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 I human insulin A-chain analogue (KR A1-15) comprised of residues 1-15 of the A-chain peptide as well as all 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 ill 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 coil formation represented as a mixed disulfide conformation. We believe these data support the presence of I 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.