Structural dissection of alkaline‐denatured pepsin

Issue title: Second International Conference on Biomedical Spectroscopy: From the Bench to the Clinic, London, UK, 5–8 July, 2003

Article type: Research Article

Authors: Kamatari, Yuji O.^{; ;} | Dobson, Christopher M.^; | Konno, Takashi

Affiliations: Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, OX1 3QT, United Kingdom | Cellular Signaling Laboratory, RIKEN Harima Institute, 1‐1‐1 Kouto, Mikazuki‐cho, Sayo‐gun, Hyogo, 679‐5148, Japan | Department of Physiology, Fukui Medical University, Yoshida, Fukui, 910‐1193, Japan

Note: [] Corresponding author: Dr. Yuji O. Kamatari, Cellular Signaling Laboratory, RIKEN Harima Institute, 1‐1‐1 Kouto, Mikazuki‐cho, Sayo‐gun, Hyogo, 679‐5148, Japan. Tel.: +81 791 58 2838/0802, ext. 3357; Fax: +81 791 58 2835; E‐mail: [email protected].

Note: [] Present address: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, United Kingdom.

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.

Keywords: Pepsin, zymogen, denaturation, partially folded state, limited proteolysis, NMR, SAXS

Journal: Spectroscopy, vol. 18, no. 2, pp. 227-236, 2004