Fig. 1

Comparative analysis of (un)folding kinetics for B4, Sb3, and their parent proteins GB and S6. (A) Sequence and topology alignment of the B4 and Sb3 constructs, differing by a single residue within the first 56 amino acids (adapted from Ruan et al., 2023). (B) Structural alignment of the B4 construct (green) with the parent IgG-binding GB domain (pink) from Streptococcal Protein G (PDB: 1PGA). (C) Structural alignment of the Sb3 construct (blue) with its parent ribosomal protein S6 (orange) from T. thermophilus (PDB: 1FKA). (D) Comparison of the chevron plots obtained for the B4 construct (green) and the GB domain (pink). Both exhibit classical V-shaped two-state folding behavior, with similar kinetic parameters except for a significantly higher unfolding rate constant in B4. (E) Comparison of the chevron plots obtained for the Sb3 construct (blue) and the S6 protein (orange). Both display classical V-shaped two-state folding behavior, with similar kinetic parameters except for a significantly higher unfolding rate constant in Sb3. The (un)folding kinetic data for the GB domain and the S6 protein were taken from Morrone et al., 2011, and Otzen and Oliveberg, 1999, respectively