Detection of Incorporation of p-Coumaric Acid into Photoactive Yellow Protein Variants in Vivo
- We report the design and characterization of photoactive yellow protein (PYP)-blue fluorescent protein (mTagBFP) fusion constructs that permit the direct assay of reconstitution and function of the PYP domain. These constructs allow for in vivo testing of co-expression systems for enzymatic production of the p-coumaric acid-based PYP chromophore, via the action of tyrosine ammonia lyase and p-coumaroyl-CoA ligase (pCL or 4CL). We find that different 4CL enzymes can function to reconstitute PYP, including 4CL from Arabidopsis thaliana that can produce similar to 100% holo-PYP protein under optimal conditions. mTagBFP fusion constructs additionally enable rapid analysis of effects of mutations on PYP photocycles. We use this mTagBFP fusion strategy to demonstrate in vivo reconstitution of several PYP-based optogenetic tools in Escherichia coli via a biosynthesized chromophore, an important step for the use of these optogenetic tools in vivo in diverse hosts.
Author details: | Katherine E. BrechunORCiD, Danlin Zhen, Anna Jaikaran, Vitali Borisenko, Masato Kumauchi, Wouter D. Hoff, Katja Maren ArndtORCiDGND, Andrew G WoolleyORCiD |
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DOI: | https://doi.org/10.1021/acs.biochem.9b00279 |
ISSN: | 0006-2960 |
Pubmed ID: | https://pubmed.ncbi.nlm.nih.gov/31117389 |
Title of parent work (English): | Biochemistry |
Publisher: | American Chemical Society |
Place of publishing: | Washington |
Publication type: | Article |
Language: | English |
Date of first publication: | 2019/06/11 |
Publication year: | 2019 |
Release date: | 2021/01/21 |
Volume: | 58 |
Issue: | 23 |
Number of pages: | 13 |
First page: | 2682 |
Last Page: | 2694 |
Funding institution: | NSERCNatural Sciences and Engineering Research Council of Canada; CIHRCanadian Institutes of Health Research (CIHR); NSFNational Science Foundation (NSF) [CHE-1413739]; Natural Sciences and Engineering Research Council of Canada (NSERC)Natural Sciences and Engineering Research Council of Canada; German Academic Exchange Service (DAAD, Deutscher Akademischer Austauschdienst)Deutscher Akademischer Austausch Dienst (DAAD); NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [R01 MH086379] |
Organizational units: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie |
DDC classification: | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
Peer review: | Referiert |