Interface ΔΔG (deep sequencing)
Binding
Gibbs Free Energy of Binding (ΔG)
kcal/mol
Illumina Sequencing
None
Under the conditions of the experiments performed (labeling concentration [L] = 1/2 Kd,wt), we can convert the fitness metric from the deep sequencing dataset to a change in binding energy (interface ΔΔGi) for each variant, i, using the following equation:
ΔΔGi (kcal//mol) = RTln [-1/2 + 3 / 2^(ζ+1)]
Where R is the gas constant and T is the temperature set to 300 K. This deep sequencing-derived interface ΔΔG is calculated using the above fitness metric representing the relative fluorescence of a variant at a single labeling concentration. This conversion results in a relatively narrow dynamic range arising from variants either at the maximum or minimum fluorescence value at the labeling conditions used. Given the sorting conditions used in the experiment, there is no discrimination among mutations with interface ΔΔG >0.5 kcal/mol.14 At the other extreme, this method can also detect stabilizing mutations, as we have designed the experiment such that no one variant takes over the entire selected binding population. This gives an upper bound of interface ΔΔG to -0.8 kcal/mol.
There are several assumptions needed to derive interface ΔΔG from sequencing counts, and any major deviations in these assumptions can substantially affect the validity of this reconstruction (see publication text for assumptions).
Statistic analysis, from cited ref 14 (Kowalski et al, J Biol Chem 2015), indicates errors reported for all experiments are STD.