Abstract
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.
Highlights
The vertebrate insulin-related superfamily consists of insulin, insulin-related growth factors (IGF-I and IGF-II),2 [1, 2], relaxin [3,4,5], and relaxin-related factors (6 –9)
As successive disulfide bridges are introduced in these equilibrium models, 1H NMR spectra exhibit a progressive increase in chemicalshift dispersion, suggesting stepwise stabilization of structure [15]
We propose that within the insulin superfamily an (A20 –B19)-related microdomain orients these thiolates for initial disulfide bond formation, stabilizes the one-disulfide intermediate once formed, and provides a platform for non-random
Summary
Its conformational propensities, foreshadowing the structure of the native state, suggest a model for an IGF-I folding nucleus This model is likely to generalize to other members of the insulin-related superfamily, including proinsulin. In the native of a one-disulfide-linked peptide model of the populated states of insulin and IGF-I (Fig. 1) this bridge connects the 18 – 61, IGF-I intermediate [14, 29]. Most chemical shifts are close to random coil values, non-random inter-residue NOEs are observed These define native-like elements of nascent secondary structure and provide evidence for the transient clustering of non-polar side chains. We propose that within the insulin superfamily an (A20 –B19)-related microdomain orients these thiolates for initial disulfide bond formation, stabilizes the one-disulfide intermediate once formed, and provides a platform for non-random
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