title: Probing the folding pathways of long R(3) insulin-like growth factor-I (LR(3)IGF-I) and IGF-I via capture and identification of disulfide intermediates by cyanylation methodology and mass spectrometry.
abstract: This report describes an integrated investigation of the refolding and reductive unfolding of insulin-like growth factor (IGF-I) and its variant, long R(3) IGF-I (LR(3)IGF-I), which has a Glu(3) to Arg(3) substitution and a hydrophobic 13-amino acid N-terminal extension. The refolding performed in glutathione redox buffer was quenched at different time points by adjusting the pH to 2.0-3.0 with a 1 N HCl solution of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, which trapped intermediates via cyanylation of free sulfhydryl groups. The disulfide structure of the intermediates was determined by chemical cleavage followed by mass mapping with mass spectrometry. Six refolding intermediates of IGF-I and three refolding intermediates of LR(3)IGF-I were isolated and characterized. Folding pathways of IGF-I and LR(3)IGF-I are proposed based on the time-dependent distribution and disulfide structure of the corresponding trapped intermediates. Similarities and differences in the refolding behavior of IGF-I and LR(3)IGF-I are discussed.
authors.affiliation: Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Yang
authors.forename: J
authors.initials: J
authors.lastname: Wu
authors.forename: J T
authors.initials: JT
authors.lastname: Watson
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003486:Cyanides / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000031:analogs & derivatives* / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D003486:Cyanides; D004220:Disulfides; D011994:Recombinant Proteins; C085700:long R(3)-insulin-like growth factor-I; D007334:Insulin-Like Growth Factor I