Team:CU-Boulder/Design
Design
Synthetic therapeutic antibodies are made up of only the single chain variable domain (scFv) of parent monoclonal antibodies, due to scFv's superior pharmacokinetic properties over the parental chains. ScFv's are protein heterodimers, and they function by having specific binding sites at the "tips" or edges of their structure. Protein structure is extremely important for retaining function. If the two chains in an scFv are not held together in a specific configuration, they would not bind to the antigen, and would lose their function.
In order to create an allosteric "off-switch" for therapeutic antibodies, we targeted the configuration of the two dimers that make up scFv's and aimed to find a way to change their conformation in order to switch off the function of the antibody. After some searching, we found a regulatory protein, AraC, that was naturally found in E. coli and dimerizes differently in the presence versus the absence of a small molecule, L-arabinose.
We decided to utilize the innate response of AraC to L-arabinose to induce scFv's to change conformation. In order to do this, AraC would have to be covalently linked to an scFv. In the natural state, this new hybrid molecule would allow for the antibody to bind to its antigen. But once L-arabinose is introduced, the homodimeric AraC will reorient, which should split the scFv in the process, preventing the antibody from binding due to its altered structure. To design the linker between AraC and a standard scFv, Pyrosetta in combination with Pymol was used. In order to simplify experimental testing, the linker was modeled to attach the AraC domain to a fluorescein-binding scFv. Once designed, the complete protein will be produced in E. coli, and a fluorescein assay will be run to demonstrate the competency of the structure.
Before the addition of arabinose, the fluorescein should be quenched and inactive. After the addition of arabinose, the assay should show fluorescein activity (glowing!).