Structural dissection of alkaline‐denatured pepsin

It has been established in a number of studies that the alkaline‐denatured state of pepsin (the I_P state) is composed of a compact C‐terminal lobe and a largely unstructured N‐terminal lobe. In the present study, we have investigated the residual structure in the I_P state in more detail, using limited proteolysis to isolate and characterize a tightly folded core region from this partially denatured pepsin. The isolated core region corresponds to the 141 C‐terminal residues of the pepsin molecule, which in the fully native state forms one of the two lobes of the structure. A comparative study using NMR and CD spectroscopy has revealed, however, that the N‐terminal lobe contributes a substantial amount of additional residual structure to the I_P state of pepsin. CD spectra indicate in addition that significant nonnative α‐helical structure is present in the C‐terminal lobe of the structure when the N‐terminal lobe of pepsin is either unfolded or removed by proteolysis. This study demonstrates that the structure of pepsin in the I_P state is significantly more complex than that of a fully folded C‐terminal lobe connected to an unstructured N‐terminal lobe.