Fig. 1: Affinity-clamp assay, resin-assisted translation workflow, conceptual overview of protein folding.
a Peptide biosensor consists of an autoinhibited TVMV protease (blue), with PDZ and FN3 domains forming the affinity clamp (AC). Binding of RGSIDTWV-fusion with the peptide of interest (X) to biosensor results in a conformational change of the AC-module that dislodges the autoinhibitory peptide (AI) from the active site of TVMV (red) leading to protease activation and subsequent cleavage of the quenched substrate (Q-F). Q and F denote the fluorescence quencher group and the fluorochrome, respectively. The calibration plot for RGS peptide is obtained by plotting the initial reaction rates against different RGS-peptide concentrations as mean ± s.d. of three independent assays. b Workflow for (poly)peptide production in resin-assisted translation reaction; PDZ and FN3 denote the respective capture domains of affinity clamp. c Combined kinetic-thermodynamic profile of protein folding. The dependence of folding/unfolding rates on denaturant concentration is illustrated by the chevron plot (modified from Kiefhaber et al.70). Blue (filled) and red (open) circles denote the values for folding (KD→N) and unfolding (KN→D) rate constants, respectively, at different denaturant concentrations. Schematic Gibbs free-energy diagrams of protein folding correspond to no denaturant (left), its highest concentration (right), and its intermediate concentration, corresponding to a point of thermodynamic equilibrium (middle). ΔG# and ΔG denote folding/unfolding transition state activation energy and the free-energy difference between folded and unfolded states, respectively. NATIVE, DENAT. and ALTERN. denote native, fully denatured, and alternative folding states, respectively. Arrows outlining the free-energy diagrams indicate the shift direction of folding/unfolding equilibrium. The yellow zone highlights the range of guanidine hydrochloride (GdnHCl) concentrations tolerated by affinity-clamp/RGS-peptide complex. The arrow along the left-hand side slope of the plot indicates a shift from kinetically controlled folding space at physiological conditions to thermodynamically controlled folding space upon an increase in denaturant concentration. Upper and lower horizontal dotted lines indicate the corresponding values for folding/unfolding rate constants at arbitrary GdnHCl concentration marked by the vertical line. Numerical ratios of rate constants are displayed at arbitrary concentration and at a point of thermodynamic equilibrium.
