title: Understanding the mechanism of prosegment-catalyzed folding by solution NMR spectroscopy.
abstract: Multidomain protein folding is often more complex than a two-state process, which leads to the spontaneous folding of the native state. Pepsin, a zymogen-derived enzyme, without its prosegment (PS), is irreversibly denatured and folds to a thermodynamically stable, non-native conformation, termed refolded pepsin, which is separated from native pepsin by a large activation barrier. While it is known that PS binds refolded pepsin and catalyzes its conversion to the native form, little structural details are known regarding this conversion. In this study, solution NMR was used to elucidate the PS-catalyzed folding mechanism by examining the key equilibrium states, e.g. native and refolded pepsin, both in the free and PS-bound states, and pepsinogen, the zymogen form of pepsin. Refolded pepsin was found to be partially structured and lacked the correct domain-domain structure and active-site cleft formed in the native state. Analysis of chemical shift data revealed that upon PS binding refolded pepsin folds into a state more similar to that of pepsinogen than to native pepsin. Comparison of pepsin folding by wild-type and mutant PSs, including a double mutant PS, indicated that hydrophobic interactions between residues of prosegment and refolded pepsin lower the folding activation barrier. A mechanism is proposed for the binding of PS to refolded pepsin and how the formation of the native structure is mediated.
authors.affiliation: From the Departments of Physics and.
authors.forename: Shenlin
authors.initials: S
authors.lastname: Wang
authors.forename: Yasumi
authors.initials: Y
authors.lastname: Horimoto
authors.forename: Derek R
authors.initials: DR
authors.lastname: Dee
authors.forename: Rickey Y
authors.initials: RY
authors.lastname: Yada
mesh_terms: D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D009154:Mutation; D010434:Pepsin A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020116:Pepsinogen A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D058849:Protein Refolding; D017434:Protein Structure, Tertiary
chemical_list: D010446:Peptide Fragments; D020116:Pepsinogen A; D010434:Pepsin A
keywords: Aspartic Protease; NMR; Peptide Interactions; Peptides; Prosegment; Protein Folding