Molecular Recognition between Genetically Engineered Streptavidin and Surface-Bound Biotin

Abstract

This study examines the binding of wild-type streptavidin and streptavidin mutants to biotinterminated self-assembled monolayers (SAMs) as a model of biomolecular recognition at solid-liquid interfaces. The types of streptavidin proteins employed in this work were wild-type, Tyr43Ala (Y43A), and Trp120Ala (W120A), which have biotin-binding affinities that span several orders of magnitude (K a varies from ∼10 13 M -1 for wild-type to 10 7 M -1 for W120A). Two types of biotin-terminated monolayers were examined: those formed by chemisorption of 11-mercaptoundecanoic-(8-biotinoylamido-3,6-dioxaoctyl) amide (1) and those formed from mixtures of 12-mercaptododecanoic-(8-biotinoylamido-3,6-dioxaoctyl) amide ( 2) and 11-mercapto undecanol (3). Our findings support two previously published studies that found that 1 forms monolayers on gold that are disordered, while 2 and mixtures of 2 and 3 form closely packed, well-organized SAMs. The kinetics of binding and desorption of wild-type streptavidin and the mutants to and from these monolayers were measured using surface plasmon resonance spectroscopy. Adsorption of the proteins was found to occur at a diffusion-limited rate and to saturate at different surface coverages depending on their biotin-binding affinity. On disordered monolayers formed from 1, only a fraction of the bound mutants could be dissociated by exposure to free biotin. The fraction of undissociated mutants correlated with the biotin-binding affinity, suggesting that the formation of nonspecific interactions depends on the residence time of the protein on the surface. On mixed SAMs formed from 2 and 3, complete dissociation of the proteins occurred upon exposure to free biotin in solution. The kinetics of desorption of streptavidin from mixed SAMs was analyzed using a model that included the possibility of bivalent protein binding to the SAM at high surface concentrations of biotin. It was found that rate constants of dissociation were larger for the dissociation of a streptavidin-biotin bond on the surface than in solution. On biotinylated SAMs, the kinetic constants of dissociation were dependent on the surface concentration of biotin. Slow dissociation rates at higher surface coverage result from attractive protein-protein interactions. The results demonstrate the importance of the preparation and the structure of the solid surface and the complexity of biomolecular recognition at solid-liquid interfaces. Molecular recognition is affected by interactions between the adsorbed proteins and the surface and also by interactions among adsorbed proteins. These conclusions have important implications for the development of reversible biosensors.

Angle-Scanning Surface Plasmon Resonance System with 3D Printed Components for Biorecognition Investigation

“…The reason was that the ureido-and tetrahydrothiophene-rings of biotin are more hydrophilic than −NH 2 terminated cysteamine SAM [26]. The contact angle of biotin SAM was smaller than the previously reported one in study [27], and this may be due to the well-organized biotin SAM. Thus, the above results showed that the SPR sensor chip was prepared successfully.…”

Steric Crowding Effects on Target Detection in an Affinity Biosensor

“…Pérez-Luna et al 13 showed that SA bound to surface linked biotin via two links at the biotin coverage where steric crowding was observed to initiate on planar surfaces. Consequently on our heterogeneous surface, SA could be assumed to bind to 57% biotin surface-coverage via two links.…”

“…It was characterized by Pérez-Luna et al 13 and with more detail by Jung et al 12 that at low biotin surface coverage (<10 %) and high biotin surface coverage (>60 %) SA binds via a single biotin-binding site. Over a median range of biotin surface coverage (10-40 %) mutant streptavidin exhibited second order off rate constants from which they concluded SA binds mainly via two biotin-binding sites.…”

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 …”