©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ligand Binding Analysis of Soluble Interleukin-2 Receptor Complexes by Surface Plasmon Resonance (*)

Zining Wu (1), Kirk W. Johnson (2), Yoon Choi (2), Thomas L. Ciardelli (1)(§)

From the (1)Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755 and The Veterans Administrations Hospital, White River Junction, Vermont 05009 and the (2)Chiron Corporation, Emeryville, California 94608-2916

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Knowledge of the kinetic binding characteristics is often critical to the development of ligand/receptor structure-activity relationships. To better understand the contribution of each of the subunits to ligand binding in the multimeric interleukin-2 receptor system, we have previously prepared stable solution complexes of the - and -subunits. In this study, we have employed surface plasmon resonance biosensor methodology (BIAcore) to evaluate both the kinetic and equilibrium binding constants for these complexes. The structural nature of the complexes facilitated immobilization on the sensor surfaces in a manner that minimized interference with ligand interactions. The interleukin-2 receptor complex surfaces displayed excellent binding capacity and stability toward regeneration. In all cases where the binding constants were measurable, the values determined for interleukin-2 were in good agreement with those previously determined by other methods. When interleukin-2 analogs with receptor subunit specific mutations were employed, the binding parameters were consistent with the nature of the mutations. The combination of coiled-coil-mediated solution assembly and surface plasmon resonance analysis of ligand binding provides a powerful approach to the study of multimeric cytokine receptor systems.


INTRODUCTION

The specificity and stoichiometry of receptor subunit association are the critical parameters that determine both the ligand binding and signaling properties for the hematopoietin receptors. In systems where both homomeric and heteromeric subunit association may occur via ligand-dependent or independent mechanisms and where multiple ligands may share a single common receptor subunit, understanding the nature of receptor aggregation is crucial to developing ligand/receptor structure-activity relationships. As a general approach to the stable solution assembly of cytokine receptors, we have employed coiled-coil molecular recognition to generate both homomeric (1, 2) and heteromeric (2) interleukin-2 receptor complexes (IL-2R()cc). These complexes bound IL-2 with characteristic cell surface affinities in competitive radioligand binding assays. Solution binding assays, however, do not reveal the kinetic characteristics of ligand binding. In earlier studies, the determination of the kinetic constants for IL-2 binding to cell surface IL-2 receptors proved to be of key importance in understanding the nature of the various physiological forms of the receptor(3, 4, 5) . In order to determine the kinetic ligand binding properties of the soluble IL-2Rcc complexes and verify their equilibrium dissociation constants, we have employed surface plasmon resonance (SPR) biosensor technology.

SPR is emerging as a sensitive and rapid method for the real time analysis of macromolecular interactions (6, 7) and has been successfully employed for quantitation of receptor-ligand interactions including human growth hormone and interleukin-5(8, 9) , members of the helical cytokine/hematopoietin receptor family. Although this method is very convenient for acquiring kinetic data, it can also be subject to a variety of experimental artifacts that may confound the analysis of data and interpretation of results(10) . In this study we have attempted to critically evaluate this technique for its ability to provide both kinetic and equilibrium constants for soluble IL-2Rcc complexes in a system where comparable information exists for cell surface receptor complexes. In addition, we have determined similar values for IL-2 analogs that contain receptor subunit specific mutations. The results of these analyses support both the utility of SPR and the feasibility of coiled-coil solution assembly of receptor complexes.


MATERIALS AND METHODS

Protein Expression and Purification

The IL-2 receptor complexes were expressed in insect cells and purified as described previously(1, 2) . Recombinant IL-2 and IL-2 analogs were expressed in Escherichia coli, refolded, and purified as described previously(4, 11, 12) . Protein concentrations were calculated from A values determined in 6 M guanidine HCl(13) .

SPR Reagents and Biosensor Surface Preparation

SPR instrumentation (BIAcore), CM5 sensor chips and amine coupling reagents containing N-hydroxysuccinimide, N-ethyl-N`-(3-diethylaminopropyl)carbodiimide, and ethanolamine HCl were obtained from Pharmacia Biotech Inc.

Purified IL-2Rcc complexes were diluted to a concentration of 30 nM in NaOAc buffer (10 mM, pH 4.5) and coupled to the dextran-modified gold surface of a CM5 sensor chip using the manufacturer's amine-coupling chemistry as described in the BIAcore systems manual. Briefly, the dextran surface of sensor chip was first activated with N-hydroxysuccinimide/N-ethyl-N`-(3-diethylaminopropyl)carbodiimide (15 µl) followed by the addition of receptor complexes (15-45 µl). The remaining activated groups were blocked by injection of ethanolamine (35 µl). Employing these conditions, surfaces containing densities of 500-2000 resonance units (RU) of IL-2Rcc complexes were generated. Prior to data collection, several methods for surface regeneration after ligand binding were evaluated. It was found that injection of 10 mM HCl (4 µl) could efficiently remove the bound proteins and preserve the binding capacity of sensor chip surface. The IL-2Rcc surfaces were stable to more than a hundred binding and regeneration cycles.

Collection and Analysis of Sensorgrams

Prior to SPR analysis, IL-2 and analogs were dialyzed against phosphate buffer (10 mM sodium phosphate, pH 7.4, 150 mM NaCl), and the protein concentrations were determined and then diluted to the desired concentrations in HBS buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% surfactant P-20) containing 100 µg/ml bovine serum albumin. Five to eight serial dilutions of each protein were injected over the IL-2Rcc surfaces at a flow rate of 8 µl/min. Sensorgrams were recorded and normalized to a base line of 0 RU. Equivalent volumes of each protein dilution were also injected over a mock, nonprotein, blocked surface to serve as blank sensorgrams for subtraction of bulk refractive index background. Each determination was repeated 3-5 times. For the analysis of association rate constants, biosensor surfaces of less than 1000 RU of IL-2Rcc complexes were employed. For the determination of dissociation rate constants, surfaces of higher receptor densities were sometimes employed, and HBS buffer containing 1 µM cc or cc complex was injected during the dissociation phase to capture released ligand and minimize rebinding to the biosensor surface.

Sensorgrams were analyzed by nonlinear least squares curve fitting using BIAevaluation 2.0 software (Pharmacia). A single-site binding model (A + B = AB) was used for analysis of interaction of IL-2 analogs to IL-2R cc and cc surfaces. The equation R = R exp(-k(t - t)) was used for dissociation phase, where R was the amount of ligand remaining bound (in RU) at time t and t was the beginning of dissociation phase. The final dissociation rate constant, k was calculated from the mean of those values of each injection in an injection series.

To analyze the association phase, the equation R = R (1 - exp(-k(t - t))) was employed where R was the amount of ligand bound in RU at equilibrium, t was the time that injection started, and k = kC + k , where C was the concentration of protein ligand injected over the sensor chip surface. The association rate constant, k, was determined from the slope of a plot of kversus C.

The two-site dissociation model, AiBj = Ai + Bj, was employed for analysis of sensorgrams on IL-2R cc surfaces. The following equation was used: R = R exp(-k (t - t)) + (R - R) exp(-k (t - t)), where binding to receptor site 1 (dissociation rate constant, k) in RU is R and to site 2 (k) is R - R. For the association phase analysis on the cc surface, the data were fitted to both a two-site and a single-site model. When low ligand concentrations were used and when IL-2 analogs with receptor subunit-directed mutations were tested, results fitted to the single-site model proved superior.

Since binding of the IL-2 analogs reached a constant equilibrium value during the injection on all three receptor complex surfaces, this equilibrium value (R) was employed for Scatchard analysis (14) of the binding data. After subtraction of the background RU due to bulk refractive index changes (determined for each dilution by injection over a blank, blocked surface), R/C was plotted versus R. The dissociation constants and surface site densities were estimated by nonlinear least squares curve fitting using the program EBDA/RADLIG (version 4, BIOSOFT) (15) employing the conversion factor 1000 RU = 1 ng IL-2/mm (BIAcore systems manual).


RESULTS AND DISCUSSION

In previous studies of coiled-coil IL-2R complexes, competitive radioligand binding assays indicated that these complexes bound IL-2 with affinities that were characteristic of cell surface receptors(1, 2) . In particular, the heteromeric cc complex contained a pseudo high affinity site having an equilibrium dissociation constant of about 300 pM. To further investigate the binding characteristics of these receptor complexes, we have employed SPR biosensor (BIAcore) analysis as a method for the determination of both the kinetic and equilibrium binding constants. In this system, the binding of a ligand to a receptor immobilized on a dextran/gold surface can be visualized in real time by surface plasmon resonance detection, and the resulting binding curves can be used to derive the kinetic parameters of the interaction(6, 7) .

Therefore, we prepared biosensor surfaces for each of the IL-2Rcc complexes (cc, cc, cc) and studied the binding of IL-2 as well as IL-2 analogs containing specific receptor subunit-directed mutations in order to evaluate this method and compare the results to similar findings determined by other techniques.

Surface Preparation and Stability

Using the standard amine coupling chemistry, the purified IL-2Rcc complexes were efficiently immobilized onto the sensor's activated dextran surface. After preparation, each surface was tested for its capacity to bind IL-2. All of the IL-2Rcc surfaces displayed a high ligand binding capacity relative to total surface RU density as well as long term stability. Typically, more than 80% of the original binding capacity was preserved after 100 cycles of ligand binding and regeneration. In contrast, surfaces prepared with the individual, uncomplexed - and -IL-2R subunit ectodomains, using the same amine coupling methods, were found to have reduced binding capacity and stability.()This is likely due to the nonspecific nature of the amine coupling technique and associated perturbation of the ligand binding site. Since the coiled-coil complexes contain an extended lysine-rich stalk (40 lysine residues on the surface of the 75-Å triple helix), covalent attachment through the coiled-coil domain is highly probable and would result in the receptor ectodomains being oriented away from the linkage site (Fig. 1). Such immobilization would offer little interference to ligand binding and provided a clear advantage over the random coupling of the individual receptor ectodomains.


Figure 1: Biosensor immobilized IL-2Rcc complexes. An illustration of IL-Rcc complexes immobilized by covalent attachment through the coiled-coil domain to the functionalized gold/dextran surface. Shown are the heteromeric cc complexes containing two -subunit ectodomains (darkgray) and one -subunit ectodomain (lightgray) on the biosensor surface. Blackshapes represent IL-2. The illustration is not drawn to scale.



SPR Analysis of Ligand Binding to IL-2R cc Surface

Sensorgrams were obtained at a variety of concentrations of IL-2 over different IL-2Rcc surface densities for each complex. For the cc surface, representative sensorgrams (100-500 nM IL-2) are shown in Fig. 2A. The association phase (340-540 s) was analyzed by nonlinear least squares curve fitting as described under ``Materials and Methods'' to yield kvalues at each concentration. A plot of kversus concentration of IL-2 provided a straight line (Fig. 2D) with a slope equal to the association rate constant (k). The value of k for IL-2 binding to the cc surface () was determined to be 1.06 ± 0.03 10 (Ms). The dissociation phase (580-700 s) was also analyzed by the nonlinear least squares curve fitting. The dissociation rate constant, k, was calculated from the fitting of the first 30 s of true dissociation. The k for IL-2 from cc surface was calculated as 3.47 ± 0.33 10 s (). The apparent equilibrium dissociation constant (K) determined from the ratio of these two kinetic constants (k/k was 32.7 nM.


Figure 2: Analysis of ligand binding to the cc surface. Sensorgrams (relative response in RU after background subtraction versus time in sec.) of IL-2 (A, lower to uppercurve: 100, 200, 300, 400, and 500 nM); D20K (B, lower to uppercurve: 50, 100, 200, and 300 nM); and T41P (C, lower to uppercurve: 100, 200, 300, 400, and 600 nM) injected over an cc surface at a flow rate of 8 µl/min. PanelD, plots of k (or kC + k) versus concentration (C) for IL-2 (), T41P (), and D20K ().



This value compared favorably to the K (30 nM) obtained from plotting the R obtained for each ligand concentration in a Scatchard analysis (see Fig. 5A). These two dissociation constants obtained from the SPR data () were also similar to that obtained in previous competitive radioligand assay (45 nM/-subunit) for the same complex(2) .


Figure 5: Scatchard plots of surface bound ligand. Plots of R/concentration (RU of ligand bound at equilibrium after background subtraction/concentration of ligand injected) versus concentration for IL-2 on the cc surface (A); IL-2 on the cc surface (B); IL-2 on the cc surface (C); and T41P on the cc surface (D). Equilibrium dissociation constants and relative binding sites determined from this data using RADLIG/EBDA as described under ``Materials and Methods.'' The results (see Table II) obtained using this analysis were as follows: for IL-2 (panelC) K = 0.56 ± 0.14 nM, K = 29.6 ± 8.8 nM, B/B = 1.17 ± 6%; for T41P (panelD) K = 11.0 ± 1.9 nM, K = 787 ± 282 nM, B/B = 0.68 ± 17%.



To check the specificity of the cc surface, we determined the binding characteristics of an IL-2 analog containing a point mutation (Pro for Thr-41, T41P) that primarily inhibits -subunit binding (12). When the T41P analog was injected over cc surface (Fig. 2C), we observed a large decrease in affinity. Kinetic analysis of T41P binding to IL-2R cc surface indicated that this mutation has little influence on the rate of association (). For analysis of the dissociation phase, 1 µM cc complex was included in the buffer to capture released ligand since binding of T41P to the -subunit alone was very weak. Nevertheless the proline for threonine 41 exchange increased the dissociation rate constant to the extent that it could not be accurately determined by this technique (>0.1 s, ). The equilibrium dissociation constant determined from Scatchard analysis of the sensorgrams (K = 650 nM, ), however, was similar to that previously determined (K = 880 nM) in solution competitive binding assays(12) .

In contrast, another IL-2 analog possessing a -subunit specific mutation (Lys for Asp-20, D20K) (16, 17) displayed little difference in binding parameters to this surface when compared with IL-2 (Fig. 2B, Tables I and II). Therefore, SPR results on the cc surface were consistent with previous findings for both the cell surface -subunit and the soluble cc complex. It should be noted that k for IL-2 binding to the cc surface was approximately 10-fold slower than we previously reported for k to the cell surface -subunit (1.2 10 (Ms))(4) . This difference could reflect real differences inherent to the SPR biosensor method or simply error in the determinations, since both association rate constants are very rapid and approach the limitations of the respective experimental procedures.

SPR Analysis of Ligand Binding to IL-2R cc Surface

We next examined the ligand binding properties to an IL-2R cc (1) surface prepared in similar fashion (Fig. 3A). Due to the lower affinity of this subunit, we examined the association phase over a concentration of 0.12-2 µM. The k for IL-2 () was approximately 5-fold slower than that to the -surface, while k was too rapid to be determined. Scatchard analysis of the R values at each concentration (see Fig. 5B) provided a K value of 407 nM. This value compares favorably with the previously determined solution K value of 300 nM(1) . These dissociation constants would imply an off-rate constant of 10 s, a value that exceeds the detection limit of the instrumentation.


Figure 3: Analysis of ligand binding to the cc surface. Sensorgrams (relative response in RU after background subtraction versus time in sec.) of IL-2 (A, lower to uppercurve: 250, 500, 750, 1000, and 1500 nM); D20K (B, 1, 2, 3, and 4 µM), and T41P (C, lower to uppercurve: 100, 250, 500, 750, and 1000 nM) injected over an cc surface at a flow rate of 8 µl/min. PanelD, plots of k (or kC + k) versus concentration (C) for IL-2 (), T41P ().



When the IL-2 analog D20K was examined, no binding was detected to the cc surface using concentrations up to 4 µM of ligand (Fig. 3B). This is consistent with the previous reports that this mutation is -receptor subunit-specific. In contrast, both the on-rate constant () and the K () for the T41P analog binding to the cc surface (Fig. 3C) were much closer to the values found for wild-type IL-2. Again, this is in accordance with the nature of this mutation being primarily -receptor subunit-directed with little influence on the binding to the -subunit. Therefore, as was the case with the cc surface, the ligand-binding parameters determined by SPR for the cc surface were in agreement with previous reports of cell surface and solution binding for IL-2 and the analogs examined.

SPR Analysis of Ligand Binding to IL-2R cc Surface

Since our interest is the solution assembly of heteromeric IL-2 receptor complexes, it was our goal to determine if SPR would be useful in the characterization of the kinetic and equilibrium binding properties of complexes containing more than one IL-2R subunit. We have prepared and characterized a heteromeric IL-2Rcc complex containing two and one IL-2R ectodomains (2). This complex was capable of binding IL-2 in solution with a K of 320 pM, a value much higher than the K values for either of the individual subunits and in the range reported for the pseudo high affinity cell surface complex(5, 18) . To determine the kinetics of binding of IL-2 to this heterocomplex, we prepared cc surfaces on a CM5 chip. Fig. 4A depicts typical sensorgrams obtained for the binding of IL-2 to an cc surface. Since this surface contains more than a single class of binding sites, we prepared a low density surface (800 RU) to analyze the high affinity site while minimizing any mass diffusion effects during the association phase. When concentrations of IL-2 from 2-20 nM were run over the surface and the association phases of the curves were analyzed, the data fit a single-site binding model with a k = 3.90 ± 0.04 10 (Ms) better than a two-site model. This value is 4-fold faster than that determined for the cc and supports our previous kinetic analysis suggesting that the pseudo high affinity IL-2R exists preformed on the cell surface and captures ligand in a cooperative fashion(4) . It is likely that with the lower IL-2 concentrations employed in the on-rate analysis, association occurs primarily to the single binding site in the complex. Therefore, the one-site binding model provided superior fitting to the data. Since the on-rate constants to both the cc and cc surfaces are within a factor of five, the result obtained from the single-site analysis may be an underestimate of the true k to the site in the complex. The on-rate to this site is at least the value reported and possibly slightly faster.


Figure 4: Analysis of ligand binding to the cc surface. Sensorgrams (relative response in RU versus time in s) of IL-2 (A, lowerto uppercurve: 20, 30, 40, 50, and 60 nM); D20K (B, lower to uppercurve: 50, 100, 300, 400, and 500 nM), and T41P (C, lower to uppercurve: 20, 30, 40, 50, and 60 nM) injected over an cc surface at a flow rate of 8 µl/min. PanelD, plots of k (or kC + k) versus concentration (C) for IL-2 (), T41P (), and D20K ().



For analysis of the dissociation phase, a higher density surface (2200 RU) was prepared. Unlike the association phase, the dissociation phase data fit a two-site binding model better than a single site. Two k values were determined from this data, a slower rate constant of 2.02 ± 0.07 10 s and a faster value of 4.72 ± 0.40 10 s (). The slower off rate corresponds to the higher affinity site and is considerably slower than the values obtained from the homomeric complex surfaces, while the faster k is similar to that previously obtained with the cc surface and is consistent with dissociation from the single site in the complex.

The dissociation constant determined from the ratio of the kinetic constants for the site was K = 0.52 nM, a value close to the K previously determined in solution(2) . Scatchard analysis of the R values at each concentration confirmed that two sites were present in the complex (Fig. 5). The K values obtained from this curvilinear plot were 0.56 and 30 nM (). Both of these dissociation constants agree with the K and K values previously obtained for the and sites, respectively (). Furthermore, the stoichiometry revealed in the Scatchard plot (Fig. 5C) indicated a ratio of high to low affinity sites of 1.17, a value that matched the subunit stoichiometry previously found(2) .

SPR analysis of the T41P analog provided similar results (Fig. 4C). The kinetic constants () and the dissociation constants () confirm that this analog has a much higher affinity to the cc surface than to either of the homomeric complex surfaces. In addition, Scatchard analysis (Fig. 5D) confirms the presence of two binding sites. The K values for both sites were in agreement with the other K values obtained for this analog at each site (). Unlike T41P, the D20K analog appeared to bind to the cc surface at a single site. The kinetic constants () and the K () were very similar to the values obtained from binding to the cc surface. These results are in accordance with the observation that this mutation disrupts -subunit binding (no binding was detected to the cc surface), and therefore binding to the cc surface is approximately equivalent to the cc surface. It should be noted that the K value (108 nM) was about 3-fold greater than obtained for the consensus K values to the cc for this analog. Since this was the single instance where these values did not closely correspond, it may reflect some small contribution of the -subunit to D20K binding on the cc surface.

By employing coiled-coil molecular recognition, we have succeeded in preparing stable homomeric and heteromeric solution complexes of IL-2R ectodomains. In this study, we have used SPR methodology (BIAcore) to examine the binding parameters of IL-2 and IL-2 analogs to cc, cc, and cc surfaces. Immobilization of these complexes proved very efficient using standard amine coupling chemistry, presumably due to the presence of the extended, lysine-rich coiled-coil domain. In addition, these surfaces displayed a high ligand binding capacity and were extremely stable to regeneration, suggesting that attachment through the coiled-coil stalk resulted in little steric interference with the binding sites.

The kinetic constants determined for IL-2 to all three surfaces were generally in accordance with literature values (where available) obtained in kinetic binding experiments of IL-2 to cell surface receptors. In particular, the association and dissociation rate constants measured for the high affinity site () were approximately the same as previously reported (5) for the pseudo high affinity cell surface receptor (k = 1.66 10 (Ms), k = 1.11 10 s). The equilibrium dissociation constants reported in that study (K = 0.67 nM and K = 0.60 nM) also matched those in .

When IL-2 analogs possessing receptor subunit specific mutations were examined, binding to surfaces differed from IL-2 according to the nature of the mutation. Primarily as a result of an increased off-rate, the analog with an -subunit specific mutation (T41P) suffered greater than a 20-fold reduction in affinity to the cc surface. In comparison, the D20K analog carrying a -subunit specific mutation bound to the cc surface in a fashion indistinguishable from IL-2. This analog, however, showed no tendency to bind to the cc surface, an observation consistent with the nature of the mutation. On this surface, the T41P analog displayed properties similar to IL-2. Binding of these analogs to the heteromeric cc surface provided further support for the conclusion that this complex is composed of single high and low affinity sites. The T41P analog bound to this surface with 80-100-fold higher affinity than to either of the homomeric subunit surfaces, and Scatchard analysis of binding confirmed the presence of two sites with 1:1 stoichiometry, as was observed for IL-2 itself. This is consistent with the observation that, although the proline exchange at position 41 greatly reduces -subunit interaction, it does not completely eliminate it(12) . The higher affinity of this analog to the cc surface also suggests that the details of IL-2-subunit interaction may differ when bound to the -subunit alone compared with the pseudo high affinity complex.

The influence of the lysine exchange at position 20 of IL-2 on -subunit binding was more severe. This analog bound to the cc surface as if the -subunit was absent from the complex. In fact, with respect to all binding parameters, the D20K analog interacted with both the cc and cc surfaces in a manner indistinguishable from the interaction of IL-2 with the cc surface.

Taken together, these results not only indicate that the IL-2Rcc complexes interact with ligand in a fashion that mimics the comparable cell surface receptors but also demonstrate the utility of SPR biosensor methodology in the ligand binding analysis of complex cytokine receptor systems.

  
Table: Kinetic rate constants

Kinetic rate constants determined as described under ``Materials and Methods'' from sensorgrams as depicted in Figs. 2-4. NA refers to values that could not be accurately determined because they exceeded the limitations of the instrumentation. ND refers to no specific binding detected. T41P and D20K are IL-2 analogs with - and -receptor subunit specific point mutations, respectively.


  
Table: Equilibrium dissociation constants

Dissociation constants as determined from the ratio of the kinetic rate constants (K = k/k) in Table I and from Scatchard analysis (K) of the equilibrium bound values R for each concentration of ligand injected as depicted in Fig. 5. NA refers to values that could not be determined due to the inability to accurately measure the kinetic constants or lack of detectable binding. Values for K in parentheses are those determined for the low affinity site in the cc complex. T41P and D20K are IL-2 analogs with - and -receptor subunit specific point mutations, respectively.



FOOTNOTES

*
This work was supported by grants form the Hitchcock Foundation, the National Institutes of Allergy and Infectious Diseases (AI34331), and the American Cancer Society (FRA-385) and also by the Norris Cotton Cancer Center and Chiron Corp. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 603-650-1474; Fax: 603-650-1129.

The abbreviations used are: IL-2R, interleukin-2 receptor; IL, interleukin; cc, coiled-coil receptor complex; RU, resonance units; SPR, surface plasmon resonance.

Z. Wu, K. W. Johnson, Y. Choi, and T. L. Ciardelli, unpublished observations.


ACKNOWLEDGEMENTS

We thank Russ Granzow and Chris Whalen (Pharmacia Biosensor) for advice and discussions.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.