Disulfide exchange folding of insulin-like growth factor I.
The disulfide exchange folding properties of insulin-like growth factor I (IGF-I) have been analyzed in a redox buffer containing reduced (10 mM) and oxidized (1 mM) glutathione. Under these conditions, the 3 disulfide bridges of the 70 amino acid peptide were not quantitatively formed. Instead, five major forms of IGF-I were detected, and these components were concluded to be in equilibrium as their relative amounts were similar starting from either reduced, native, or a mismatched variant of IGF-I containing two non-native disulfides. The different components in the mixtures were trapped by thiol alkylation using vinylpyridine and subsequently isolated by reverse-phase HPLC. The purified variants were further characterized using plasma desorption mass spectrometry and peptide mapping. Two of the five different forms were identified as native and mismatched IGF-I. One form was a variant with only one disulfide bond, and the other two major components had two disulfides formed. In a separate experiment, early refolding intermediates were trapped by pyridylethylation after only 90 s of refolding in the glutathione buffer, starting from reduced IGF-I. The intermediates were identical to the components observed at equilibrium, but at different relative concentrations. On the basis of the disulfide bond patterns of the different components in the equilibrium mixtures, we conclude that the disulfide between cysteines-47 and -52 in IGF-I is an unfavorable high-energy bond that may exist in the native molecule in a strained configuration.
Hober
S
S
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Forsberg
G
G
Palm
G
G
Hartmanis
M
M
Nilsson
B
B
D000478:Alkylation; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D005978:Glutathione / Q000737:chemistry; D007334:Insulin-Like Growth Factor I / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000737:chemistry
D000596:Amino Acids; D004220:Disulfides; D013438:Sulfhydryl Compounds; D007334:Insulin-Like Growth Factor I; D005978:Glutathione