Interface ΔΔG (clonal titrations)
Binding
Gibbs Free Energy of Binding (ΔG)
kcal/mol
Flow Cytometry , Yeast Surface Display
None
Yeast clonal titrations were done according to Chao et al. (Nat Protoc 2017: 1:755-768) to determine the binding affinity of individual mutants. Briefly, colonies were inoculated in 1 mL SDCAA overnight at 30C, and protein expressed inducted in SGCAA at OD600 = 1.0 and 22C for 18 h. 1 X 10^5 cells were labeled with different concentrations of the respective biotinylated binding protein (range 64 pM to 400 nM) for 30 min at room temperature in DPBSF (DPBS with 1 g/L fraction V BSA). Cells were centrifuged at 17,000g for 1 min and labeled with anti-cmyc-FITC (Miltenyi Biotec, San Diego, CA) and streptavidin–phycoerythrin (Thermo Fisher, Waltham, MA) on ice for 5 min. Cells were pelleted and washed twice with DPBSF before evaluating on a Accuri C6 flow cytometer. Clonal titrations were done at least in triplicate on two independent days. Point mutants were made by the method of Kunkel. Binding to biotinylated MBP was assessed as a negative control, with no binding observed at 400 nM.
Statistical analysis, from cited ref 14 (Kowalski et al, J Biol Chem 2015), indicates errors reported for all experiments are STD.
Table S2 and SIFig.4a show different values (and errors) for same mutants.