Evaluating the intrinsic cysteine redox-dependent states of the A-chain of human insulin using NMR spectroscopy, quantum chemical calculations, and mass spectrometry.

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 lysine-arginine 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 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(gamma)) 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 redox-dependent mechanism for regulating the activity of human insulin and provide a better understanding of redox chemistry that may be extended to other protein systems.