Binding and Dissociation Kinetics of Wild-Type and ... - ACS Publications

Langmuir 2000, 16, 9421-9432

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Binding and Dissociation Kinetics of Wild-Type and Mutant Streptavidins on Mixed Biotin-Containing Alkylthiolate Monolayers Linda S. Jung,† Kjell E. Nelson,‡ P. S. Stayton,‡ and Charles T. Campbell*,† Department of Chemistry and Department of Bioengineering, University of Washington, Seattle, Washington 98195 Received February 2, 2000. In Final Form: July 31, 2000 The kinetics of adsorption and competitive desorption of wild-type streptavidin (WT SA) and three genetically engineered mutants (S27A, N23E, and W120A) was studied at gold surfaces functionalized with mixed alkylthiolates, some terminated with biotin headgroups and the rest with oligo(ethylene oxide) using surface plasmon resonance (SPR). The saturation coverage of the protein varied strongly with surface biotin concentration (XBAT) and was independent of mutation (except at very low and very high XBAT, where a weak dependence was seen). Initial adsorption rates were nearly diffusion-limited except at extremely low XBAT, where the rate varied weakly between mutants in accordance with their differing strengths of binding to biotin. Initial sticking probabilities were estimated to be between ∼1-6 × 10-6 per collision with the surface. The adsorbed SA desorbs upon introduction of solution-phase biotin. For XBAT below 1%, the desorption rate constants of the SA variants closely follow their off-rate constants measured in homogeneous solution (which at 25 °C are WT ) 4 × 10-6 sec-1, N23E ) 1.6 × 10-3 sec-1, S27A ) 1.2 × 10-3 sec-1, and W120A estimated to be ca. 23 s-1). This proves that SA is mainly bound to the surface by a single SA-biotin link at very low XBAT. Importantly, for XBAT between 10 and 40%, where desorption is 30- to >1000-fold slower and the saturation coverage maximizes, the ratios of off-rate constants between mutants (W120A/N23E and W120A/S27A) are approximately the square of their ratios for XBAT below 1%. This squaring strongly suggests that the dominant species at these coverages is doubly bounded SA (i.e., immobilized via two surface biotins). The kinetics are explained with a mechanism involving only two first-order rate constants, that is, for (1) the slow dissociation of any bond between an SA site and a surface-immobilized biotin and (2) the fast reforming of this bond in the special case that it was released from a doubly bonded SA whose other site is still linked to one surface-immobilized biotin. The rate constant for (2) is almost independent of the SA mutant, as it is for adsorption. For XBAT > 60%, the desorption rates again approach the singly bound SA values, and the ratios of rate constants for the SA variants drop to slightly less than below 1% biotin. This is due to the dominance of singly bonded SA, plus a contribution from nonspecific binding, consistent with structural studies of these alkylthiolate films.

I. Introduction The streptavidin-biotin couple is of interest due to its extremely high binding affinity (Ka ∼1013 M-1)1 and the fact that each streptavidin (SA) has four equivalent sites for biotin (two on one side of the protein and two on the opposite). These sites can be used to link streptavidin nearly irreversibly to up to four biotinylated molecules. This often can be done with minimal impact on the biological activity (e.g., recognition specificity, catalytic activity, etc.) of the secondary molecules. Streptavidin is thus used as a molecular linker apparatus in many assays, sensors, purifications, and syntheses of importance in biotechnology. Protein engineering approaches have further tailored streptavidin for specific applications. For example, site-directed mutagenesis techniques have been used to alter most of the direct binding contacts to biotin.2-4 These streptavidin mutants have increased off-rates and lower binding affinities that allow more facile capture and release properties than the native streptavidin. They * To whom correspondence should to be addressed. † Department of Chemistry. ‡ Department of Bioengineering. (1) Green, N. M. Adv. Protein Chem. 1975, 29, 85. (2) Klumb, L. A.; Chu, V.; Stayton, P. S. Biochemistry 1998, 37, 7657. (3) Chilkoti, A.; Tan, P. H.; Stayton, P. S. Proc. Natl. Acad. Sci. 1995, 92, 1754. (4) Freitag, S.; Chu, V.; Penzotti, J.; Klumb, L. A.; To, R.; Trong, I. L.; Lybrand, T.; Stenkamp, R. E.; Stayton, P. S. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 8384.

may also prove useful in pretargeting strategies for delivering cancer therapeutics, where the faster dissociation kinetics could allow exchange of blocking endogenous biotin with the biotinylated therapeutic. Knoll’s group has shown that, by dosing an ethanolic solution of biotin-terminated and hydroxyl-terminated alkylthiols onto gold surfaces, biotin-containing binary alkylthiolate monolayers (BTMs) can be prepared that immobilize SA with high coverage, specificity, and activity in such as way as to expose two of its binding sites away from the surface.5-8 These sites have been shown to be useful for immobilizing secondary molecules chemically modified with biotin, again with minimal impact on the biological activity (e.g., specificity) of the immobilized molecules.8,9 This makes these SA monolayers very convenient substrates for the development of biosensors. Pe´rez-Luna et al.10 characterized in more detail the same BTMs that were found by Knoll’s group to optimize (5) Haussling, L.; Ringsdorf, H. Langmuir 1991, 7, 1837. (6) Knoll, W.; Angermaier, L.; Batz, G.; Fritz, T.; Fujisawa, S.; Furuno, T.; Guder, H. J.; Hara, M.; Liley, M.; Niki, K.; Spinke, J. Synth. Met. 1993, 61, 5. (7) Spinke, J.; Liley, M.; Schmitt, F. J.; Guder, H. J.; Angermaier, L.; Knoll, W. J. Chem. Phys. 1993, 99, 7012. (8) Spinke, J.; Liley, M.; Guder, H. J.; Angermaier, L.; Knoll, W. Langmuir 1993, 9, 1821. (9) Jordan, C. E.; Frutos, A. G.; Thiel, A. J.; Corn, R. M. Anal. Chem. 1997, 69, 4939. (10) Perez-Luna, V. H.; O’Brien, M. J.; Opperman, K. A.; Hampton, P. D.; Stayton, P. S.; Klumb, L.; Lopez, G. P. J. Am. Chem. Soc. 1999, 121, 6469.

10.1021/la000144r CCC: $19.00 © 2000 American Chemical Society Published on Web 10/12/2000

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Figure 1. (a) Idealized depiction of mixed BAT/PEO BTMs and streptavidin (not to scale). Note that the thiol molecules are not expected to display such high order and rigid orientation as shown here; a more accurate description of the composition, orientation, and order within these mixed BTMs is presented elsewhere.23 (b) Bond-line formulas for PEO and BAT.

streptavidin immobilization. They performed contact angle measurements and X-ray photoelectron spectroscopy (XPS) of the surface composition and SPR measurements of the adsorption amounts and competitive desorption kinetics of the surface-bound wild-type (WT) SA and two of its genetically engineered mutants for BTMs of several different compositions. In competitive desorption, the desorption (i.e., dissociation of the surface biotin-SA bond) is initiated by switching from pure buffer above the surface to a buffer containing an excess of free biotin. This biotin competes with the surface biotin for the SA binding sites, thus driving the desorption process. The mutants they studied bind biotin much more weakly and therefore allowed faster and more accurate off-rate measurements. (The off-rate for WT SA is extremely slow and therefore difficult to accurately measure.) Desorption rate comparisons between mutants allowed them a very direct proof of the specificity of the SA binding to the surface. They found that the desorption curves were well-fitted by a double exponential with faster rates at lower surface biotin concentration and concluded that the SA adsorbed to the surface through either one or two biotin linkages, which we will denote here as “-SB” and “BSB”, respectively. The present paper is complementary to the initial study of Pe´rez-Luna et al.10 and provides new insight through the use of a more extensive set of mutants and biotin surface concentrations. We examine the adsorption and competitive desorption of wild-type streptavidin and three site-directed mutants at the biotin-functionalized gold surfaces shown schematically in Figure 1 using SPR. These BTMs contain various concentrations of biotin-terminated alkylthiolate (BAT) in mixed monolayers, wherein the remainder of the surface is saturated with oligo(ethylene oxide)-terminated alkylthiolate (PEO). Figure 1 also depicts the biotin moieties of two BATs inserting into the binding pockets of a streptavidin. The depth of this insertion is ∼1.4 nm in bulk crystals.11,12 The BTMs studied (11) Hendrickson, W. A.; Pahler, A.; Smith, J. L.; Satow, Y.; Merritt, E. A.; Phizackerley, R. P. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 2190. (12) Weber, P. C.; Ohlendorf, D. H.; Wendoloski, J. J.; Salemme, F. R. Science 1989, 243, 85.

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here differ slightly from those studied by Pe´rez-Luna et al. and Knoll’s group in that PEO replaces the simple hydroxyl-terminated alkylthiolate. In addition, the BAT contains a slightly longer oligo(ethylene oxide) spacer than used in those earlier studies. Both these changes may offer some advantages in certain applications since oligo(ethylene oxide) coatings show excellent resistance to nonspecific protein and DNA binding, biocompatibility, and nonfouling properties,13-19 and the longer spacer may improve specificity. Both the specificity with which SA binds to the biotin on such films and the number of biotin links with which it adsorbs are of interest. These issues are clarified here as a detailed function of the BAT/PEO ratio in the monolayer. Further, we find that these changes in the BTMs relative to those used by Pe´rez-Luna et al. result in ligand-protein binding (at low ligand surface concentrations) with off-rates closer to those seen in homogeneous solution. The present paper extends the study by Pe´rez-Luna et al. by including much more detailed kinetic measurements of the adsorption and competitive desorption of SA, involving also many more surface ratios of BAT/PEO. (Only three ratios were studied in ref 10.) In addition, three genetically engineered mutants (N23E, S27A, and W120A) are studied here, along with the WT SA. The N23E and S27A mutants were not studied in ref 10. The side-chains of Asn 23 and Ser 27 contribute hydrogen bonds to the ureido oxygen of biotin. These hydrogen bonding interactions have thus been perturbed in N23E and removed in S27A. The effects of these mutations on the biotin binding free energy and dissociation rates are less than the W120A alteration.2 The W120A mutation perturbs the only dyad-related side-chain that interacts with biotin from the adjacent subunit, and this residue has been shown to be the most energetically significant of the four tryptophan contacts.20 The biotin dissociation constants in homogeneous solution at 25 °C are 1.2 × 10-3 s-1 and 1.6 × 10-3 s-1 for S27A and N23E, respectively.2 The off-rate constant of biotin from WT SA was found to be ∼103 times slower (3.8 × 10-6 s-1), and that for the weakest binding of these four variants, W120A, was indirectly estimated to be 23 s-12. Here, in-depth analysis of the desorption kinetics of SA as a function of mutation and surface mole fraction of the biotinylated alkylthiolate (XBAT) clarifies how the SA is binding to the surface. The results further support earlier work, which suggested that the quality of the bound SA layer is strongly dependent on the composition of the BAT/ PEO thiolate monolayer.7,10 A few of these results were presented in a preliminary report.16 A new mechanism for the competitive desorption of the SA variants from these monolayers is introduced here that simplifies the Pe´rez-Luna mechanism. It also explains the observed kinetics over the full range of surface compositions, including the interesting new observation that, with XBAT (13) Prime, K.; Whiteside, G. M. J. Am. Chem. Soc. 1993, 115, 10714. (14) Pale-Grosdemange, C.; Simon, E. S.; Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 12. (15) Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E. J. Phys. Chem. B 1998, 102, 426. (16) Jung, L. S.; Nelson, K. E.; Campbell, C. T.; Stayton, P. S.; Yee, S. S.; Perez-Luna, V.; Lopez, G. P. Sens. Actuators, B 1999, 54, 137. (17) Bamdad, C. Biophys. J. 1998, 75, 1997. (18) Shumaker-Parry, J. S.; Campbell, C. T.; Stormo, G. D.; Silbaq, F. S.; Aebersold, R. H. Probing Protein: DNA Interactions Using a Uniform Monolayer of DNA and Surface Plasmon Resonance. Proceedings of the SPIE Photonics West Conference, joint with the International Biomedical Optics Symposium, San Jose, CA, 2000. (19) Jung, L. S.; Shumaker-Parry, J.; Campbell, C. T.; Yee, S. S.; Gelb, M. H. J. Am. Chem. Soc. 2000, 122, 4177. (20) Chilkoti, A.; Boland, T.; Ratner, B. D.; Stayton, P. S. Biophys. J. 1995, 69, 2125.

Kinetics of Wild-Type and Mutant Streptavidins

between 10 and 40%, where SA adsorption maximizes, the ratios of off-rate constants between mutants (W120A/ N23E and W120A/S27A) are almost the square of their ratios with XBAT below 1% (which are similar to homogeneous phase ratios). This proves that SA binds ligandspecifically to the BTMs of Figure 1 mainly by a single biotin at low XBAT and mainly by two biotins at moderate XBAT. The results also show that the SA again predominately binds to the surface through a single biotin at high XBAT (>55%), which was not suggested previously. The rate constants for all the elementary steps in this mechanism are estimated for the SA mutants. Finally, a detailed study of the adsorption kinetics of the SA onto these BTMs is performed here, and it reveals some conditions under which adsorption is not strictly diffusion-limited. (Earlier studies found only diffusionlimited kinetics.7,10) This allows the first study of the effect of mutations on the SA adsorption kinetics. It is found that mutants that bind to biotin much more weakly adsorb slightly more slowly. Sticking probabilities are also estimated for SA adsorption from these data. The sticking probability, defined as the probability of adsorption upon collision with the surface, directly reflects the difficulty in scaling the free energy barrier to adsorption. This is one of the few times a sticking probability has been reported for any protein’s adsorption and, to the best of our knowledge, the first time its coverage dependence has been determined. Previous reports of protein sticking probabilities have used a much less direct approach to analyze the data,21,22 which does not allow the coverage dependence to be determined (that is, first-order Langmuir adsorption kinetics were assumed). The surface structure of the BTMs studied here (Figure 1) have been characterized in detail previously using element-specific measurements by angle-resolved XPS, near-edge X-ray absorption fine structure (NEXAFS), and SPR.23 That study showed that, when the ratio of BAT/ PEO in the monolayer is 1.5), the BATs are orientationally disordered, and their biotin headgroups are uniformly spread throughout the film thickness rather than being exposed to solution. Those results provide the structural framework for the interpretation of the kinetics presented here. II. Experimental Section The SPR instrument used in this study is based on a planar prism (Kretschmann) configuration, described and characterized in more detail elsewhere.24 It can detect changes in bulk solution refractive index down to ∼2 × 10-6 (corresponding to ∼1 × 10-3 monolayers of protein for a system such as the one studied here, with a monolayer packing density of ∼230 ng/cm2). White light is directed at the gold-coated substrate through the prism at a fixed angle (78° from normal), and adsorption-induced changes in the refractive index near the Au sensor surface are observed by automated monitoring of the changes in wavelength where the SPR reflectance minimum occurs. These shifts were converted into coverages of adsorbed streptavidin on the biotinylated Au surface using a formalism for quantitative SPR described (21) Weaver, D. R.; Pitt, W. G. Biomaterials 1992, 13, 577. (22) Pitt, W. G.; Weaver, D. R.; Cooper, S. L. J. Biomater. Sci. Polymer 1993, 4, 337. (23) Nelson, K. E.; Jung, L. S.; Gamble, L.; Boeckl, M.; Naeemi, E.; Campbell, C. T.; Golledge, S. L.; Sasaki, T.; Castner, D. G.; Stayton, P. S. Langmuir, submitted for publication. (24) Jung, L. S.; Campbell, C. T.; Chinowsky, T. M.; Mar, M. N.; Yee, S. S. Langmuir 1998, 14, 5636.

Langmuir, Vol. 16, No. 24, 2000 9423 elsewhere,24 which involves a sensitivity calibration based on the sensor response to changes in bulk index of refraction, an exponential probe-depth estimated from Fresnel equations, and the known index of refraction for this class of proteins (1.57).24 Glass microscope slides (Corning) were cleaned and coated with ∼20 Å of chromium followed by ∼500 Å of gold (Alfa Aesar, 99.9999% metals basis) using an electron beam evaporator to form the SPR sensor surfaces. These were functionalized as described previously16,23 by immersion for up to 4 days in solutions of absolute ethanol with varying ratios of the two alkylthiols shown in Figure 1 (i.e., BAT:PEO), using a total alkylthiol concentration (BAT + PEO) of 0.1 mM. This treated surface was used to immobilize SA, as shown schematically in Figure 1. X-ray photoelectron spectroscopy (XPS) was used to measure the surface elemental composition of films prepared in an identical manner, as described in ref 23. Those results confirm the purity of these monolayers. Their total surface coverage of thiolates was found by XPS to be about 4 × 1014 thiolates cm-2 and was only weakly dependent upon XBAT. Since nitrogen is present in the biotin moiety of the BAT only, and not in the PEO, the mole fraction of nitrogen detected by XPS was found to provide a useful quantitative measure of the concentration of biotin on the surface. The surface biotin mole fraction (XBAT) was measured in this way and was found to vary with the biotin mole fraction in the preparation solution (Xsoln) approximately as XBAT ) 4.2 Xsoln/(1 + 3.2 Xsoln). (These XPS measurements are discussed in more detail in ref 23.) Here, Xsoln refers to the BAT mole fraction relative to total thiols only (i.e., not counting the ethanol solvent). This relationship was used for all the results below to estimate the resulting surface biotin concentration based on the biotin concentration in the preparation mixture. The number of BATs per unit area in any such monolayer can be determined by multiplying XBAT by the total surface coverage of thiolates (∼4 × 1014 cm-2). This treated glass slide was refractive index-matched to the prism and fitted with an 80-µL flow cell. This flow cell was connected to a stop-flow system that included a syringe pump (Cavro) and two valves (VICI), which allow rapid switching between buffer and sample loops with a time constant of ∼1 s.16 A steady baseline in the SPR response (i.e., wavelength of reflectance minimum) was first established under phosphatebuffered saline solution (PBS, 0.1 M, pH 7.4). Then, a solution of the same buffer containing streptavidin (typically, 0.05 mg of SA per milliliter, or ∼0.9 µM) was injected from the sample loop to initiate SA adsorption. After adsorption for 10 min, during which time the coverage appeared to reach saturation, competitive desorption was initiated by injecting the same buffer, containing a large molar excess of biotin (1 mM biotin in PBS) but no SA, into the flow cell. Pure PBS was injected again at the end of the experiments to determine the baseline shift of the SPR response resulting from the small difference in bulk refractive index between pure PBS and the biotin-containing PBS. The thiols used to functionalize the gold surfaces were synthesized and purified as described in ref 23 and were verified for purity with proton NMR. Recombinant wild-type core streptavidin was expressed and purified as previously described.3,16 The mutants (S27A, N23E, and W120A, molecular weight ) 52,800 g/mol) were constructed as described in refs 2 and 3.

III. Results III.1. Saturation Coverages of SA. The surface coverage of adsorbed streptavidin was monitored by following the SPR wavelength shift versus time (t) upon injection of SA-containing buffer (0.05 mg/mL) into the flow cell, as described above. Typical curves of coverage versus exposure time were already presented in ref 16. In all cases, the coverage increased rapidly (half-life ∼10 s), but by ∼1 min, the rate of adsorption had decreased dramatically, and the coverage thereafter increased only extremely slowly. By 10 min, the rate of adsorption was ∼1/1000 of the initial rate, and we therefore define the coverage after 10 min as the “saturation coverage”. It is

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Figure 2. The adsorption of wild-type streptavidin onto mixed BAT/PEO monolayers. Saturation SPR wavelength shift observed upon exposure of these surfaces to 0.05 mg/mL of WT SA in PBS buffer at room temperature is plotted versus the mole fraction of BAT in the original BAT + PEO alkylthiol mixture (in ethanol solution) used to prepare the monolayer.

possible, however, that slow rearrangements in the monolayer could allow even higher coverages to build up on the 24-hour time scale. The relative amount of SA bound to the BAT/PEO monolayers at saturation varied with the surface biotin concentration. Figure 2 shows the saturation SPR wavelength shift (which is proportional to the SA coverage) observed for various monolayer compositions upon exposure to the buffer solution containing WT SA at a concentration of 0.05 mg/mL. The monolayer composition here is indicated as the mole fraction of BAT in the original BAT + PEO alkylthiol mixture used to prepare the monolayer (0.1 mM total alkylthiols in ethanol solution). The same data are shown in Figure 3a, where, instead, the SPR wavelength shift has been converted to units of absolute SA coverage (ng/cm2). This conversion was made as described previously.24 The ordinate in Figure 3a has also been changed compared to Figure 2. The biotin concentration is expressed in terms of the surface biotin concentration (i.e., the mole fraction BAT in the BAT/ PEO mixed monolayer, estimated as described above) instead of its concentration in the solution used to prepare the monolayer. All further references to the biotin composition will refer to this mole fraction of biotin on the surface in the BAT/PEO mixed thiolate monolayer, XBAT. As can be seen in Figure 3a, the amount of SA adsorbed on the surface at saturation (θsat) is < 3.8 ng/cm2 at 0% surface biotin (i.e., 100% PEO). This is not surprising since oligo(ethylene oxide) surfaces are resistant to most protein adsorption13 (and see below). The amount of SA adsorption increases rapidly with XBAT, to a give a broad maximum between 10 and 50% surface biotin. As XBAT increases further, the amount of bound SA drops. These trends match previous results of others using somewhat different molecules to attach the biotin functionalities to the surface and a simple OH-terminated alkylthiolate instead of one containing an oligo(ethylene oxide) spacer as used here.6-8,10 The maximum coverage of SA (∼230 ng of SA per cm2 or ∼2.4 × 1012 molecules/cm2) is within 20% of that reported for a two-dimensional crystalline phase of SA grown at the air-water interface.25 At 0.34% BAT, the saturation coverage of WT SA is ∼47 ng/cm2 or ∼5 × 1011 molecules/cm2. This is ∼3-fold

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smaller than the BAT coverage (∼14 × 1011 molecules/ cm2), showing that only one in every ∼3 BATs is used to anchor an SA to the surface. (Below, it is shown that each SA is held to the surface by a single biotin link at this coverage.) The other two biotins are apparently not available for bonding to an SA, possibly because they are hidden under the footprint of an existing SA molecule or too poorly located within the gaps between existing SAs to be able to insert into the pocket of any incoming SAs that approach the surface. This implies a lack of mobility of the surface BATs or islanding of the BATs. At higher XBAT, the biotin:SA ratio on the surface is even higher. Figure 3b-3d shows the saturation SA coverage versus surface biotin concentration for the adsorption of the mutants of SA from solutions of this same concentration (0.05 mg/mL): W120A (Figure 3b), N23E (Figure 3c), and S27A (Figure 3d). The saturation coverage of each SA mutant exhibits the same trend with XBAT as does WT SA (Figure 3a). The saturation coverage of all SA variants is lowest at the lowest surface biotin concentration studied (0.34%), but as XBAT increases, the SA coverage increases to a maximum of 200-230 ng/cm2 in the range 10-40% BAT. This maximum coverage is nearly independent of mutant. As XBAT continues to increase above ∼40%, the saturation amount of these mutants also decreases, even more so than that of WT SA. The W120A mutant shows the least adsorption at low and high BAT concentrations, as expected since it binds most weakly to biotin. This implies that this mutant can desorb slowly, even in the absence of solution-phase biotin (at least near saturation), on low and high BAT coverages, so that its saturation coverages are really determined by dynamic equilibrium rather than by the size of the SA or by the availability of surface BATs. However, desorption of WT SA and the other mutants is so slow (see below) that their saturation coverages are, instead, mainly determined by the availability of surface BATs and the size of the SA. This would explain why they show similar coverages despite dramatic differences in their off-rates (see below). The same is true, even on the weakest binding mutant (W120A), for a BAT concentration of 10-50%, where it reaches the same saturation coverages as the other variants. In this coverage range, desorption is very slow, even for this weak mutant (see below), showing that even it bonds to the surface strongly enough to truly occupy all available surface sites. III.2. SA Adsorption Kinetics. The kinetics of adsorption of WT SA onto surfaces containing ∼30% biotin were monitored for several SA concentrations in the buffer. The observed half-lives for adsorption are plotted versus SA concentration as the solid circles in Figure 4. The squares show the adsorption half-life that would be observed if the rate were completely diffusion-limited. This is given by26,27 t1/2,diff ) (1/2) [θsat2/(C2D)], where C is the SA concentration in the buffer and D is the diffusion constant for SA (reported to be 7.4 × 10-7 cm2/s based on light scattering measurements).7 As can be seen, the measured half-life is quite similar to the diffusion-limited value, especially at low concentrations, where it decreases as 1/C2, as expected for diffusion-limited kinetics. The adsorption rates of SA on similar BTMs, wherein the diluent thiolate (PEO here) was, instead, OH-terminated alkylthiolate, also have been measured at various con(25) Darst, S. A.; Ahlers, M.; Meller, P. H.; Kubalek, E. W.; Blankenburg, R.; Ribi, H. O.; Ringsdorf, H.; Kornberg, R. D. Biophys. J. 1991, 59, 387. (26) Andrade, J. D. Surface and Interfacial Aspects of Biomedical Polymers; Plenum Press: New York, 1985; Vol. 2. (27) Ward, A. F. H.; Tordai, L. J. Chem. Phys. 1946, 14, 453.

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Figure 3. The saturation coverage of adsorbed protein versus surface biotin mole fraction for SA variants: (a) WT SA, (b) W120A, (c) N23E, and (d) S27A.

Figure 4. Measured half-life (t1/2) for the adsorption of WT SA versus SA concentration (C) in the buffer (filled circles and solid curve). This half-life decreases as 1/C2 at lower concentrations, as expected for diffusion-limited adsorption, but it decreases more slowly at the highest concentrations, as expected for reaction-limited adsorption. Shown for comparison is the adsorption half-life that would be observed if the rate were completely diffusion-limited (t1/2,diff, squares).

centrations by Spinke et al.,7 and also were found to be mainly diffusion-limited. At the highest concentration, on the other hand, the half-life in Figure 4 is substantially longer than t1/2,diff, and it decreases more slowly with

concentration, as expected for reaction-limited adsorption rates. The SA concentration used in all the remaining experiments to be discussed in this paper was 0.05 mg/mL, which is near the cutoff between purely diffusion-limited and reaction-limited rates. Proof that the rates are not completely diffusion-limited at this concentration comes from the fact that the rate depends weakly on XBAT and the mutant (see below) and more strongly on SA coverage than would be expected for purely diffusion-limited growth (where coverage varies simply as t1/2.26,27) The kinetics of the SA adsorption to the BAT/PEO surfaces show variations with surface biotin composition, as summarized in Figure 5. The initial rate of adsorption (defined here as the rate averaged over the first ∼50% of saturation; 1/2 θsat/t1/2 ) is plotted versus XBAT. Since the initial rate of adsorption followed similar trends for all four variants, the rates presented here are averaged over all four variants. At the lowest biotin concentration (0.34%), adsorption is the slowest. This is attributed to the low availability of biotin for binding. Thus, some collisions of SA with the surface are unproductive because they occur in an area where no biotins can be found. As the surface biotin composition increases, the adsorption rate rapidly increases until ∼4% biotin is reached. Thereafter, the rate remains nearly constant until the surface biotin composition exceeds ∼65%, where the adsorption rate increases slowly up to 100% surface biotin. (The reason for this final increase is addressed below.) Figure 6 shows how the initial adsorption rate depends on the protein mutation. The solid points show the initial

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Figure 7. Sticking probability of WT SA versus SA coverage for a mixed BAT/PEO surface containing ∼29% surface biotin.

Figure 5. The effect of surface biotin concentration on the “initial” rate of adsorption (i.e., the rate averaged over the first ∼50% of saturation) averaged over all four SA variants (dots). The squares show the “initial” sticking probability, S0, averaged over all four SA variants.

Figure 6. The differences between the SA variants in their initial adsorption rates. Filled circles: rates averaged over all those biotin concentrations exceeding 1% where all 4 mutants were studied. These rates appear nearly independent of SA variant. Squares: rates at 0.34% surface biotin, which provides a better comparison of the relative rates of the different mutants since these rates are not diffusion-limited (see text).

rate for each variant, averaged over all biotin concentrations above 1%. This average rate is nearly independent of the variant of SA, although this may be dictated by the fact that these conditions give nearly diffusion-limited rates. A better comparison is made at the lowest biotin coverage (XBAT ) 0.34%), where the rates and saturation coverages are the lowest, and, therefore, the diffusion limitations are the least severe. Indeed, the observed onrates gave half-lives of 2-6 s here, much slower than the diffusion-limited rates, which would have half-lives below 1/2 s at these conditions. (Note that the diffusion-limited half-life, t1/2,diff, varies as θsat2 and that θsat is 5-10-fold lower at XBAT ) 0.34% than under the conditions of Figure 4 (see Figures 2-3), so that t1/2,diff is < 0.5 s.) The initial

rates at this biotin concentration are plotted as the squares in Figure 6. As can be seen, WT SA has the fastest onrate, W120A is the slowest, and S27A and N23E are intermediate. The relative on-rate in homogeneous solution for WT, W120A, S27A, and N23E can be estimated from their off-rate constants divided by their equilibrium dissociation constants, which have been measured previously.28 Unfortunately, the on-rate constants estimated in this way for homogeneous solutions are indistinguishable among the different SA variants when the resulting error bars (factor of ∼3) are properly considered. This is not inconsistent with the statistically significant variations between mutants seen here in Figure 6, since those variations are relatively small. The reverse trend is seen in their off-rates both from these surfaces (see below) and in homogeneous solution.2 Thus, more weakly bound SA variants (i.e., those with faster off-rates) display slower adsorption rates. This implies that the energetic destabilization of the biotin-SA bond induced by protein mutation is partially manifested also in the transition state for forming that bond. This is a typical effect known as a “linear-free-energy relationship” in kinetics.29 The sticking probabilities versus coverage were calculated from the rate data by a method previously described in detail.30 Briefly, the observed rate of adsorption versus time from the SPR measurement was applied as a boundary condition in a numerical (finite differences) solution to Fick’s Law, which provides the SA concentrations versus distance from the surface and time as adsorption proceeds. The resulting concentration nearest the surface, Cs, allows calculation of the collision frequency of SA with the surface, which is just Cs [kBT/(2πm)]1/2, where kB is Boltzmann’s constant and m is the mass of SA.30 The rate divided by this collision frequency is the sticking probability, S. The initial sticking probability, S0, was estimated from the resulting value of S averaged over the first 20% of saturation. The value of S0, averaged over the variants as with the initial adsorption rates, is also shown in Figure 5. It varies with surface biotin composition similarly to those initial rates. A typical result showing the complex dependence of the sticking probability of WT SA on coverage is shown in Figure 7 for a surface containing ∼30% BAT. The apparent slight increase seen at the lowest coverages (shortest times) probably is an artifact of the time response of our (28) Hyre; Stayton, P. S. Unpublished results. (29) Gardner, W. C. Rate and Mechanisms of Chemical Reactions; W. A. Benjamin Inc.: Menlo Park, CA, 1972. (30) Jung, L. S.; Campbell, C. T. Phys. Rev. Lett. 2000, 84, 5164.

Kinetics of Wild-Type and Mutant Streptavidins

Figure 8. Competitive desorption of W120A from mixed BAT/ PEO monolayers of increasing BAT concentration (3.3%, 16%, 29%, 65%, 85%, and 100%) induced by injecting biotincontaining buffer at time 0. The amount of adsorbed SA, measured by SPR and normalized to the initial value, is plotted versus time. Each curve is labeled with the appropriate surface biotin composition. Concentrations 5 × 10-5 s-1. Despite these large errors, it is obvious that the correlation between k and kb is very weak. The fast rate of kb and the lack of correlation between kb and k strongly support the two-step mechanism we propose here for desorption of a doubly bound SA, reaction 3. Since the site on this SA created by the loss of the BAT in the first step is not yet bound with an aqueous biotin competitor, and since this surface BAT that was lost is very close by and is oriented properly for rebinding, then the rate of the reverse reaction for this first step should be much like an adsorption rate measured at very high SA concentration: extremely fast and weakly dependent on mutation (relative to off-rates). This is just how the values of kb determined within this model behave. The off-rate constants for desorption of singly bound WT SA and W120A were also estimated by Pe´rez-Luna et al.10 from similar measurements at a very low surface BAT concentration (0.7%) but using a slightly different mixed monolayer. (Their BAT had a shorter oligo(ethylene

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glycol) spacer, and their diluent thiolate had no oligo(ethylene glycol) spacer at all between its alkyl chain and its terminal OH group.) They found values for WT SA and W120A of ∼3.3 × 10-4 and 2.4 s-1, respectively, which are ∼15-fold faster than our values (k in Table 1). This difference is partially because they used only the fastest exponential component of a double exponential fit to get these values, whereas we used the full desorption curve’s half-life. However, careful inspection of the desorption curves shows that this does not explain most of this difference. This indicates that it is mainly due to the abovementioned differences in the monolayer structures. The diluent and the length of oligo(ethylene glycol) spacer in the BAT could alter the adsorption energetics of the singly bound SA directly or indirectly (e.g., by influencing the lateral distribution of the two thiolates across the surface). In Figure 9, a weak decrease is seen in the SA off-rate between 60 and 100% BAT for all the mutants (but not WT). Also, the off-rate is 10- to 25-fold faster at 0.34% BAT than at 100% BAT for all three mutants, although singly bound SA dominates at both these mole fraction extremes. We attribute both these observations to an increase in relatively strong nonspecific attractions of singly bound SA to the surface when in contact with other BATs (as opposed to PEOs). Note in Figure 9 that the same straight line can fit reasonably well the data point at XBAT ) 0.34% and those at XBAT ) 60-100%, and this line has a positive slope for the 3 mutants. These singly bonded SAs are surrounded mainly by PEO at 0.34% BAT (Figure 10) but mainly by BATs above 60% BAT (Figure 12). If we assume that the sides of these surrounding BATs interact more attractively with the surfaces of the mutant proteins than do PEOs, this positive slope is explained. This is consistent with ARXPS and NEXAFS results, which showed that the BATs are much more poorly oriented on the surface at these high BAT concentrations.23 An increasing extent of nonspecific binding between 60% and 100% BAT would also explain the increase in SA adsorption rate observed in this range (see above). For the WT SA, this positive slope is not observed, which suggests that this nonspecific binding is weaker (i.e., has a faster off-rate) than the specific binding in the special case of WT SA. This could occur since WT SA has by far the strongest specific binding. V. Conclusions The adsorption and desorption behavior of WT and SA mutants on these mixed BAT/PEO layers confirm that surface layers can be prepared that allow rapid, quantitative testing of the effects of protein mutations on their binding strengths to ligands. Adsorption is nearly diffusion-rate-limited, but subtle differences in the adsorption rate can be seen between the SA variants and as a function of XBAT and SA coverage. The desorption kinetics vary strongly between variants and as a function of XBAT. The dependences on XBAT suggests strong heterogeneity in the surface layer. At low coverages (0.34% surface biotin), the surface is composed primarily of singly bound SA, which

Jung et al.

desorbs with rate constants for the SA variants that are similar to those from solution measurements. As the concentration of BAT increases to ∼29%, the desorption rates slow and the amount bound increases. These observations, in addition to the change in off-rates, point to a surface dominated by doubly bound species of SA. A mechanism for desorption that models these data well is proposed, and the two key rate constants for elementary steps in this mechanism are determined for each SA variant. They are tabulated in Table 1 and correspond to the first-order rate constants for (1) the competitive cleavage of a bond between an SA and a surface-bound biotin and (2) the rapid reforming of this bond to the same surface biotin in the special case that it was temporarily cleaved from a doubly bonded species whose other site is still bound to a surface-immobilized biotin. The kinetics of desorption also suggest significant heterogeneity or lateral interactions in the SA monolayer. For XBAT above 40%, the SA desorption rates increase and the amount adsorbed decreases. This is attributed to the lack of protruding biotin headgroups in the BAT-rich monolayers. Due to poor orientational order of the BATs at high XBAT, most of the biotin headgroups do not protrude from the film, and therefore, they are not able to bind deep enough into the pocket of an SA. This prevents SA from forming bonds to two surface BATs, so instead, it appears to link to the surface via a single BAT as if the BAT concentration were very low. By 100% BAT, nonspecific attractions to the surface become stronger, which correlates with XPS and NEXAFS measurements showing that many of the biotin headgroups are now buried in the adlayer, exposing other parts of the molecule (possibly aliphatic regions) at the surface that appear to interact nonspecifically with SA. These results show that an XBAT of ∼30% should be used in hybrid monolayers designed to create the most dense and stable SA linker layers. The trends in Figure 9, if continued for WT SA, indicate that WT SA would have a surface lifetime of >107 s at this condition, even in 1 mM biotin solutions. On the other hand, an XBAT of