title: Reduction of the C191-C220 disulfide of α-chymotrypsinogen A reduces nucleation barriers for aggregation.
abstract: Proper disulfide formation can be essential for the conformational stability of natively folded proteins. For proteins that must unfold in order to aggregate, disruption of native disulfides may therefore promote aggregation. This study characterizes differences in the aggregation process for wild-type (WT) α-chymostrypsinogen A (aCgn) and the same molecule with one of its native disulfides (C191-C220) reduced to free thiols (aCgnSH) at acidic pH, where WT aCgn forms semi-flexible amyloid polymers. Loss of the disulfide leads to no discernable differences in folded monomer secondary or tertiary structure based on circular dichroism (CD) or intrinsic fluorescence (FL), and causes a small decrease in the free energy change upon unfolding. After unfolding-mediated aggregation, the resulting amyloid morphology and structure are similar or indistinguishable for aCgn and aCgnSH by CD, FL, ThT binding, multi-angle laser light scattering, and transmission electron microscopy. Aggregates of aCgn and aCgnSH are also able to cross-seed with monomers of the other species. However, aggregates of aCgnSH are more resistive than aCgn aggregates to urea-mediated dissociation, suggesting some degree of structural differences in the aggregated species that was not resolvable in detail without higher resolution methods. Mechanistic analyses of aggregation kinetics indicate that the initiation or nucleation of new aggregates from aCgnSH involves a mono-molecular rate limiting step, possibly the unfolding step. In contrast, that for aCgn involves an oligomeric intermediate, suggesting native disulfide linkages help to hinder non-native protein aggregation by providing conformational barriers to key nucleation event(s).
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716, United States.
authors.forename: William F
authors.initials: WF
authors.lastname: Weiss
authors.affiliation: Department of Chemical Engineering, University of Virginia, Charlottesville, VA 22904, United States.
authors.forename: Aming
authors.initials: A
authors.lastname: Zhang
authors.affiliation: Molecular Biology Institute, University of California, Los Angeles, CA 90095, United States.
authors.forename: Magdalena I
authors.initials: MI
authors.lastname: Ivanova
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716, United States.
authors.forename: Erinc
authors.initials: E
authors.lastname: Sahin
authors.affiliation: Department of Chemical Engineering, University of Virginia, Charlottesville, VA 22904, United States.
authors.forename: Jacob L
authors.initials: JL
authors.lastname: Jordan
authors.affiliation: Department of Chemical Engineering, University of Virginia, Charlottesville, VA 22904, United States.
authors.forename: Erik J
authors.initials: EJ
authors.lastname: Fernandez
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716, United States. Electronic address: cjr@udel.edu.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Roberts
mesh_terms: D000682:Amyloid / Q000737:chemistry*; D002919:Chymotrypsinogen / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011487:Protein Conformation; D058767:Protein Unfolding
chemical_list: D000682:Amyloid; D004220:Disulfides; D002919:Chymotrypsinogen
keywords: Aggregate structure; Disulfide bond; Protein aggregation; Protein unfolding