Role of native disulfide bonds in the structure and activity of insulin-like growth factor 1: genetic models of protein-folding intermediates.
Insulin and insulin-related proteins contain three motif-specific disulfide bonds. Here we examine the role of these disulfide bonds in the folding and function of one family member, human insulin-like growth factor 1 (IGF-1). Analogues containing pariwise Cys-->Ser or Cys-->Ala substitutions were expressed in Escherichia coli, purified, and analyzed with respect to receptor-binding, solution structure, and thermodynamic stability. An analogue lacking all three disulfide bonds (designated des-Cys-IGF-1) is inactive and unfolded. Introduction of the [18-61] disulfide bond, previously shown to occur in an early intermediate in oxidative refolding [Miller, J. A., Owers-Narhi, L., Hua, Q. X., Rosenfeld, R., Arakawa, T., Rohde, M., Prestrelski, S., Lauren, S., S. Stoney, K. S., Tsai, L., & Weiss, M. A. (1993) Biochemistry (preceding paper in this issue)], results in a compact partially folded state with low but significant biological activity. Additional but incomplete structural organization and biological activity are observed following introduction of either the [6-48] or the [47-52] disulfide bonds. Native function, structure, and stability require the presence of all three disulfide bonds. These analogues provide genetic models of IGF-1 protein-folding intermediates. Their characterization suggests that bifurcation of the IGF-1 folding pathway reflects alternative late steps in the folding of a molten-globule intermediate.
Amgen Inc., Amgen Center, Thousand Oaks, California 91320.
L O
LO
Narhi
Q X
QX
Hua
T
T
Arakawa
G M
GM
Fox
L
L
Tsai
R
R
Rosenfeld
P
P
Holst
J A
JA
Miller
M A
MA
Weiss
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014443:Tyrosine / Q000737:chemistry
D004220:Disulfides; D012996:Solutions; D014443:Tyrosine; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1