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Identification of the ubiquitin interfacial residues in a ubiquitin-E2 covalent complex

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Abstract

One of the key intermediates formed during the protein ubiquitination cycle is a covalent complex between ubiquitin (Ub) and the conjugation enzyme, UBC1. In order to probe the interface between these two proteins we have formed the covalent complex in situ (in the NMR tube) using Ub, the catalytic domain of UBC1, UBC1Δ450, an activation enzyme, E1, and Mg2+-ATP. The size of the Ub-UBC1Δ450 complex (25 kDa) and its relatively short lifetime (∼ 4 h) makes assignment of the backbone resonances in the covalent species difficult. In order to monitor the formation and identify the interface in the complex we have used fast 1H-15N HSQC spectra to monitor the decay of 1H-15N correlations as a function of time until the complex formed reached about 90%. The residual peak intensities were used to probe the surface of interaction between Ub and UBC1Δ450 and provided a clear surface of interaction on Ub.

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Authors and Affiliations

  1. Department of Biochemistry, University of Alberta, Edmonton, AB, Canada, T6G 2H7

    Katherine S. Hamilton & Michael J. Ellison

  2. Department of Biochemistry and McLaughlin Macromolecular Structure Facility, University of Western Ontario, London, ON, Canada, N6A 5C1

    Gary S. Shaw

Authors
  1. Katherine S. Hamilton
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  2. Michael J. Ellison
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  3. Gary S. Shaw
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Hamilton, K.S., Ellison, M.J. & Shaw, G.S. Identification of the ubiquitin interfacial residues in a ubiquitin-E2 covalent complex. J Biomol NMR 18, 319–327 (2000). https://doi.org/10.1023/A:1026773008237

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  • DOI: https://doi.org/10.1023/A:1026773008237

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