An official website of the United States government
Abstract Protein‐polymer bioconjugates present a way to make enzymes more efficient and robust for industrial and medicinal applications. While much work has focused on mono‐functional conjugates, that is, conjugates with one type of polymer attached such as poly(ethylene glycol) or poly(N‐isopropylacrylamide), there is a practical interest in gaining additional functionality by synthesizing well‐defined bifunctional conjugates in a hetero‐arm star copolymer architecture with protein as the core. Using ubiquitin as a model protein, a synthetic scheme is developed to attach two different polymers (oligo(ethylene oxide) methacrylate and N,N‐dimethylacrylamide) directly to the protein surface, using orthogonal conjugation chemistries and grafting‐from by photochemical living radical polymerization techniques. The additional complexity arising from attempts to selectively modify multiple sites led to decreased polymerization performance and indicates that initiators for continuous activator regeneration atom transfer radical polymerization and reversible addition‐fragmentation chain transfer polymerization are not well‐suited to bifunctional bioconjugates applications under the studied conditions. Nonetheless, the polymerization conditions preserve the native fold of the ubiquitin and enable production of a hetero‐arm star protein‐polymer bioconjugate.
more »
« less
Regulation of the endocytosis and prion-chaperoning machineries by yeast E3 ubiquitin ligase Rsp5 as revealed by orthogonal ubiquitin transfer
More Like this
-
-
Ubiquitin and ubiquitin like proteins (UBLs) play key roles in eukaryotes. These proteins are attached to their target proteins through an E1-E2-E3 cascade and modify the functions of these proteins. Since the discovery of ubiquitin, several UBLs have been identified, including Nedd8, SUMO, ISG15, and Atg8. Ubiquitin and UBLs share a similar three-dimensional structure: β -grasp fold and an X-X-[R/A/E/K]-X-X-[G/X]-G motif at the C-terminus. We have previously reported that ubiquitin, Nedd8, and SUMO mimicking peptides which all contain the conserved motif X-X-[R/A/E/K]-X-X-[G/X]-G still retained their reactivity toward their corresponding E1, E2, and E3 enzymes. In our current study, we investigated whether such C-terminal peptides could still be transferred onto related pathway enzymes to probe the function of these enzymes when they are fused with a protein. By bioinformatic search of protein databases, we selected eight proteins carrying the X-X-[R/A/E/K]-X-X-[G/X]-G motif at the C-terminus of the β -grasp fold. We synthesized the C-terminal sequences of these candidates as short peptides and found that three of them showed significant reactivity with the ubiquitin E1 enzyme Ube1. We next fused the three reactive short peptides to three different protein frames, including their respective native protein frames, a ubiquitin frame and a peptidyl carrier protein (PCP) frame, and measured the reactivities of these peptide-fused proteins with Ube1. Peptide-fused proteins on ubiquitin and PCP frames showed obvious reactivity with Ube1. However, when we measured E2 UbcH7 transfer, we found that the PCP-peptide fusions lost their reactivity with UbcH7. Taken together, these results suggested that the recognition of E2 enzymes with peptide-fused proteins depended not only on the C-terminal sequences of the ubiquitin-mimicking peptides, but also on the overall structures of the protein frames.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1016/j.chembiol.2021.02.005
