Molecular Recognition between Genetically Engineered Streptavidin and Surface-Bound Biotin. Pérez-Luna, V. H., O'Brien, M. J., Opperman, K. A., Hampton, P. D., López, G. P., Klumb, L. A., & Stayton, P. S. Journal of the American Chemical Society, 121(27):6469--6478, July, 1999. ![Molecular Recognition between Genetically Engineered Streptavidin and Surface-Bound Biotin [link]](https://bibbase.org/img/filetypes/link.svg "link")
Paper doi abstract bibtex This study examines the binding of wild-type streptavidin and streptavidin mutants to biotin-terminated 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 (Ka varies from ?1013 M-1 for wild-type to 107 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.
@article{perez-luna_molecular_1999,
title = {Molecular {Recognition} between {Genetically} {Engineered} {Streptavidin} and {Surface}-{Bound} {Biotin}},
volume = {121},
issn = {0002-7863},
url = {http://dx.doi.org/10.1021/ja983984p},
doi = {10.1021/ja983984p},
abstract = {This study examines the binding of wild-type streptavidin and streptavidin mutants to biotin-terminated 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 (Ka varies from ?1013 M-1 for wild-type to 107 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.},
number = {27},
urldate = {2015-05-05TZ},
journal = {Journal of the American Chemical Society},
author = {Pérez-Luna, Víctor H. and O'Brien, Michael J. and Opperman, Kimberly A. and Hampton, Philip D. and López, Gabriel P. and Klumb, Lisa A. and Stayton, Patrick S.},
month = jul,
year = {1999},
pages = {6469--6478}
}
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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. 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