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Structural dissection of alkaline-denatured pepsin
Yuji O Kamatari1, Christopher M Dobson, Takashi Konno
1Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, Oxford OX1 3QT, UK. kamatari@spring8.or.jp
Protein Science : a Publication of the Protein Society
|March 22, 2003
Summary
The alkaline-denatured state of pepsin (I(P) state) has more residual structure than previously thought. Further analysis reveals complex structural contributions from both pepsin lobes.
Area of Science:
- Biochemistry
- Structural Biology
- Protein Chemistry
Background:
- The alkaline-denatured state of pepsin (I(P) state) was previously understood as having a compact C-terminal lobe and an unstructured N-terminal lobe.
- This model suggested limited residual structure in the denatured state.
Purpose of the Study:
- To investigate the residual structure in the alkaline-denatured state of pepsin (I(P) state) in greater detail.
- To characterize the structural contributions of pepsin's lobes in this partially denatured state.
Main Methods:
- Limited proteolysis was used to isolate and characterize a core region of pepsin.
- Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD) spectroscopy were employed for comparative structural analysis.
Main Results:
- A tightly folded core region, comprising the C-terminal 141 residues, was isolated.
- NMR and CD spectroscopy revealed significant residual structure in the N-terminal lobe, contrary to previous assumptions.
- CD spectra indicated nonnative alpha-helical structures in the C-terminal lobe when the N-terminal lobe was unfolded or removed.
Conclusions:
- The structure of pepsin in the I(P) state is more complex than a simple model of a folded C-terminal lobe and unstructured N-terminal lobe.
- Both lobes of pepsin contribute substantial residual structure to the I(P) state.
- Nonnative helical structures are present in the C-terminal lobe under specific conditions.