Foldase and inhibitor functionalities of the pepsinogen prosegment are encoded within discrete segments of the 44 residue domain.

Pepsin is initially produced as the zymogen pepsinogen, containing a 44 residue prosegment (PS) domain. When folded without the PS, pepsin forms a thermodynamically stable denatured state (refolded pepsin, Rp). To guide native folding, the PS binds to Rp, stabilizes the folding transition state, and binds tightly to native pepsin (Np), thereby driving the folding equilibrium to favor the native state. It is unknown whether these functionalities of the PS are encoded within the entire sequence or within discrete segments. PS residues 1p-29p correspond to a highly conserved region in pepsin-like aspartic proteases and we hypothesized that this segment is critical to PS-catalyzed folding. This notion was tested in the present study by characterizing the ability of various truncated PS peptides to bind Rp, catalyze folding from Rp to Np, and to inhibit Np. Four PS truncations were examined, corresponding to PS residues 1p-16p (PS1-16), 1p-29p (PS1-29), 17p-44p (PS17-44) and 30p-44p (PS30-44). The three PS functionalities could be ascribed primarily to discrete regions within the highly conserved motif: 1p-16p dictated Rp binding, 17p-29p dictated Np binding/inhibition, while the entire 1p-29p dictated transition state binding/catalyzing folding. Conversely, PS30-44 played no obvious role in PS-catalyzed folding; it is hypothesized that this more variable region may serve as a linker between PS1-29 and the mature domain. The high sequence conservation of PS1-29 and its role in catalyzing pepsin folding strongly suggest that there is a conserved PS-catalyzed folding mechanism shared by pepsin-like aspartic proteases with this motif.

Dynamics of thermodynamically stable, kinetically trapped, and inhibitor-bound states of pepsin.

The pepsin folding mechanism involves a prosegment (PS) domain that catalyzes folding, which is then removed, resulting in a kinetically trapped native state. Although native pepsin (Np) is kinetically stable, it is irreversibly denatured due to a large folding barrier, and in the absence of the PS it folds to a more thermodynamically stable denatured state, termed refolded pepsin (Rp). This system serves as a model to understand the nature of kinetic barriers and folding transitions between compact states. Quasielastic neutron scattering (QENS) was used to characterize and compare the flexibility of Np, as a kinetically trapped state, with that of Rp, as a thermodynamically stable fold. Additionally, the dynamics of Np were compared with those of a partially unfolded form and a thermally stabilized, inhibitor-bound form. QENS revealed length-scale-dependent differences between Np and Rp on a picosecond timescale and indicated greater flexibility in Np, leading to the conclusion that kinetic stabilization likely does not correspond to reduced internal dynamics. Furthermore, large differences were observed upon inhibition, indicating that QENS of proteins in solution may prove useful for examining the role of conformational entropy changes in ligand binding.