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Molecular basis of ubiquitin chain synthesis and recognition Open Access


Other title
Type of item
Degree grantor
University of Alberta
Author or creator
Markin, Craig J
Supervisor and department
Spyracopoulos, Leo (Department of Biochemistry)
Examining committee member and department
Sykes, Brian (Department of Biochemistry)
Glover, Mark (Department of Biochemistry)
McIntosh, Lawrence (Department of Biochemistry and Molecular Biology and Department of Chemistry, University of British Columbia)
LaPointe, Paul (Department of Cell Biology)
Department of Biochemistry

Date accepted
Graduation date
Doctor of Philosophy
Degree level
K63-linked polyubiquitin is synthesized by the E2 Ubc13, and plays non-degradative roles in immunity and the DNA damage response, yet a full molecular understanding of its synthesis and subsequent recognition remains incomplete. Although previously considered a slow enzyme, we demonstrate that Ubc13 is able to achieve a significant rate enhancement in synthesis of K63-linked Ub2, even in a putative off-state. Using a non-steady state kinetic approach to measure K63-linked polyUb formation, we are able to measure a true, rather than apparent, kcat for Ub2 formation. Accurate knowledge of this value provides mechanistic insight that would otherwise remain obscure. These non-steady-state approaches provide the groundwork for quantitative measurement of the activated E2 in the presence of E3 enzymes, the latter currently thought to function in activating their cognate E2s. In response to DNA double strand breaks, K63-linked polyUb is synthesized on adjacent histones. RAP80 recognizes these chains using ubiquitin interacting motifs (UIMs) and recruits downstream repair proteins. The presence of tandem UIMs and multiple Ub molecules tethered together in the chains leverages multivalency to increase the affinity. We use a novel combination of NMR methods and thermodynamic binding models to dissect these complex interactions to develop a molecular basis for signal amplification through multivalency. For interactions characterized by relatively fast kinetics, NMR is a powerful method for determining binding thermodynamics. We developed two novel methodologies which increase both the accuracy and precision of thermodynamic values obtained from NMR chemical shift titrations. These are sampling schemes in which the concentrations of analyte and titrant are varied simultaneously. Simulations are used to demonstrate the potential for increased accuracy and precision while titrations of Mms2 with ubiquitin demonstrate the experimental feasibility. Whereas chemical shifts provide thermodynamic information, the full line-shapes conceal kinetic information. We demonstrate that classical line-shape analysis allows for determination of kinetics over a broad range of biologically relevant exchange rates, which can be widened using our sampling methods. This provides the opportunity of accurately and precisely quantifying both thermodynamics and kinetics from a single NMR chemical shift titration.
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
C.J. Markin, L.F. Saltibus, and L. Spyracopoulos. Dynamics of the RING domain from human TRAF6 by 15N NMR spectroscopy: implications for biological function. Biochemistry, 47(38):10010-10017, 2008C.J. Markin, W. Xiao, and L. Spyracopoulos. Mechanism for recognition of polyubiquitin chains: balancing affinity through interplay between multivalent binding and dynamics. Journal of the American Chemical Society, 132(32):11247-11258, 2010C.J. Markin, L.F. Saltibus, M.J. Kean, R.T. McKay, W. Xiao, and L. Spyracopoulos. Catalytic proficiency of ubiquitin conjugation enzymes: balancing pKa suppression, entropy, and electrostatics. Journal of the American Chemical Society, 132(50):17775-17786, 2010C.J. Markin, and L. Spyracopoulos. Increased precision for analysis of protein-ligand dissociation constants determined from chemical shift titrations. Journal of Biomolecular NMR, 53(2): 125-138, 2012C.J. Markin, and L. Spyracopoulos. Accuracy and precision of protein-ligand interaction kinetics determined from chemical shift titrations. Journal of Biomolecular NMR, 54(4): 355-376, 2012

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