1 Ubiquitin (Ub) is a small 76 amino acid protein that is covalently attached to substrates through an isopeptide bond between the C‐terminal glycine of Ub and the ε‐amino group of lysine residues. A PTM of particular interest, due to its involvement in virtually every biochemical pathway, is protein ubiquitination. Understanding the functional consequences of PTMs on a molecular level requires access to modified proteins for structural and biochemical studies-a challenge that is driving research in numerous labs. Post‐translational modifications (PTMs) dramatically expand the functions of proteins. Considering the significant challenges associated with building topologically diverse native Ub chains, our results illustrate that chains harboring non‐native linkages can serve as surrogate substrates for explorations of Ub function. Through steady‐state kinetic analyses, we demonstrate that different families of deubiquitinases hydrolyze native and non‐native isopeptide linkages with comparable efficiency and selectivity.
An important application of non‐native Ub oligomers is to probe the activity and selectivity of deubiquitinases. Moreover, using an experimental structural library and atomistic simulations to fit the experimental SAXS profiles, we find that the two types of Ub dimers can be matched to analogous structures. Using small‐angle X‐ray scattering (SAXS) analysis, we show that scattering profiles for the two types of dimers are similar. Here, we compare the structure and function of Ub dimers bearing native and non‐native linkages.
A number of chemical approaches have been used to generate Ub oligomers connected by non‐native linkages however, few studies have examined the extent to which non‐native linkages recapitulate the structural and functional properties associated with native isopeptide bonds. To understand how distinct Ub chains orchestrate different biochemical events, we and other investigators have developed enzymatic and non‐enzymatic methods to synthesize Ub chains of well‐defined length and connectivity. Ubiquitin (Ub) chains regulate a wide range of biological processes, and Ub chain connectivity is a critical determinant of the many regulatory roles that this post‐translational modification plays in cells.
0 kommentar(er)
