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. 2017 Mar 22;139(11):4157-4167.
doi: 10.1021/jacs.7b00540. Epub 2017 Mar 9.

Controlling Multivalent Binding through Surface Chemistry: Model Study on Streptavidin

Galina V Dubacheva  1   2 , Carolina Araya-Callis  1 , Anne Geert Volbeda  3 , Michael Fairhead  4 , Jeroen Codée  3 , Mark Howarth  4 , Ralf P Richter  1   5   6
Affiliations

Controlling Multivalent Binding through Surface Chemistry: Model Study on Streptavidin

Galina V Dubacheva et al. J Am Chem Soc. .

Abstract

Although multivalent binding to surfaces is an important tool in nanotechnology, quantitative information about the residual valency and orientation of surface-bound molecules is missing. To address these questions, we study streptavidin (SAv) binding to commonly used biotinylated surfaces such as supported lipid bilayers (SLBs) and self-assembled monolayers (SAMs). Stability and kinetics of SAv binding are characterized by quartz crystal microbalance with dissipation monitoring, while the residual valency of immobilized SAv is quantified using spectroscopic ellipsometry by monitoring binding of biotinylated probes. Purpose-designed SAv constructs having controlled valencies (mono-, di-, trivalent in terms of biotin-binding sites) are studied to rationalize the results obtained on regular (tetravalent) SAv. We find that divalent interaction of SAv with biotinylated surfaces is a strict requirement for stable immobilization, while monovalent attachment is reversible and, in the case of SLBs, leads to the extraction of biotinylated lipids from the bilayer. The surface density and lateral mobility of biotin, and the SAv surface coverage are all found to influence the average orientation and residual valency of SAv on a biotinylated surface. We demonstrate how the residual valency can be adjusted to one or two biotin binding sites per immobilized SAv by choosing appropriate surface chemistry. The obtained results provide means for the rational design of surface-confined supramolecular architectures involving specific biointeractions at tunable valency. This knowledge can be used for the development of well-defined bioactive coatings, biosensors and biomimetic model systems.

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Conflict of interest statement

The authors declare the following competing financial interest(s): M.H. is an author on a patent for monovalent streptavidin (US8586708 B2).

Figures

Figure 1
Figure 1
Tunable model system to study SAv binding to biotinylated surfaces. (A) Table of tunable parameters. (B) SAv constructs having different valencies. On the left, the structure of the SAv tetramer (ribbon diagram with each monomer in distinct color) with biotins attached to its binding pockets (ball-and-stick model) is shown; on the right, the SAv constructs used are listed schematically. (C–E) Fluorescence measurements in the presence of biotin in solution reveal the number of binding sites in SAv constructs. (C) Relative fluorescence emission intensity of tryptophan located in the binding pockets of rSAv, tSAv, dSAv-trans, mSAv (filled squares, each data point represents a single measurement) and dSAv-cis (empty triangle, mean of 2 measurements with standard error) upon biotin binding to saturation. All data fall onto a straight line (linear fit) that crosses the y axis at 100%. (D) Example of tryptophan relative intensity change upon biotin binding to SAv (the moment of biotin injection is indicated by an arrow). This data set corresponds to the last point in (C). (E) Examples of fluorescence spectra. Here, tryptophan fluorescence emission spectra of rSAv solution in the absence of biotin (blue) and 45 min after biotin injection (red) are shown, and the maxima in these spectra correspond to the first and last points, respectively, in (D). A negative control (i.e., biotin without SAv) is also shown (black).
Figure 2
Figure 2
Biotinylated surfaces with controlled lateral mobility and biotin content. (A) Schematic representation of a b-SLB, chemical structure of lipids and a characteristic QCM-D signature of b-SLB formation using SUVs made of DOPC (1) and DOPE-CAP-biotin (2) at a 19:1 molar ratio. The time of SUV exposure is indicated by an arrow. (B) Schematic representation of b-SAM, chemical structure of thiols and characteristic images of water drops placed on a bare Au surface and on Au surfaces functionalized with HS-(CH2)11-EG4-OH (3) and its mixture with HS-(CH2)11-EG6-biotin (4) at a 9:1 molar ratio with contact angles indicated.
Figure 3
Figure 3
Reporters for residual SAv valency. Schematic representation of biotinylated probes, b-ZZ and b-oHA, used as reporters of the residual valency of SAv bound to a biotinylated surface (in this case b-SLB). The reporter probes are drawn approximately to scale with the rSAv and the SLB thickness.
Figure 4
Figure 4
Binding of monovalent SAv to biotinylated surfaces. (A,B) QCM-D responses recorded during the binding of mSAv, and subsequently b-ZZ, to b10%-SAM (A) and b5%-SLB (B). Insets in A and B illustrate reversible mSAv binding and extraction of biotinylated lipids by mSAv, respectively. (C) QCM-D responses obtained upon exposure of mSAv and subsequently rSAv and b-ZZ to a b1%-SLB (squares) together with a control in which the exposure to mSAv was omitted (circles). The data indicate SAv-induced depletion of biotinylated lipids from the SLB, as illustrated schematically. (D) Representative measurement of changes in tryptophan fluorescence emission intensity upon injection of b5%-SUVs to a solution of mSAv (injection time is indicated by an arrow). The inset shows mean and standard deviation of the relative intensity decrease at equilibrium; the decrease is similar to that of free biotin in solution (Figure 1C) and indicates specific and quantitative binding of mSAv to biotinylated lipids through the biotin-binding pocket of mSAv.
Figure 5
Figure 5
Binding of multivalent SAv constructs to densely biotinylated surfaces. Shown are QCM-D responses recorded during the binding of SAv constructs, and subsequently b-ZZ, to b10%-SAM (A) and b5%-SLB (B). Insets in A and B illustrate the surfaces and molecules used, and the schematic representations in C illustrate the binding features—residual valency, reported by b-ZZ, and orientation—of the different SAv constructs. Data are marked with distinct symbols for each SAv construct: rSAv (squares), tSAv (circles), dSAv-cis (upward-pointing triangles) and dSAv-trans (downward-pointing triangles).
Figure 6
Figure 6
SAv binding and residual valency on densely biotinylated surfaces, quantified by SE. (A) Example of binding curves obtained by SE, here for rSAv and b-oHA on a b5%-SLB. The inset illustrates the SE setup. (B) Table of surface densities of SAv, ΓSAv, and biotinylated reporter probes (b-ZZ or b-oHA), Γb, and the mean residual valency, Γb/ΓSAv. Values are presented as mean ± error, where the latter is the sum of the reproducibility error (4%; averaged from 4 independent sets of SE measurements with 2 to 4 samples in each set, cf. column 3 in Table S1) and the detection limit of the SE (1 ng/cm2). aBinding was below the detection limit of SE; bmSAv removed biotinylated lipids from b-SLB (cf. Figure 4B); cMeasured before buffer rinsing, as binding was not stable (cf. Figure 4A); db-ZZ accelerated displacement of mSAv from the surface. Conditions: SAv adsorption time = 90 min, biotinylated probe adsorption time = 15–60 min. All values were determined after adsorption and buffer rinsing once the SE response was stabilized, except for mSAv on SAMs, where the equilibrium bound amount before rinsing is given (as rinsing provokes mSAv detachment, Figure 4A).
Figure 7
Figure 7
Effect of SAv coverage, and biotin surface density on SAv binding to biotinylated surfaces. The surface density of the biotinylated reporter probe vs the surface density of rSAv (squares) and tSAv (circles) was quantified by SE for immobile (A) and fluid (B) surfaces. The SAv surface density was tuned by varying either biotin surface density (incubating SAv to saturation or equilibrium; blue), or SAv adsorption time (on b10%-SAMs and b5%-SLBs, respectively; red). All values were determined after adsorption and buffer rinsing once the SE response was stabilized. The ratio Γb/ΓSAv (insets) corresponds to the mean residual valency of SAv. Lines are parabolic or linear fits; dashed lines indicate the range where SAv binding is not stable precluding quantification of residual valency. Conditions: biotin surface fraction = 10% (A, red), 5% (B, red), tuned from 1 to 10% (A, blue) or from 0.45 to 5% (B, blue); SAv adsorption time–tuned from 10 s to 90 min (A and B, red), 90 min (A, blue), 30 min (B, blue); biotinylated probe adsorption time = 15 to 60 min; see Table S1 for full details on conditions and determined values.
Figure 8
Figure 8
SAv binding to sparsely biotinylated surfaces. (A) QCM-D responses obtained during the binding of rSAv (squares), tSAv (circles) and mSAv (lozenges) to b0.1%-SAMs. Binding is followed by partial detachment, as illustrated schematically. (B) The QCM-D response for the exposure of mSAv to b0.05%-SAM reveals rapid unbinding upon rinsing in buffer (as illustrated schematically) and repeatable binding.
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