title: Folding disulfide-containing proteins faster with an aromatic thiol.
abstract: The traditional method for in vitro folding of disulfide-containing proteins is slow and involves a redox buffer of glutathione and glutathione disulfide. To increase the folding rate and to gain insight into the folding process, we replaced glutathione, an aliphatic thiol, with a commercially available aromatic thiol, 4-mercaptobenzeneacetate (1). Aromatic thiol 1 was selected due to its enhanced nucleophilicity and its enhanced leaving-group ability relative to glutathione at pH 7.7. To demonstrate the advantages of 1, the folding of reduced and scrambled RNase A at pH 7.0 and 7.7 in the presence of 1 and glutathione was investigated. For each set of folding conditions, the optimum concentration of each thiol was initially determined and then the folding rates in the presence of each thiol were measured concurrently. In all cases examined, the folding rate enhancement with the aromatic thiol was 5- 6-fold. Furthermore, under similar conditions folding rates were almost identical with either reduced or scrambled RNase A. In addition the 5-6-fold folding rate enhancement varied only slightly with pH, 7.0 vs 7.7.
authors.affiliation: Department of Chemistry, Syracuse University, Syracuse, New York 13244, USA.
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Gough
authors.forename: Rhondye H
authors.initials: RH
authors.lastname: Williams
authors.forename: Anthony E
authors.initials: AE
authors.lastname: Donofrio
authors.forename: Watson J
authors.initials: WJ
authors.lastname: Lees
mesh_terms: D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism; D019803:Glutathione Disulfide / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D010648:Phenylacetates / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism
chemical_list: D010648:Phenylacetates; D013438:Sulfhydryl Compounds; D012259:Ribonuclease, Pancreatic; D005978:Glutathione; C456415:4-mercaptobenzeneacetic acid; D019803:Glutathione Disulfide