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

Center for Vascular Biology Research, Division of Molecular and Vascular Medicine, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
The Journal of Physical Chemistry B (Impact Factor: 3.3). 12/2009; 114(1):585-91. DOI: 10.1021/jp908729h
Source: PubMed


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.

Download full-text


Available from: David A Hafler
  • [Show abstract] [Hide abstract]
    ABSTRACT: Computational investigations of spectroscopic observables can help many experimental studies and provide an important venue for the structural investigations of proteins. Here we report the first detailed quantum chemical investigation of the hydrogen-bonding effect on Mössbauer spectroscopic properties of metalloproteins, using various active site models of oxymyoglobin. The hydrogen bond between O2 and the distal His residue was found to strengthen the binding of oxygen, highlighting the role of protein environment on its biological function. The hydrogen bonding also entails more FeIII– O2−character. These structural effects result in clear differences in the predicted Mössbauer properties, with those of the lowest energy, hydrogen-bonded, Weiss-type, open-shell singlet state, in best agreement with the experiment. These results suggest that the use of quantum chemical calculations of Mössbauer properties can help identify and assess the effect of hydrogen bonding in the protein active site.
    No preview · Article · Jan 2010 · Annual Reports in Computational Chemistry
  • [Show abstract] [Hide abstract]
    ABSTRACT: The geometries and relative energies of new N,N carbonyl dipyrrinone-derived oxime molecules (E/Z-s-cis 4a and E/Z-s-cis 4b) have been investigated. The calculated energies, molecular geometries, and (1) H/(13) C NMR chemical shifts agree with experimental data, and the results are presented herein. The E-s-cis conformations of 4a and 4b and the Z-s-cis conformation of 5b were found to be the thermodynamically most stable isomers with the oxime hydrogen atom or the methyl functional group adopting an anti-orientation with respect to the dipyrrinone group. This conformation was unambiguously supported by a number of 2D NMR experiments.
    No preview · Article · May 2011 · Magnetic Resonance in Chemistry