SI Methods and Materials Protein Mutagenesis, Expression, and Purification. To allow for single-molecule FRET experiments, an AK enzyme from E. coli was appended with a (His)6 tag for immobilization at the C terminus (1). Using site-directed mutagenesis, cysteine mutations

Protein Mutagenesis, Expression, and Purification. To allow for single-molecule FRET experiments, an AK enzyme from E. coli was appended with a (His)6 tag for immobilization at the C terminus (1). Using site-directed mutagenesis, cysteine mutations were introduced in the Lid (A127C) and the Core (A194C) domains for site-specific labeling. A further mutation was introduced (C77A) to remove a single native cysteine from the gene. The mutant AK gene was cloned into pET-21a (Novagen) and induced in BL21(DE3)pLysS cells at 37°C for 3 h. Unless otherwise noted, all work with the expressed AK was done in AK reaction buffer: 100 mM Tris HCl (pH 7.5), 100 mM KCl, and 2 mM MgCl2. All chemicals were purchased from Sigma and used as received unless specified. After sonication and centrifugation, the protein was purified over a Ni affinity column (HisTrap HP, Amersham Pharmacia) according to the manufacturer’s instructions. The protein was further purified over a Q-Sepharose anion exchange column (Amersham Pharmacia) with a linear gradient from 0–500 mM KCl. Finally, aggregates were removed from the sample by running it over a 1-m S-200 gel filtration column (Amersham Pharmacia) (2).