A split methyl halide transferase AND gate that reports by synthesizing an indicator gas
| Autor: | Jonathan J. Silberg, Caroline A. Masiello, Margaret Lie, Dongkuk Huh, Emily M Fulk, Joshua T. Atkinson |
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| Rok vydání: | 2020 |
| Předmět: |
0106 biological sciences
Biomedical Engineering Sequence (biology) Tacrolimus Binding Protein 1A 01 natural sciences Biochemistry Genetics and Molecular Biology (miscellaneous) 03 medical and health sciences Transferases Protein-fragment complementation assay 010608 biotechnology Pentosyltransferases Protein Interaction Maps 030304 developmental biology Sirolimus 0303 health sciences Molecular interactions Chemistry TOR Serine-Threonine Kinases General Medicine Protein engineering Methyl halide transferase Combinatorial chemistry FKBP Gases Biosensor Dimerization Protein Processing Post-Translational Competitive inhibitor AND gate Binding domain |
| DOI: | 10.1101/2020.06.11.146191 |
| Popis: | It is challenging to detect microbial reactions in highly opaque or autofluorescent environments like soils, seawater, and wastewater. To develop a simple approach for monitoring post-translational reactions within microbes situated in environmental matrices, we designed a methyl halide transferase (MHT) fragment complementation assay that reports by synthesizing an indicator gas. We show that backbone fission within regions of high sequence variability in the Rossmann-fold domain yields split MHT (sMHT) AND gates whose fragments cooperatively associate to synthesize CH3Br. Additionally, we identify a sMHT whose fragments require fusion to pairs of interacting partner proteins for maximal activity. We also show that sMHT fragments fused to FKBP12 and the FKBP-rapamycin binding domain of mTOR display significantly enhanced CH3Br production in the presence of rapamycin. This gas production is reversed in the presence of the competitive inhibitor of FKBP12/FKPB dimerization, indicating that sMHT is a reversible reporter of post-translational reactions. This sMHT represents the first genetic AND gate that can report on protein-protein interactions via an indicator gas. Because indicator gases can be measured in the headspaces of complex environmental samples, this protein fragment complementation assay should be useful for monitoring the dynamics of diverse molecular interactions within microbes situated in hard-to-image marine and terrestrial matrices. |
| Databáze: | OpenAIRE |
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