Biochemical Analysis Leads to Improved Orthogonal Bioluminescent Tools

Autor: Sierra J. Williams, Jordan A. Gewing‐Mullins, Whitney K. Lieberman, Bethany Kolbaba‐Kartchner, Reema Iqbal, Hana M. Burgess, Clair M. Colee, Marya Y. Ornelas, Edison S. Reid‐McLaughlin, Jeremy H. Mills, Jennifer A. Prescher, Aaron M. Leconte
Rok vydání: 2023
Předmět:
Zdroj: ChemBioChem. 24
ISSN: 1439-7633
1439-4227
DOI: 10.1002/cbic.202200726
Popis: Engineered luciferase-luciferin pairs have expanded the number of cellular targets that can be visualized in tandem. While light production relies on selective processing of synthetic luciferins by mutant luciferases, little is known about the origin of selectivity. The development of new and improved pairs requires a better understanding of the structure-function relationship of bioluminescent probes. In this work, we report a biochemical approach to assessing and optimizing two popular bioluminescent pairs: Cashew/D-luc and Pecan/4'-BrLuc. Single mutants derived from Cashew and Pecan revealed key residues for selectivity and thermal stability. Stability was further improved through rational addition of beneficial residues. Additionally, adding mutations known to improve stability provided extra stability and, surprisingly, improved selectivity dramatically. The resultant improved pair of luciferases is100-fold selective for their respective substrates and highly thermally stable. Collectively, this work highlights the importance of mechanistic insight for improving bioluminescent pairs and provides significantly improved Cashew and Pecan enzymes which should be immediately suitable for multi-component imaging applications.
Databáze: OpenAIRE
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