Mechanistic comparison of artificial-chaperone-assisted and unassisted refolding of urea-denatured carbonic anhydrase B
| Autor: | Peter Hanson, Samuel H. Gellman |
|---|---|
| Rok vydání: | 1998 |
| Předmět: |
Protein Denaturation
Protein Folding Kinetics Biochemistry refolding 03 medical and health sciences chemistry.chemical_compound detergent artificial chaperone Animals Urea Carbonic Anhydrases 030304 developmental biology chemistry.chemical_classification 0303 health sciences Cyclodextrin biology 030302 biochemistry & molecular biology Cationic polymerization food and beverages carbonic anhydrase B Small molecule Enzyme cyclodextrin chemistry Chaperone (protein) biology.protein Molecular Medicine Cattle Protein folding Lysozyme Molecular Chaperones |
| Zdroj: | Folding and Design. 3:457-468 |
| ISSN: | 1359-0278 |
| DOI: | 10.1016/s1359-0278(98)00063-7 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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