Mechanism of insulin chain combination. Asymmetric roles of A-chain alpha-helices in disulfide pairing.
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.
Department of Biochemistry, Case Western Reserve School of Medicine, Cleveland, Ohio 44106-4935, USA.
Qing-Xin
QX
Hua
Ying-Chi
YC
Chu
Wenhua
W
Jia
Nelson F B
NF
Phillips
Run-Ying
RY
Wang
Panayotis G
PG
Katsoyannis
Michael A
MA
Weiss
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004220:Disulfides / Q000032:analysis*; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives* / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D004220:Disulfides; D007328:Insulin; D010446:Peptide Fragments