Artificial chaperone-assisted refolding of denatured-reduced lysozyme: modulation of the competition between renaturation and aggregation.
Conditions that promote renaturation of an unfolded protein also promote protein aggregation, in many cases, because these competing intramolecular and intermolecular processes are driven by similar networks of noncovalent interactions. The GroEL/GroES system and related biological chaperones facilitate the renaturation of substrate proteins by minimizing the aggregation pathway. We have devised a two-step method in which small molecules, "artificial chaperones," facilitate protein refolding from a chemically denatured state. In the first step, the protein is captured by a detergent as guanidinium chloride is diluted to a non-denaturing concentration; formation of a protein-detergent complex prevents both protein aggregation and proper refolding. In the second step, a cyclodextrin strips detergent from the protein, allowing the protein to refold. Here we describe the first application of this method to a protein that must form disulfides in the native state. Lysozyme (hen egg white) can be refolded from the Gdm-denatured, DTT-reduced state in good yields at final protein concentrations as high as 1 mg/mL with the artificial chaperone method. Several mechanistic aspects of artificial chaperone-assisted refolding have been probed, and a detailed mechanism for the kinetically controlled stripping step is proposed.
Rozema
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Department of Chemistry, University of Wisconsin, Madison 53706, USA.
Gellman
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D000218:Adamantane / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000494:pharmacology; D002645:Chickens; D002942:Circular Dichroism; D003505:Cyclodextrins / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004529:Egg White; D005260:Female; D005453:Fluorescence; D005978:Glutathione / Q000378:metabolism / Q000494:pharmacology; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D018832:Molecular Chaperones / Q000494:pharmacology*; D009113:Muramidase / Q000378:metabolism*; D010636:Phenols / Q000494:pharmacology; D011489:Protein Denaturation; D017510:Protein Folding
D002593:Cetrimonium Compounds; D003505:Cyclodextrins; D003902:Detergents; D006146:Guanidines; D018832:Molecular Chaperones; D010636:Phenols; C080417:4-octylphenol; C012698:adamantanecarboxylic acid; D009113:Muramidase; D005978:Glutathione; D019791:Guanidine; D000218:Adamantane; D004229:Dithiothreitol; D000077286:Cetrimonium