Figure 1.

Stabilized mutant identification using second-site saturation suppressor mutagenesis (SSSM). Schematic representation of second-site saturation suppressor mutagenesis. Proteins exist in an equilibrium between folded and unfolded states. (A) Generally, in the case of WT proteins, equilibrium is shifted toward the folded conformation. Such proteins show high levels of folded expressed protein when expressed on the yeast cell surface and bound to their cognate partner. However, upon introduction of a (B) parent inactive mutation (PIM), the equilibrium is shifted toward the unfolded state and the extent of equilibrium shift will be determined by the destabilizing effect of the PIM. Such PIMs show a lower level of expressed as well as folded proteins on the yeast cell surface. (C) The equilibrium between folded and unfolded states of the PIM can be shifted toward the folded state if a suppressor mutation is introduced. Such double mutants show higher levels of folded expressed proteins on yeast cell surface compared to the PIM alone. (D) Such suppressor mutants generally have a higher amount of folded fraction at the equilibrium than the WT protein. However, the amount of expressed protein on the yeast cell surface will be similar to WT. Normalized MFI of suppressor mutant (E) expression and (F) binding of individually analyzed putative suppressors in the background of the PIM. The MFI of double mutants was normalized with the MFI of PIM, and a twofold cutoff was used to differentiate between putative suppressors and non-suppressor mutations. (G) ΔT_m of purified single mutants identified from suppressor analysis, measured by nano-DSF. The mutants were categorized into stabilized, WT-like, and destabilized mutants indicated by light gray, dark gray, and back bars, respectively. Binding MFI of double mutants relative to PIM (E) robustly identifies stabilizing suppressors. (H) ΔT_m of multi-mutants which were generated by combining multiple stabilizing mutations, purified and in vitro characterized for thermal stability by nano-DSF. Details of mutations present in multi-mutants are provided in the Table 1 . (I) mRBD1-3.2 has A348P, Y365W, and P527L mutations. The positions of A348 and Y365 are highlighted in green color. The C-terminal residues (521–526) are highlighted in red color indicating the absence of the electron density of residue 527 in PDB 6M0J. These stabilizing mutations are distant from the receptor-binding motif, highlighted in blue color. (J) Neutralizing antibodies can bind to different regions of RBD. The binding epitopes of CR3022, S309, and ACE2 are highlighted by green, red, and blue, respectively.