Mechanistic comparison of artificial-chaperone-assisted and unassisted refolding of urea-denatured carbonic anhydrase B.
BACKGROUND
We have previously described a method for the refolding of chemically denatured proteins in which small molecules ('artificial chaperones', a detergent and cyclodextrin) assist renaturation. In a previous analysis of lysozyme refolding from the GdmCl-denatured, DTT-reduced state, we found that enzymatic activity is regained at indistinguishable rates for unassisted (absence of additives) and artificial-chaperone-assisted refolding. While unassisted and artificial-chaperone-assisted refolding rates could also be directly compared for GdmCl-denatured bovine carbonic anhydrase B (CAB), only cationic detergents could be used as assistants. We therefore set out to determine whether artificial chaperones could assist the refolding of urea-denatured CAB, whether the charge and structure of the detergent used affects refolding assistance, and, if so, whether the assistance is mechanistically similar to that observed for GdmCl-denatured CAB.


RESULTS
Our results indicate that CAB can be refolded from the urea-denatured state via the artificial chaperone process, using both anionic and cationic detergents. There is a distinctive product-determining step early in the artificial-chaperone-assisted refolding mechanism, but the rate-determining steps of the unassisted and artificial-chaperone-assisted processes are indistinguishable.


CONCLUSIONS
Because the rate-determining steps of unassisted and artificial-chaperone-assisted refolding are indistinguishable, we conclude that the rate-determining step of CAB refolding is unaffected by the use of artificial chaperones. Our observations also suggest that denatured CAB undergoes a slow partial folding in concentrated urea solution.
Hanson
P E
PE
Department of Chemistry, University of Wisconsin, Madison 53706-1396, USA.
Gellman
S H
SH
D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry*; D002417:Cattle; D007700:Kinetics; D018832:Molecular Chaperones / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D014508:Urea
D018832:Molecular Chaperones; D014508:Urea; D002256:Carbonic Anhydrases