Structural dissection of alkaline-denatured pepsin.
It has been established in a number of studies that the alkaline-denatured state of pepsin (the I(P) 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 I(P) 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 I(P) 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 I(P) state is significantly more complex than that of a fully folded C-terminal lobe connected to an unstructured N-terminal lobe.
Kamatari
Yuji O
YO
Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, Oxford OX1 3QT, UK. kamatari@spring8.or.jp
Dobson
Christopher M
CM
Konno
Takashi
T
D000595:Amino Acid Sequence; D002918:Chymotrypsin / Q000378:metabolism; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation / Q000502:physiology*; D013696:Temperature
D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D010434:Pepsin A