Abstract
Pepsin, a gastric aspartic proteinase, is a zymogen‒derived protein that undergoes irreversible alkaline denaturation at pH 6–7. Detailed knowledge of the structure of the alkaline‒denatured state is an important step in understanding the mechanism of the formation of the active enzyme. It has been established in a number of studies that the alkaline‒denatured state of pepsin (the IP 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 IP 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 IP state of pepsin. CD spectra indicate in addition that significant non‒native 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 IP state is significantly more complex than that of a fully folded C‒terminal lobe connected to an unstructured N‒terminal lobe. The “misfolding” in this state could inhibit the proper refolding of the protein when returned to conditions that stabilize the native state.
Highlights
In order to obtain more detailed structural information on the IP state of pepsin we have carried out experiments designed to isolate the folded core of the molecule, by purifying the material obtained from limited proteolysis of the alkaline denatured
This result strongly supports the conclusion that the IP state has a structure consisting of both of fully folded and highly denatured regions. This proteolytic experiment reveals definitively that the tightly folded fragment corresponds to the C-terminal lobe of pepsin. This finding, together with the similarity of the thermal denaturation curve above 25◦C for the IP state and the C fragment monitored by farUV circular dichroism (CD) spectroscopy (Fig. 6B), indicates that the folded cooperative core of the IP state corresponds primarily to that of the C-terminal lobe of intact pepsin
Analysis of the far-UV CD spectra indicates that the IP state and the C fragment have a larger α-helical and a smaller β-sheet content than that observed in the native state of pepsin (Fig. 6A and Table 2)
Summary
In order to obtain more detailed structural information on the IP state of pepsin we have carried out experiments designed to isolate the folded core of the molecule, by purifying the material obtained from limited proteolysis of the alkaline denatured At pH 8.0, where pepsin is denatured, the spectrum indicates a partial loss of secondary structure and the emergence of intensity characteristic of random coil regions of structure (Fig. 2A, bold solid line; [22,32,35]).
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