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Protein Science (2003), 12:717-724.
Copyright © 2003 The Protein Society

Structural dissection of alkaline-denatured pepsin

Yuji O. Kamatari1,3, Christopher M. Dobson1,4 and Takashi Konno2

1 Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, Oxford OX1 3QT, UK
2 Department of Physiology, Fukui Medical University, Yoshida, Fukui 910-1193, Japan

Reprint requests to: Yuji O. Kamatari, Cellular Signaling Laboratory, RIKEN Harima Institute, 1-1-1 Kouto, Mikazuki-cho, Sayo-gun, Hyogo 679-5148, Japan; e-mail: kamatari{at}spring8.or.jp; fax: 81-791-58-2835.

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 nonnative {alpha}-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.

Keywords: Pepsin; zymogen; denaturation; partially folded state; limited proteolysis

Abbreviations: CD, circular dichroism • UV, ultraviolet • NMR, nuclear magnetic resonance • ppm, parts per million • DSS, 2,2-dimethyl-2-silapentane-5-sulfonic acid • IP, the alkaline-denatured state of pepsin at pH 8.0 and 25°C • C fragment, the pepsin fragment designated by {alpha}-chymotrypsin and chromatographically purified • {alpha}LP, {alpha}-lytic protease • TLCK, N-{alpha}-p-tosyl-L-lysine chloromethyl ketone hydrochloride • SDS, sodium dodecylsulfate • PAGE, polyacrylamide gel electrophoresis • HPLC, high performance liquid chromatography


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