Disulfide exchange folding of disulfide mutants of insulin-like growth factor I in vitro.
We have previously concluded that insulin-like growth factor-I (IGF-I) is thermodynamically unable to quantitatively form its disulfide bonds under reversible redox conditions in vitro. From detailed analyses it was hypothesized that the 47-52 disulfide is energetically unfavorable in the native IGF-I structure [Hober et al. (1992) Biochemistry 31, 1749-1756]. In this paper, this hypothesis has been tested by refolding of IGF-I mutant proteins lacking either the 47-52 or 6-48 disulfide bond. The disulfide exchange folding equilibrium behavior of these mutated IGF-I variants were examined in a glutathione redox buffer. The mutant protein IGF-I(C47A,C52A) was demonstrated to form both remaining native disulfide bonds. In contrast, IGF-I(C6A,C48A) was unable to quantitatively form both of its disulfides and was shown to accumulate a one disulfide variant lacking the 47-52 disulfide bond. These folding data corroborate the hypothesis that the 47-52 disulfide bond of IGF-I is energetically unfavorable also in the absence of the 6-48 disulfide bond. The two IGF-I variants were purified in oxidized forms where both native disulfides are formed. Both variants were suggested to be structurally perturbed compared with the native molecule as determined by circular dichroism spectroscopy. Further, binding affinities to the IGF binding protein 1 and a soluble IGF type I receptor, respectively, were severely lowered in both disulfide mutant proteins compared to the native IGF-I molecule. Interestingly, the binding affinity toward the IGF type I receptor is higher for IGF-I(C6A,C48A) than for IGF-I(C47A,C52A) while the binding affinity to IGFBP-1 is higher for IGF-I(C47A,C52A) than for IGF-I(C6A,C48A). Thus, the structural changes due to removal of the 6-48 or 47-52 disulfide bonds, respectively, yield structural changes in different regions of the IGF-I molecule reflected in the different binding activities.
Hober
S
S
Department of Biology, Pharmacia & Upjohn, Stockholm, Sweden.
Uhlén
M
M
Nilsson
B
B
D000409:Alanine / Q000235:genetics; D002942:Circular Dichroism; D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000737:chemistry*; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000737:chemistry; D007334:Insulin-Like Growth Factor I / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D017510:Protein Folding; D017526:Receptor, IGF Type 1 / Q000737:chemistry
D004220:Disulfides; D018970:Insulin-Like Growth Factor Binding Protein 1; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1; D003545:Cysteine; D000409:Alanine