Dataset Information

A Highly Efficient Synthesis of Polyubiquitin Chains.

ABSTRACT: A robust, microwave-assisted, highly efficient, solid-phase peptide synthesis method for preparing isopeptide-linked 62-mer and 76-mer isoubiquitins and polyubiquitin is developed. The strategy avoids the use of costly resins and pseudoprolines, and the isopeptide-linked building blocks can be assembled with high initial purity within 1 day. All seven diubiquitins are successfully synthesized on a multi-milligram scale; a four-segment, three-ligation method is used to obtain a K33-/K11-linked mixed triubiquitin in excellent yield. Circular dichroism and crystallographic analyses are used to verify the structures of the well-folded, synthetic polyubiquitin chains. The facile synthetic strategy is expected to be generally applicable for the rapid synthesis of isopeptide-linked isoUbs and to pave the way for the study of longer polyubiquitin chains.

SUBMITTER: Qu Q

PROVIDER: S-EPMC6051384 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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A Highly Efficient Synthesis of Polyubiquitin Chains.

Qu Qian Q Pan Man M Gao Shuai S Zheng Qing-Yun QY Yu Yuan-Yuan YY Su Jia-Can JC Li Xiang X Hu Hong-Gang HG

Advanced science (Weinheim, Baden-Wurttemberg, Germany) 20180514 7

A robust, microwave-assisted, highly efficient, solid-phase peptide synthesis method for preparing isopeptide-linked 62-mer and 76-mer isoubiquitins and polyubiquitin is developed. The strategy avoids the use of costly resins and pseudoprolines, and the isopeptide-linked building blocks can be assembled with high initial purity within 1 day. All seven diubiquitins are successfully synthesized on a multi-milligram scale; a four-segment, three-ligation method is used to obtain a K33-/K11-linked mi ...[more]

PMID: 30027052

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Project description:Polymeric chains of a small protein ubiquitin are involved in regulation of nearly all vital processes in eukaryotic cells. Elucidating the signaling properties of polyubiquitin requires the ability to make these chains in vitro. In recent years, chemical and chemical-biology tools have been developed that produce fully natural isopeptide-linked polyUb chains with no need for linkage-specific ubiquitin-conjugating enzymes. These methods produced unbranched chains (in which no more than one lysine per ubiquitin is conjugated to another ubiquitin). Here we report a nonenzymatic method for the assembly of fully natural isopeptide-linked branched polyubiquitin chains. This method is based on the use of mutually orthogonal removable protecting groups (e.g., Boc- and Alloc-) on lysines combined with an Ag-catalyzed condensation reaction between a C-terminal thioester on one ubiquitin and a specific ε-amine on another ubiquitin, and involves genetic incorporation of more than one Lys(Boc) at the desired linkage positions in the ubiquitin sequence. We demonstrate our method by making a fully natural branched tri-ubiquitin containing isopeptide linkages via Lys11 and Lys33, and a (15)N-enriched proximal ubiquitin, which enabled monomer-specific structural and dynamical studies by NMR. Furthermore, we assayed disassembly of branched and unbranched tri-ubiquitins as well as control di-ubiquitins by the yeast proteasome-associated deubiquitinase Ubp6. Our results show that Ubp6 can recognize and disassemble a branched polyubiquitin, wherein cleavage preferences for individual linkages are retained. Our spectroscopic and functional data suggest that, at least for the chains studied here, the isopeptide linkages are effectively independent of each other. Together with our method for nonenzymatic assembly of unbranched polyubiquitin, these developments now provide tools for making fully natural polyubiquitin chains of essentially any type of linkage and length.

Dataset Information

A Highly Efficient Synthesis of Polyubiquitin Chains.

Publications

A Highly Efficient Synthesis of Polyubiquitin Chains.

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