%0 Journal Article
%T Structural dissection of alkaline-denatured pepsin
%A Yuji O. Kamatari
%A Christopher M. Dobson
%A Takashi Konno
%J Spectroscopy: An International Journal
%D 2004
%I Hindawi Publishing Corporation
%R 10.1155/2004/769354
%X 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.
%U http://www.hindawi.com/journals/spectroscopy/2004/769354/abs/