affinity constants derived from SPR measurements (Table I); kcat, GTPase rate constants derived from single turnover GTPase measurements (Fig. 2).
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FOOTNOTES

*   This work was supported by National Institute on Aging Grant AG 12288.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    Recipient of a fellowship from the Deutsche Forschungsgemeinschaft.
§   These authors contributed equally to this work.
   To whom correspondence should be addressed: Division of Biology, 147-75, California Inst. of Technology, Pasadena, CA 91125. Tel.: 818-395-3944; Fax: 818-796-7066; E-mail: simonm{at}starbase1.caltech.edu.
1   The abbreviations used are: TD (alpha , beta , gamma ), transducin and its subunits; PDE (alpha , beta , gamma 2), retinal cGMP phosphodiesterase and its subunits; GAP, GTPase-activating protein; G protein, heterotrimeric guanine nucleotide-binding protein; RGS-r, mouse retinal specific RGS protein; GTPgamma S, guanosine 5'-(gamma -thio)triphosphate; SPR, surface plasmon resonance; PAGE, polyacrylamide gel electrophoresis; ROS, rod outer segment(s); Ni2+-NTA, nickel-nitrilotriacetic acid; RU, SPR response unit(s).

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