Abstract
The A and B chains of insulin combine to form native disulfide bridges without detectable isomers. The fidelity of chain combination thus recapitulates the folding of proinsulin, a precursor protein in which the two chains are tethered by a disordered connecting peptide. We have recently shown that chain combination is blocked by seemingly conservative substitutions in the C-terminal alpha-helix of the A chain. Such analogs, once formed, nevertheless retain high biological activity. By contrast, we demonstrate here that chain combination is robust to non-conservative substitutions in the N-terminal alpha-helix. Introduction of multiple glycine substitutions into the N-terminal segment of the A chain (residues A1-A5) yields analogs that are less stable than native insulin and essentially without biological activity. (1)H NMR studies of a representative analog lacking invariant side chains Ile(A2) and Val(A3) (A chain sequence GGGEQCCTSICSLYQLENYCN; substitutions are italicized and cysteines are underlined) demonstrate local unfolding of the A1-A5 segment in an otherwise native-like structure. That this and related partial folds retain efficient disulfide pairing suggests that the native N-terminal alpha-helix does not participate in the transition state of the reaction. Implications for the hierarchical folding mechanisms of proinsulin and insulin-like growth factors are discussed.
Highlights
The A and B chains of insulin combine to form native disulfide bridges without detectable isomers
The fidelity of chain combination recapitulates the folding of proinsulin, a precursor protein in which the two chains are tethered by a disordered connecting peptide
Whereas chain combination has enabled the synthesis of many novel insulin analogs [11], including the first commercial recombinant DNA human insulin [12, 13], disulfide pairing can be blocked by specific amino acid substitutions [14, 15]
Summary
Materials—4-Methylbenzhydrylamine resin (0.6 mmol of amine/g; Star Biochemicals, Inc.) was used as solid support for synthesis of A chain analogs; (N-butoxycarbonyl,O-benzyl)-threonine-PAM resin (0.56 mmol/g; Bachem, Inc.) was used as solid support for synthesis of the DKP B chain analog. tert-Butoxycarbonyl-amino acids and derivatives were obtained from Bachem and Peninsula Laboratories; N,NЈ-dicyclohexylcarbodiimide and N-hydroxybenzotriazole (recrystallized from 95% ethanol) were from Fluka. X is the concentration of guanidine hydrochloride, and A and B are base-line values in the native and unfolded states These base lines were approximated by pre- and post-transition lines A(x) ϭ AH2O ϩ mAx and B(x) ϭ BH2O ϩ mBx. Fitting CD data and base lines simultaneously circumvents artifacts associated with linear plots of ⌬G as a function of denaturant according to ⌬Go(x) ϭ ⌬GH o 2O ϩ mBx (for review, see Ref. 36). Activity is defined by affinity for the human placental insulin receptor relative to human insulin (100%); number of assays is given in parenthesis with standard deviations provided Under these conditions the Kd for native insulin is 0.48 Ϯ 0.06 nM. Distance geometry (DG)/ simulated annealing calculations were performed using the program DG-II; restrained molecular dynamics (RMD) calculations were performed using X-PLOR
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