Engineering and exploiting synthetic allostery of NanoLuc luciferase

Zhong Guo, Rinky D. Parakra, Ying Xiong, Wayne A. Johnston, Patricia Walden, Selvakumar Edwardraja, Shayli Varasteh Moradi, Jacobus P. J. Ungerer, Hui-wang Ai, Jonathan J. Phillips () and Kirill Alexandrov ()
Additional contact information
Zhong Guo: ARC Centre of Excellence in Synthetic Biology
Rinky D. Parakra: University of Exeter
Ying Xiong: University of Virginia
Wayne A. Johnston: ARC Centre of Excellence in Synthetic Biology
Patricia Walden: ARC Centre of Excellence in Synthetic Biology
Selvakumar Edwardraja: The University of Queensland
Shayli Varasteh Moradi: ARC Centre of Excellence in Synthetic Biology
Jacobus P. J. Ungerer: Pathology Queensland
Hui-wang Ai: University of Virginia
Jonathan J. Phillips: University of Exeter
Kirill Alexandrov: ARC Centre of Excellence in Synthetic Biology

Nature Communications, 2022, vol. 13, issue 1, 1-12

Abstract: Abstract Allostery enables proteins to interconvert different biochemical signals and form complex metabolic and signaling networks. We hypothesize that circular permutation of proteins increases the probability of functional coupling of new N- and C- termini with the protein’s active center through increased local structural disorder. To test this we construct a synthetically allosteric version of circular permutated NanoLuc luciferase that can be activated through ligand-induced intramolecular non-covalent cyclisation. This switch module is tolerant of the structure of binding domains and their ligands, and can be used to create biosensors of proteins and small molecules. The developed biosensors covers a range of emission wavelengths and displays sensitivity as low as 50pM and dynamic range as high as 16-fold and could quantify their cognate ligand in human fluids. We apply hydrogen exchange kinetic mass spectroscopy to analyze time resolved structural changes in the developed biosensors and observe ligand-mediated folding of newly created termini.

Date: 2022
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DOI: 10.1038/s41467-022-28425-2

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