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OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy
Published on: February 5, 2020
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A modular toolbox to generate complex polymeric ubiquitin architectures using orthogonal sortase enzymes.
Maximilian Fottner1,2, Maria Weyh1, Stefan Gaussmann3,4
1Department of Chemistry, Lab for Synthetic Biochemistry, Technical University of Munich, Institute for Advanced Study, TUM-IAS, Lichtenberg Str. 4, 85748, Garching, Germany.
Nature Communications
|November 12, 2021
Summary
Researchers developed Ubl-tools, a modular system for creating complex ubiquitin (Ub) and Ub-like modifier (Ubl) chains. This tool facilitates studying how these protein modifications regulate cellular processes like DNA repair.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Post-translational modification with ubiquitin (Ub) and Ub-like modifiers (Ubls) is crucial for eukaryotic cell regulation.
- Distinct topologies of polymeric Ub/Ubl chains control protein activity, stability, and localization.
- Characterizing Ub/Ubl chain roles is limited by the difficulty in preparing specific chain topologies.
Purpose of the Study:
- To introduce a flexible and user-defined modular toolbox (Ubl-tools) for assembling Ub/Ubl chains.
- To enable the generation of distinct homo-, hetero-typic, and branched Ub/Ubl chains.
- To provide a method for studying the functional impact of complex Ub/Ubl modifications on cellular processes.
Main Methods:
- Development of a modular toolbox (Ubl-tools) utilizing orthogonal sortase enzymes.
- Stepwise assembly of Ub/Ubl chains with defined linkages and topologies.
- Generation of Ub/Ubl hybrid chains and investigation of their role in DNA damage repair.
Main Results:
- Demonstrated the universality and applicability of Ubl-tools for creating diverse Ub/Ubl chain structures.
- Successfully generated distinctly linked Ub/Ubl hybrid chains.
- Investigated the role of these custom chains in DNA damage repair pathways.
Conclusions:
- Ubl-tools provides straightforward access to site-specifically modified proteins with complex Ub/Ubl chains.
- This powerful approach facilitates the study of Ub/Ubl chain function in various cellular processes.
- The tool enables deeper understanding of the biological outputs controlled by Ub/Ubl modifications.
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