Targeted Preparation and NMR Spectroscopic Characterization of Lys11‐Linked Ubiquitin Trimers

Abstract

Ubiquitylation refers to the attachment of mono‐ or poly‐ubiquitin molecules to a substrate protein. To shield ubiquitin chains against potential hydrolysis, a facile, click‐chemistry based approach was recently established for the generation of site‐specifically conjugated ubiquitin dimers relying on triazole‐linkage. Here, the preparation of such ubiquitin chains was advanced by the generation of homotypic Lys11‐linked ubiquitin trimers considering an isotopic labeling scheme in a moiety‐wise manner. The structural and dynamical impact on the ubiquitin unit at proximal, central, or distal position that is potentially invoked by the respective other two moieties was systematically probed by heteronuclear high‐resolution NMR spectroscopic approaches. As a result, conjugating a third ubiquitin moiety to the proximal or distal site of a ubiquitin dimer does not alter structural and dynamical characteristics as it has been seen for ubiquitin dimers. This observation suggests that recognition of a homotypically assembled ubiquitin chain by a potential substrate is primarily done by screening the length of a ubiquitin chain rather than relying on subtle changes in structure or dynamic properties of single ubiquitin moieties composing the chain.