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Permanent link (DOI): https://doi.org/10.7939/R3SP5S

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

Descriptions

Other title
Subject/Keyword
protein metal binding
streptavidin biotin cooperativity
blackbody infrared radiative dissociation
gas phase stability
collisional induced dissociation
ion mobility seperation
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Deng, Lu
Supervisor and department
Klassen, John S (Chemistry)
Examining committee member and department
Klobukowski, Mariusz (Chemistry)
Gibbs-Davis, Julianne M (Chemistry)
Cairo, Christopher W (Chemistry)
Douglas, Don J (Chemistry, The University of British Columbia)
Li, Xinfang (Laboratory Medicine and Pathology)
Department
Department of Chemistry
Specialization

Date accepted
2013-03-28T16:02:42Z
Graduation date
2013-06
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
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.
Language
English
DOI
doi:10.7939/R3SP5S
Rights
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication
Deng L, Sun N, Kitova EN, Klassen JS. “Direct Quantification of Protein-Metal Ion Affinities by Electrospray Ionization Mass Spectrometry” Anal. Chem. 2010, 82, 2170 - 2174Deng L, Kitova EN, Klassen JS. “Dissociation Kinetics of the Streptavidin-Biotin Interaction Measured using Direct Electrospray Ionization Mass Spectrometry Analysis” J. Am. Soc. Mass Spectrom. 2013, 24, 49-56Deng L, Broom A, Kitova EN, Richards MR, Zheng RB, Shoemaker GK, Meiering EM, Klassen JS. “Kinetic Stability of the Streptavidin-Biotin Interaction Enhanced in the Gas Phase” J. Am. Chem. Soc. 2012, 134, 16586-16596Deng L, Kitova EN, Klassen JS. “Mapping Protein-Ligand Interactions in the Gas Phase Using a Functional Group Replacement Strategy. Comparison of CID and BIRD Activation Methods” J. Am. Soc. Mass Spectrom. 2013, Submitted for publication

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