title: Unassisted refolding of urea unfolded rhodanese.
abstract: In vitro refolding after urea unfolding of the enzyme rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) normally requires the assistance of detergents or chaperonin proteins. No efficient, unassisted, reversible unfolding/folding transition has been demonstrated to date. The detergents or the chaperonin proteins have been proposed to stabilize folding intermediates that kinetically limit folding by aggregating. Based on this hypothesis, we have investigated a number of experimental conditions and have developed a protocol for refolding, without assistants, that gives evidence of a reversible unfolding transition and leads to greater than 80% recovery of native enzyme. In addition to low protein concentration (10 micrograms/ml), low temperatures are required to maximize refolding. Otherwise optimal conditions give less than 10% refolding at 37 degrees C, whereas at 10 degrees C the recovery approaches 80%. The unfolding/refolding phases of the transition curves are most similar in the region of the transition, and refolding yields are significantly reduced when unfolded rhodanese is diluted to low urea concentrations, rather than to concentrations near the transition region. This is consistent with the formation of "sticky" intermediates that can remain soluble close to the transition region. Apparently, nonnative structures, e.g. aggregates, can form rapidly at low denaturant concentrations, and their subsequent conversion to the native structure is slow.
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
authors.forename: J A
authors.initials: JA
authors.lastname: Mendoza
authors.forename: E
authors.initials: E
authors.lastname: Rogers
authors.forename: G H
authors.initials: GH
authors.lastname: Lorimer
authors.forename: P M
authors.initials: PM
authors.lastname: Horowitz
mesh_terms: D066298:In Vitro Techniques; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D013696:Temperature; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D014508:Urea / Q000737:chemistry
chemical_list: D014508:Urea; D013884:Thiosulfate Sulfurtransferase