Fig. 2: MAX and Pho4 differ in folding-and-binding to CACGTG.
a Binding isotherms for WT (teal, n = 20) and R36A (red, n = 3) MAX variants binding to cognate DNA (left). Reproducibility of ΔΔG measurements across two microfluidic devices (right, median ± SEM per device). Light gray markers indicate mutants un-resolvable from background binding for which reported Kds represent a lower limit. b Affinities for MAX mutants binding CACGTG (median ± SEM). Red markers denote mutations to DNA-contacting residues, gray markers with red outlines denote mutations to phosphate backbone-contacting residues, and arrows denote Kd limits. c Binding isotherms for WT MAX (n = 51), MAX L31V (n = 10), WT Pho4, and Pho4 A258V (left) and comparison of ΔΔG measurements for aligned substitutions to MAX and Pho4 (right); marker size indicates residue conservation across the bHLH family (Methods). d Thermodynamic model for a three-state system such that observable Kd depends on the folding equilibrium (Kfold) and true binding affinity (Kd, true). e Measured change in cognate affinity (median ± SEM) versus changes in helical propensity39 for mutations to non-DNA contacting basic region residues in MAX (teal) and Pho4 (orange); dashed line indicates fitted thermodynamic model with indicated fitted values of Kfold and Kd. Replicate counts for all Kd measurements are contained in Supplementary Data 1. Source data are provided as a Source Data file.
