The folding nucleus of the insulin superfamily: a flexible peptide model foreshadows the native state.
Oxidative folding of insulin-like growth factor I (IGF-I) and single-chain insulin analogs proceeds via one- and two-disulfide intermediates. A predominant one-disulfide intermediate in each case contains the canonical A20-B19 disulfide bridge (cystines 18-61 in IGF-I and 19-85 in human proinsulin). Here, we describe a disulfide-linked peptide model of this on-pathway intermediate. One peptide fragment (19 amino acids) spans IGF-I residues 7-25 (canonical positions B8-B26 in the insulin superfamily); the other (18 amino acids) spans IGF-I residues 53-70 (positions A12-A21 and D1-D8). Containing only half of the IGF-I sequence, the disulfide-linked polypeptide (designated IGF-p) is not well ordered. Nascent helical elements corresponding to native alpha-helices are nonetheless observed at 4 degrees C. Furthermore, (13)C-edited nuclear Overhauser effects establish transient formation of a native-like partial core; no non-native nuclear Overhauser effects are observed. Together, these observations suggest that early events in the folding of insulin-related polypeptides are nucleated by a native-like molten subdomain containing Cys(A20) and Cys(B19). We propose that nascent interactions within this subdomain orient the A20 and B19 thiolates for disulfide bond formation and stabilize the one-disulfide intermediate once formed. Substitutions in the corresponding region of insulin are associated with inefficient chain combination and impaired biosynthetic expression. The intrinsic conformational propensities of a flexible disulfide-linked peptide thus define a folding nucleus, foreshadowing the structure of the native state.
Hua
Qing-xin
QX
Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106, USA.
Mayer
John P
JP
Jia
Wenhua
W
Zhang
Jingwen
J
Weiss
Michael A
MA
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007334:Insulin-Like Growth Factor I / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D004220:Disulfides; D010446:Peptide Fragments; D007334:Insulin-Like Growth Factor I