Previous functional studies have proposed that solution-phase loading of human insulin A-chain peptides into cell surface Class II molecules may be limited by the redox state of intrinsic cysteine residues within the A-chain peptide. T cell functional studies of a human insulin A-chain analogue (KR A1-15) comprised of residues 1-15 of the A-chain peptide as well as an amino-terminal lysinearginine extension have been carried out in a reducing environment. These data suggest that free thiol moieties within this peptide may participate in major histocompatibility complex (MHC) II/ peptide interactions. Two-dimensional 1 H NMR spectroscopy data partnered with quantum chemical calculations identified that KR A1-15 exists in a conformational flux sampling heterogeneous redox dependent conformations including: one reduced and two oxidized states. These findings were further supported by mass spectrometry analysis of this peptide that confirmed the presence of a redox state dependent conformational equilibrium. Interestingly, the presence of a free thiol ( 1 H γ ) resonance for cysteine 8 in the oxidized state supports the existence of the third redox dependent conformation represented as a mixed disulfide conformation. We believe these data support the presence of a redoxdependent mechanism for reg ulating the a ctivity of huma n insulin and provides a better understanding of redox chemistry that may be extended to other protein systems.
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