Development and Application of ESI-MS Based Techniques to Study Non-Covalent Protein-Ligand Complexes in Solution and the Gas Phase

  • Author / Creator
    Deng, Lu
  • This thesis describes the development and application of electrospray ionization mass spectrometry (ESI-MS) based techniques to study noncovalent protein-ligand complexes in solution and the gas phase.
    The application of the direct ESI-MS assay for quantifying the stoichiometry and absolute affinity of protein-metal ion binding in vitro was described. The direct ESI-MS assay was also used to determine the dissociation rate constants (koff) for the model high affinity interaction between biotin (B) and the homotetramer of natural core streptavidin (S4) at pH 7 and temperatures ranging from 15 to 45 °C. Importantly, the dissociation activation energies determined by ESI-MS agree, within 1 kcal mol-1, with the reported value using a radiolabeled biotin assay. In addition to providing a quantitative measure of koff, the results of the ESI-MS measurements revealed that sequential binding of B to S4 occurs in a non-cooperative fashion with the four ligand binding sites being kinetically and thermodynamically equivalent and independent.
    The structure and stability of the gaseous protonated ions of the (S4 + 4B) complexes were investigated using various experimental and computational methods. Rate constants were determined for the dissociation of (S4 + 4B)n+ ions using time-resolved blackbody infrared radiative dissociation (BIRD). These kinetic data, together with results of ion mobility measurements and molecular dynamics simulations suggest that significant structural changes do not occur upon transfer of the complexes from solution to the gas phase by ESI and at least some of the specific intermolecular interactions are preserved in the gas phase. Comparison of the dissociation kinetics for the gaseous (S4 + 4B)13+ ions with those determined for the (S4 + 4B) complexes in solution provides evidence for a late dissociative transition state and the rehydration of B and protein binding cavity in solution during dissociation. Intermolecular interactions in (S4 + 4B)13+ and (scFv+L1)10+ ions were investigated using a collision-induced dissociation (CID)-functional group replacement (FGR) strategy. Comparison of the results obtained by the CID-FGR approach with those determined by the BIRD-FGR approach suggests that the CID-FGR method does not represent a reliable approach for identifying interactions in the gaseous protein-ligand complexes.

  • Subjects / Keywords
  • Graduation date
    Spring 2013
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
  • Supervisor / co-supervisor and their department(s)
  • Examining committee members and their departments
    • Cairo, Christopher W (Chemistry)
    • Klobukowski, Mariusz (Chemistry)
    • Douglas, Don J (Chemistry, The University of British Columbia)
    • Li, Xinfang (Laboratory Medicine and Pathology)
    • Gibbs-Davis, Julianne M (Chemistry)