Figure 2
From: Conformational folding and disulfide bonding drive distinct stages of protein structure formation
Folding of strands C to H drives the formation of the intra-subunit disulfide bond. (A) Inclusion bodies (IB) formed by CRP wt in E. coli cytoplasm were separated and solubilized by 8 M urea. After passing through a desalting column to remove urea, the proteins were incubated in 20 mM Tris, 140 mM NaCl, pH 7.4 at 4 °C overnight (left panel). Alternatively, IB without urea solubilization were incubated overnight (right panel). Samples with or without DTT reduction were analyzed by non-reducing SDS-PAGE and immunoblotting with 3H12 mAb. Oxidized subunits migrated faster than their reduced counterparts14,28. Urea-solubilized CRP subunits in IB were in reduced state, but were oxidized spontaneously following urea removal. (B) Crystal structure of CRP subunit (PDB 1B09). The hydrophobic core of the subunit is composed of a two-layered β sheet comprising strands A to K. Cys36 and Cys97 are located on strands C and H, respectively. They form an intra-subunit disulfide bond that is covered by a.a. 168–176 helix. (C) Secretory CRP mutants with Cys36 and Cys97 mutated to serines and disulfide bonds introduced between the indicated strands were expressed in E. coli cells (Table S1). Two strands were considered to be in close proximity only when the introduced disulfide bond formed more efficiently than that of CRP wt (in periplasm; left panel) or the threshold value (in conditioned media; right panel). The threshold value (indicated by the red dotted line) was assigned as the averaged ratio of disulfide bonding between distant elements plus 3 times S.D. to account for the stochastic oxidation. Strands C to H appeared to be in near native configuration before Cys36-Cys97 bonding. (D) The spontaneous formation of the intra-subunit disulfide bond in purified CRP wt and Δ1-31 mutant with strep tags. The absence of 1–31 (strands A and B) drastically reduced the speed of Cys36-Cys97 bonding. (E) The intra-subunit disulfide bond formation in CRP wt and 168–176 helix mutants secreted into conditioned media by E. coli cells. Mutating D169 and E170 (D169A/E170A), deleting a.a. 168–176 (Δ168-176), or replacing a.a. 168–176 with alanines (168–176 A) all dramatically impaired the intra-subunit disulfide bonding. Data were presented as mean +/− S.E. Each data point represents the mean of data obtained from at least 3 independent experiments.
