Rewiring the Genetic Code for Global Protein Footprinting

Proteins are molecular machines that generally function in large assemblies to carry out most biological processes. Although NMR and crystal structures have enhanced our understanding of protein function, they only provide a “snapshot” of one transient state and thus fail to reveal conformational changes and partner interactions that are key to the operation of molecular machines. Alternative chemical footprinting techniques can give time‑resolved pictures of which residues in a protein are solvent accessible. Patterns of residue accessibility are characteristic signatures of different protein conformational states, allowing characterization of conformational changes in real time. Although cysteine’s unique chemical properties are ideal for footprinting, cysteine’s low abundance in naturally occurring proteins limits applicability. One way to overcome this problem is to rewire the genetic code so that, under the control of a genetic switch, any codon can be translated into cysteine. Previous work has shown that non‑cysteine codons can be rewired to cysteine by expression of a mutant cysteine-tRNA with an anticodon complementary to the desired non‑cysteine codon, but in many cases this has been inefficient. I have designed a pulse‑chase genetic selection to find mutant cysteine-tRNA and cysteine-tRNA synthetases that rewire the genetic code with high efficiency.  Preliminary experiments involved constructing a yellow fluorescent protein with an intein insertion to serve as a reporter for cysteine misincorporation. Currently, mutant cysteine-tRNA and tRNA synthetase libraries are being screened for efficient misincorporation. Each mutant cys-tRNA/synthetase pair discovered will provide a valuable tool for analyzing protein structure and "action" in protein complex mixtures.