Productive and nonproductive intermediates in the folding of denatured rhodanese.
The competition between protein aggregation and folding has been investigated using rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) as a model. During folding from a urea-denatured state, rhodanese rapidly forms associated species or intermediates, some of which are large and/or sticky. The early removal of such particles by filtration results in a decreased refolding yield. With time, a portion of the smaller aggregates can partition back first to intermediates and then to refolded protein, while a fraction of these irreversibly form unproductive higher aggregates. Dynamic light scattering measurements indicate that the average sizes of the aggregates formed during rhodanese folding increase from 225 to 325 nm over 45 min and they become increasingly heterogeneous. Glycerol addition or the application of high hydrostatic pressure improved the final refolding yields by stabilizing smaller particles. Although addition of glycerol into the refolding mixture blocks the formation of unproductive aggregates, it cannot dissociate them back to productive intermediates. The presence of 3.9 M urea keeps the aggregates small, and they can be dissociated to monomers by high hydrostatic pressure even after 1 h of incubation. These studies suggest that early associated intermediates formed during folding can be reversed to give active species.
Panda
M
M
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284, USA.
Gorovits
B M
BM
Horowitz
P M
PM
D000818:Animals; D002417:Cattle; D005990:Glycerol / Q000494:pharmacology; D006874:Hydrostatic Pressure; D008027:Light; D008956:Models, Chemical; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D012542:Scattering, Radiation; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013884:Thiosulfate Sulfurtransferase / Q000235:genetics / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology
D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate; D014508:Urea; D013884:Thiosulfate Sulfurtransferase; D005990:Glycerol