Comparison of solution structures and stabilities of native, partially unfolded and partially refolded pepsin

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DOIResolve DOI: http://doi.org/10.1021/bi061270i
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TypeArticle
Journal titleBiochemistry
ISSN0006-2960
Volume45
Issue47
Pages1398213992; # of pages: 11
AbstractA zymogen-derived protein, pepsin, appears to be incapable of folding to the native state without the presence of the prosegment. To better understand the nature of the irreversible denaturation of pepsin, the present study reports on the characterization of the stability and low-resolution tertiary and secondary structures of native, alkaline unfolded and acid refolded porcine pepsin. Through a combination of small-angle neutron scattering (SANS), CD, and DSC, acid refolded pepsin (Rp) was shown to have secondary and tertiary structures intermediate between the alkaline denatured and native forms but was found to be thermodynamically stable relative to the native state. It was also observed that the acid refolded state of pepsin was dependent on the protein concentration during refolding because CD and SANS data revealed that both the secondary and tertiary structures of concentrated-refolded pepsin (>10 mg/mL) (CRp) were native-like, in contrast to the intermediate nature of Rp, refolded under dilute concentration (<10 mg/mL). Despite a native-like conformation, CRp was more stable and had substantially reduced activity compared to that of the native state, suggesting that the protein was misfolded. It is proposed that the stable but misfolded, acid-refolded states are evidence that pepsin in its native conformation was metastable. Furthermore, the disruption of the active site cleft in the denatured states could be discerned by modeling of the SANS data.
Publication date
AffiliationNational Research Council Canada; NRC Canadian Neutron Beam Centre
Peer reviewedNo
Identifier10052306
NPARC number12328456
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Record identifier705f429f-8d85-4dd8-8d16-d137d1df0df3
Record created2009-09-10
Record modified2016-05-09
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