The Length of a Ubiquitin Chain: A General Factor for Selective Recognition by Ubiquitin-Binding Proteins.

The attachment of ubiquitin (Ub) chains of various length to proteins is a prevalent posttranslational modification in eukaryotes. The fate of a modified protein is determined by Ub-binding proteins (UBPs), which interact with Ub chains in a linkage-selective manner. However, the impact and functional consequences of chain length on the binding selectivity of UBPs remain mostly elusive. We have generated Ub chains of defined length and linkage by using click chemistry and GELFrEE fractionation. These defined polymers were used in affinity-based enrichment assays to identify length- and linkage-selective interaction partners on a proteome-wide scale. For the first time, it is revealed that the length of a Ub chain generally has a major impact on its ability to be selectively recognized by UBPs.

Ubiquitin Designer Proteins as a New Additive Generation toward Controlling Crystallization.

Proteins controlling mineralization in vivo are diverse, suggesting that there are various ways by which mineralization can be directed in bioinspired approaches. While well-defined three-dimensional (3D) structures occur in biomineralization proteins, the design of synthetic, soluble, bioinspired macromolecules with specific, reproducible, and predictable 3D arrangements of mineral-interacting functions poses an ultimate challenge. Thus, the question of how certain arrangements of such functions on protein surfaces influence mineralization and in what ways specific alterations subsequently affect this process remains elusive. Here we used genetically engineered ubiquitin (Ub) proteins in order to overcome the limitations of generic bioinspired additive systems. By advancing existing protocols, we introduced an unnatural amino acid and subsequently mineral-interacting functions via selective-pressure incorporation and click chemistry, respectively, without affecting the Ub secondary structure. Indeed, as-obtained Ub with three phosphate functions at defined positions shows unique effects based on a yet-unmatched capability toward the stabilization of a film of a dense liquid mineral phase visible even with the naked eye and its transformation into amorphous nanoparticles and afterward crystals with complex shapes. We thereby demonstrate that Ub designer proteins pose a unique new generation of crystallization additives where the 3D arrangement of mineral-interacting functions can be designed at will, promising their future use for programmable, target-oriented mineralization control.

Artificially Linked Ubiquitin Dimers Characterised Structurally and Dynamically by NMR Spectroscopy.

As one of the most prevalent post-translational modifications in eukaryotic cells, ubiquitylation plays vital roles in many cellular processes, such as protein degradation, DNA metabolism, and cell differentiation. Substrate proteins can be tagged by distinct types of polymeric ubiquitin (Ub) chains, which determine the eventual fate of the modified protein. A facile, click chemistry based approach for the efficient generation of linkage-defined Ub chains, including Ub dimers, was recently established. Within these chains, individual Ub moieties are connected through a triazole linkage, rather than the natural isopeptide bond. Herein, it is reported that the conformation of an artificially K48-linked Ub dimer resembles that of the natively linked dimer, with respect to structural and dynamic characteristics, as demonstrated by means of high-resolution NMR spectroscopy. Thus, it is proposed that artificially linked Ub dimers, as generated by this approach, represent potent tools for studying the inherently different properties and functions of distinct Ub chains.

Identification of Proteins Interacting with Ubiquitin Chains.

Ubiquitylation, the modification of proteins with ubiquitin (Ub), is one of the most versatile post-translational modifications in eukaryotic cells. Since Ub also serves as its own substrate, proteins can be modified by numerous different Ub chains, in which the individual moieties are linked via one or several of the seven lysines of Ub. Homogeneous Ub chains, in which the moieties are sequentially linked via the same residue, have been most extensively studied. However, due to their restricted availability, the functions of Ub chains linked via K27, K29, or K33 are poorly understood. We have developed an approach that, for the first time, allows the generation of all seven homogeneous Ub chains in large quantities. The potential of our approach is demonstrated by the identification of previously unknown interaction partners of K27-, K29-, and K33-linked Ub chains by affinity-based proteomics.