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A Rapid and Versatile Method for Generating Proteins with Defined Ubiquitin Chains

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Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas 78712, United States
Department of Molecular Biosciences, Northwestern University, Evanston, Illinois 60208, United States
*E-mail: [email protected]. Phone: 512-232-4045.
Cite this: Biochemistry 2016, 55, 12, 1898–1908
Publication Date (Web):March 4, 2016
https://doi.org/10.1021/acs.biochem.5b01310
Copyright © 2016 American Chemical Society

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    Abstract

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    Ubiquitin and polyubiquitin chains target proteins for a wide variety of cellular processes. Ubiquitin-mediated targeting is regulated by the lysine through which the ubiquitins are linked as well as the broader ubiquitin landscape on the protein. The mechanisms of this regulation are not fully understood. For example, the canonical proteasome targeting signal is a lysine 48-linked polyubiquitin chain, and the canonical endocytosis signal is a lysine 63-linked polyubiquitin chain. However, lysine 63-linked polyubiquitin chains can also target substrates for degradation. Biochemical studies of ubiquitinated proteins have been limited by the difficulty of building proteins with well-defined polyubiquitin chains. Here we describe an efficient and versatile method for synthesizing ubiquitin chains of defined linkage and length. The synthesized ubiquitin chains are then attached to any protein containing a ubiquitin moiety. These proteins can be used to study ubiquitin targeting in in vitro assays in the tightly controlled manner required for biochemical studies.

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.5b01310.

    • Materials and methods and results for ubiquitin chain conformation by mass spectrometry, a gel demonstrating that GFP proteins run as two species unboiled folded and denatured (Figure S1), amino acid sequences of target proteins to which ubiquitin chains are attached (Table S1), mass spectrometric conformations of ubiquitin chain linkages (Table S2), and yields of ubiquitin chain synthesis and attachment reactions (Table S3) (PDF)

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