Evolutionary adaptation of the protein folding pathway for secretability

A

Highly stabilized native contacts that are dissimilar between PpiA and PpiB are indicated and labelled as spheres (C_α, Fig 4C) on the 3D structure of PpiB (PDB 1LOP) and PpiA (PDB 1V9T) together with the initial foldons coloured (data from 4°C, Fig 3).

B

Mutant derivatives of PpiA (orange) or PpiB (green) with grafted residues from PpiB and PpiA (labelled PpiA>B and PpiB>A, respectively, Fig 4E) are displayed as mutations as squares below the linear map of secondary structure (PpiB, green; PpiA, orange) with annotations at the bottom.

C

Mutational differences dddG values from in silico mutational scanning using Rosetta cartesian‐ddG application. dddGs are subtracted residue‐wise ddG values of PpiA and PpiB to compare the stability of residues between proteins.

D

Equilibrium constant K₁ of the refolding PpiB>A and PpiA>B derivatives at 4°C between the unfolded and intermediate state are shown as bar plots in rows for the grafted triplets (T) and sixplet (S) mutants compared with the wildtype (WT) proteins (calculated from Fig 4E). n = 2–4 biological repeats, s.d.

E

In vivo protein expression in the E. coli strain MC4100 at 30°C during in vivo secretion assay detected by immunostaining with α‐PhoA antibodies on western blots (Fig 4F, See Materials and Methods, PhoA secretion activity in Dataset EV9B). ppiX‐phoA fusions carried on vector pBAD501 (ara promoter) were expressed in the cell (13.3 μM arabinose) to monitor PpiX secretion in the presence of secY _prlA4 EG encoded on plasmid pET610 (lac promoter; expressed with 0.05 mM IPTG). Expression of secY _prlA4 is required for secretion of proteins that have no signal peptide (Derman et al, 1993). Left, purified PhoA protein loaded at the indicated amounts was used for quantification of protein expression (Dataset EV9).

Figure EV4. Grafting of native contacts between PpiA and PpiB (related to Fig 4).