Evaluating the Intrinsic Cysteine Redox-Dependent States of the A-Chain of Human Insulin Using NMR Spectroscopy, Quantum Chemical Calculations, and Mass Spectrometry

Yan Ling, University of Southern Mississippi
Yong Zhang, University of Southern Mississippi

Abstract

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.