Oxidative refolding of insulin-like growth factor 1 yields two products of similar thermodynamic stability: a bifurcating protein-folding pathway.
Can one protein sequence encode two structures? Oxidative folding of human insulin-like growth factor 1 (IGF-1), a globular protein of 70 residues, is shown to yield two products of similar thermodynamic stability. This observation is of particular interest in light of the recent demonstration that two of the three disulfide bonds in native IGF-1 rearrange in the presence of dithiothreitol [Hober, S., et al. (1992) Biochemistry 31, 1749-1756]. Kinetics of the IGF-1 folding pathway were monitored by high-performance liquid chromatography (rp-HPLC). Disulfide-pairing schemes of intermediates and products were established by peptide mapping. Two disulfide isomers were obtained as products: one with native insulin-like pairing [6-48; 18-61; 47-52] (designated native IGF-1; 60% yield) and the other with alternative pairing [6-47; 18-61; 48-52] (designated IGF-swap; 40% yield). The predominant early intermediate contains the single disulfide 18-61, which is shared in common by the two products. Relative yields of native IGF-1 and IGF-swap are independent of protein concentration under dilute conditions. In the absence of an added thiol reagent, each isomer is stable indefinitely at neutral pH; in the presence of an added thiol reagent, the two isomers interconvert with an Arrhenius activation barrier of 12 kcal/mol. Interconversion does not require complete reduction and yields the same ratio of products as initial folding, demonstrating thermodynamic control. Spectroscopic studies using circular dichroism (CD), infrared spectroscopy (FTIR), two-dimensional 1H-NMR (2D-NMR), and photochemical dynamic nuclear polarization (photo-CIDNP) suggest that IGF-1 and IGF-swap adopt similar secondary structures but distinct tertiary folds. Implications of these observations for understanding the topology of protein-folding pathways are discussed.
Amgen Inc., Amgen Center, Thousand Oaks, California 91320.
J A
JA
Miller
L O
LO
Narhi
Q X
QX
Hua
R
R
Rosenfeld
T
T
Arakawa
M
M
Rohde
S
S
Prestrelski
S
S
Lauren
K S
KS
Stoney
L
L
Tsai
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D004355:Drug Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007334:Insulin-Like Growth Factor I / Q000737:chemistry*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D013055:Spectrophotometry, Infrared; D013816:Thermodynamics
D004220:Disulfides; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I