Artificial chaperone-assisted refolding of carbonic anhydrase B.
We recently reported a new approach to protein refolding that utilizes a pair of low molecular weight folding assistants, a detergent and a cyclodextrin (Rozema, D., and Gellman, S. H. (1995) J. Am. Chem. Soc. 117, 2373-2374). Here, we provide a detailed study of carbonic anhydrase B (CAB) refolding assisted by these "artificial chaperones." When CAB is heated in the presence of a competent detergent, or when guanidinium-denatured CAB is diluted to nondenaturing guanidinium concentration in the presence of such a detergent, the detergent forms a complex with the non-native protein, thereby preventing aggregation. CAB is unable to refold from the detergent-complexed state, but folding can be induced by introduction of a cyclodextrin, which strips the detergent away from the protein. Use of artificial chaperones provides excellent yields of reactivated CAB under conditions that lead to little or no reactivation in the absence of the refolding assistants. Our studies show that the detergent can capture the unfolded protein even at submicellar concentrations, but that not all CAB-detergent complexes lead efficiently to refolded enzyme upon introduction of the stripping agent. Effective refolding appears to require that detergent stripping occur as rapidly as possible; intrinsically slow methods of detergent removal (dialysis or use of macroscopic adsorbents) are less effective than cyclodextrin at inducing renaturation upon detergent removal. The detailed characterization of artificial chaperone-assisted CAB refolding reported here should guide the application of this strategy to other proteins.
Rozema
D
D
Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Gellman
S H
SH
D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000378:metabolism*; D000077286:Cetrimonium; D002593:Cetrimonium Compounds; D018833:Chaperonins; D002942:Circular Dichroism; D003505:Cyclodextrins; D003902:Detergents; D019791:Guanidine; D006146:Guanidines; D006358:Hot Temperature; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012967:Sodium Dodecyl Sulfate; D013816:Thermodynamics; D047392:beta-Cyclodextrins
D002593:Cetrimonium Compounds; D003505:Cyclodextrins; D003902:Detergents; D006146:Guanidines; D047392:beta-Cyclodextrins; D012967:Sodium Dodecyl Sulfate; D018833:Chaperonins; D002256:Carbonic Anhydrases; D019791:Guanidine; C031215:betadex; D000077286:Cetrimonium