Figure 1.
Zymogen-derived proteins deviate from common trends in protein folding.
(A) Comparison of the non- and PS-catalyzed folding of αLP [8], SGPB [5] and pepsin [7]. (B) Relation between topology and folding rate for a number of two- and three-state folding proteins (circles, data taken from [3], [21], [22]). The folding rate of αLP (squares), SGPB (triangles) and pepsin (stars) is accelerated to the value (hollow points) expected based on the topology, only when the PS is included. (C) Reaction scheme of pepsin PS-catalyzed folding. The PS binds Rp and catalyzes its conversion to Np at pH 5.3, where the PS is a strong inhibitor of Np. The PS dissociates from Np at pH<3.
Figure 2.
Effects of PS point mutants on binding and catalyzing pepsin folding.
(A) Structure of pepsinogen (PDB code: 3PSG) with the PS (pink) located between the N- and C-terminal lobes, forming part of a six-stranded β-sheet, and K36 of the PS interacts with the catalytic residues, D32 and D215 (red). PS residues selected for mutation to Ala are shown in space-filling form and coloured according to type (grey-hydrophobic, orange-polar, blue-basic, red-acidic). (B) Comparison of wild-type and mutant PS-catalyzed folding of pepsin. The rate of PS-catalyzed folding (kf) was determined by adding PS to Rp, at pH 5.3, 15°C (see Text S2: folding rate followed Arrhenius temp-dependence from 0—15°C, shown in Fig S5), and measuring the formation of Np based on enzyme activity measured at pH 1.2, 25°C. The data were fit according to a monoexponential function to obtain kf. (C) Comparison of wild-type and mutant PS affinity for Rp. PS-Rp binding was determined by following the increase in Trp-fluorescence of pepsin as a function of [PS]. The data were fit according to eq 1 to determine the dissociation constant, Kd, at 20°C, pH 5.3. (d) Comparison of wild-type and mutant PS affinity for Np. The reduction in Np activity was measured as a function of [PS]. The data were fit according to a competitive inhibitor model, eq 2, to determine the inhibition (dissociation) constant, Ki, at 20°C, pH 5.3. All data are reported as the average ± SD of 3-5 measurements for each PS peptide.
Table 1.
Changes in binding and folding constantsa and associated free energiesb upon mutation of the PS.
Figure 3.
Changes in the PS-catalyzed folding energy landscape upon mutation of the PS peptide.
(A) The changes in energy of each conformation were determined as changes in binding energies. (B) Φ-values calculated from the ratio of the changes in activation energy (ΔΔG‡) and free energy difference between PS-Np and PS-Rp (ΔΔGPS(Np-Rp)). Error bars show ± SD derived by propagation of errors.
Figure 4.
A comparison of the mutation effects on the folding activation energy as a function of the change in equilibrium stability. Dashed lines indicate the trend lines for ΔΔG values that would give rise to Φ-values of 0 or 1 and error bars show ± SD.