Latent Bioreactive Uaa

Latent Bioreactive Uaa and proximity-enabled bioreactivity

Protein side chains can spontaneously form a covalent linkage via cysteines only. This unique disulfide bond is crucial for the folding and function of various proteins, yet its reversibility and redox sensitivity impose limitations on protein expression, engineering, and applications. We broke this natural barrier by adding new covalent bonds into proteins in live cells.

It had been infeasible to engineer a new covalent bond for proteins in live systems: introduction into cells a Uaa bioreactive toward a natural amino acid will result in nonspecific linkages due to ubiquitous presence of latter, and a bioreactive Uaa might not go through the translational machinery for genetic incorporation. Indeed, Uaas genetically incorporated hitherto are either chemically inert or bioorthogonal.

We overcame this challenge by designing a Uaa to react with a natural amino acid through proximity-enabled bioreactivity. The reactivity of the bioreactive Uaa is fine tuned so that it remains intact inside cells and through protein translation; only when the Uaa is placed proximal to the target natural amino acid residue will the reaction occur specifically forming a new covalent bond.

new covalent bonds

Figure 2.1: Generation of new covalent bonds within and between proteins through proximity-enabled bioreactivity. Bioreactive Uaa Ffact was designed to react with Cys only when they are in proximity.

We demonstrated that the resultant new bond enables infinite binding affinity to proteins, enhances optical properties challenging to engineer, and allows us precisely probe ligand-GPCR interaction in live mammalian cells. This new concept of proximity-enabled bioreactivity thus opens the door to genetically encoding a new class of Uaas, the bioreactive Uaas, which will afford novel avenues toward generating protein properties and functions previously inaccessible, with broad applications in biological studies, protein therapeutics, and synthetic biology.