Comparison of solution structures and stabilities of native, partially unfolded and partially refolded pepsin.
A 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.
Dee
Derek
D
Department of Food Science, University of Guelph, Guelph, ON N1G 2W1, Canada.
Pencer
Jeremy
J
Nieh
Mu-Ping
MP
Krueger
Susan
S
Katsaras
John
J
Yada
Rickey Y
RY
D000818:Animals; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D017666:Deuterium Oxide; D008958:Models, Molecular; D009502:Neutrons; D010434:Pepsin A / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012542:Scattering, Radiation; D013552:Swine; D014867:Water
D014867:Water; D010434:Pepsin A; D017666:Deuterium Oxide