Forging isopeptide bonds using thiol-ene chemistry: site-specific coupling of ubiquitin molecules for studying the activity of isopeptidases.

ABSTRACT: Chemical methods for modifying proteins can enable studies aimed at uncovering biochemical function. Herein, we describe the use of thiol-ene coupling (TEC) chemistry to report on the function of branched (also referred to as forked) ubiquitin trimers. We show how site-specific isopeptide (N?-Gly-L-homothiaLys) bonds are forged between two molecules of Ub, demonstrating the power of TEC in protein conjugation. Moreover, we demonstrate that the N?-Gly-L-homothiaLys isopeptide bond is processed to a similar extent by deubiquitinases (DUBs) as that of a native N?-Gly-L-Lys isopeptide bond, thereby establishing the utility of TEC in the generation of Ub-Ub linkages. TEC is then applied to the synthesis of branched Ub trimers. Interrogation of these branched derivatives with DUBs reveals that the relative orientation of the two Ub units has a dramatic impact on how they are hydrolyzed. In particular, cleavage of K48C-linkages is suppressed when the central Ub unit is also conjugated through K6C, whereas cleavage proceeds normally when the central unit is conjugated through either K11C or K63C. The results of this work presage a role for branched polymeric Ub chains in regulating linkage-selective interactions.

SUBMITTER: Valkevich EM 

PROVIDER: S-EPMC4373626 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature