Wong, Sui-LamNg, KennethMarangoni, Jesse M2022-11-152020-07-22Marangoni, J. M. (2020). Engineering Streptavidin with Switchable Ligand Binding Affinity Using Disulfide Bonds at the Biotin Entry Gateway (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.http://hdl.handle.net/1880/115471https://dx.doi.org/10.11575/PRISM/40438Streptavidin is widely used in biotechnological applications for its specific, high-affinity interaction with its natural ligand biotin, a small vitamin that can be easily conjugated to biomolecules of interest. However, its utility is often limited since many applications require reversibility in binding whereas others require higher affinity than that offered by wild-type streptavidin. For some applications, it would even be beneficial to allow extremely tight binding and subsequent ligand release within a single protocol. To combine both extremely tight and reversible binding into a single protein, two streptavidin muteins, designated M88 and M112, were engineered to each contain a distinct disulfide on opposite sides of a flexible loop critical for ligand binding. Each disulfide bond has markedly different effects on protein structure and binding kinetics. While the disulfide in M112 caused a detrimental conformational change which decreased biotin binding affinity, oxidized M88 showed a ~250-fold decrease in off-rate constant at 21°C and increased thermostability when compared to wild-type streptavidin. Furthermore, reduction of the disulfide bond increased the off-rate constant ~19,000-fold compared to the oxidized form, reducing the half-life for dissociation from 50 years to 1 day. Increasing the temperature to 50°C allows ligand release from the reduced form with a half-life of 9 minutes. M88 thus displays redox and temperature dependent ligand binding, both of which can be used to switch between high- and low-affinity states. M88 coupled to a matrix can be used to capture and release biotinylated biomolecules. For applications where increased temperature is not viable, further engineering of M88 has been used to reduce ligand binding affinity. The relative ease of controlling protein disulfide bond redox state with mild chemical agents allows switchable affinity of M88 towards biotin.enUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.Biology--Moleculardoctoral thesis