Binding and Dissociation Kinetics of Wild-Type and Mutant Streptavidins on Mixed Biotin-Containing Alkylthiolate Monolayers

Abstract

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.

XPS, TOF-SIMS, NEXAFS, and SPR Characterization of Nitrilotriacetic Acid-Terminated Self-Assembled Monolayers for Controllable Immobilization of Proteins

“…[40][41][42][43][44] The calculation of the protein surface coverage from the SPR response is described elsewhere. 15,16 The home-built SPR liquid biosensing system used in this study has been described and characterized in more detail elsewhere. 45,46 A dual-channel Teflon flow cell with two independent, low volume parallel flow channels was used.…”

“…One attractive way to anchor proteins involves preparing a surface of dispersed bioactive ligands (e.g. biotin 15,16 ) in a protein resistant background (e.g. oligo(ethylene glycol) [17][18][19] ).…”

Optimisation of a multivalent Strep tag for protein detection

“…3C) were 1.49 ± 0.01µM and 260 ± 0.18nM respectively (Fig. 3D) [34,35,36], a configuration demonstrably useful in building up surface structures through accessing the initially unused streptavidin sites pointing away from the surface.…”

“…The increase in tag number results, specifically, in both a higher local concentration of binding points, and a greater receptive surface area at which the large 52.8 kDa streptavidin molecules can be accommodated. The added advantage of streptavidin having four binding pockets increases the potential network of interactions that may occur, though it is likely that only 1-2 binding pockets per streptavidin are bound to any one SQM receptor at any single point in time [34][35][36]. An approximately linear increase in affinity is achieved on adding more tags through the Strep1-Strep5 …”

A Highly Sensitive and Selective Surface-Enhanced Nanobiosensor

“…These propagating surface plasmon polariton (SPP) sensors, which operate on real-time refractive index changes, offer a label-free method for the detection of biomolecules [11][12][13].…”