title: The prosegment catalyzes pepsin folding to a kinetically trapped native state.
abstract: Investigations of irreversible protein unfolding often assume that alterations to the unfolded state, rather than the nature of the native state itself, are the cause of the irreversibility. However, the present study describes a less common explanation for the irreversible denaturation of pepsin, a zymogen-derived aspartic peptidase. The presence of a large folding barrier combined with the thermodynamically metastable nature of the native state, the formation of which depends on a separate prosegment (PS) domain, is the source of the irreversibility. Pepsin is unable to refold to the native state upon return from denaturing conditions due to a large folding barrier (24.6 kcal/mol) and instead forms a thermodynamically stable, yet inactive, refolded state. The native state is kinetically stabilized by an unfolding activation energy of 24.5 kcal/mol, comparable to the folding barrier, indicating that native pepsin exists as a thermodynamically metastable state. However, in the presence of the PS, the native state becomes thermodynamically stable, and the PS catalyzes pepsin folding by stabilizing the folding transition state by 14.7 kcal/mol. Once folded, the PS is removed, and the native conformation exists as a kinetically trapped state. Thus, while PS-guided folding is thermodynamically driven, without the PS the pepsin energy landscape is dominated by kinetic barriers rather than by free energy differences between native and denatured states. As pepsin is the archetype of a broad class of aspartic peptidases of similar structure and function, and many require their PS for correct folding, these results suggest that the occurrence of native states optimized for kinetic rather than thermodynamic stability may be a common feature of protein design.
authors.affiliation: Biophysics Interdepartmental Group, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
authors.forename: Derek R
authors.initials: DR
authors.lastname: Dee
authors.forename: Rickey Y
authors.initials: RY
authors.lastname: Yada
mesh_terms: D000595:Amino Acid Sequence; D002151:Calorimetry; D002384:Catalysis; D018360:Crystallography, X-Ray; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D020116:Pepsinogen A / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding
chemical_list: D010446:Peptide Fragments; D020116:Pepsinogen A; D010434:Pepsin A