Engineering and exploiting synthetic allostery of NanoLuc luciferase

Zhong Guo,Ying Xiong,Kirill Alexandrov,Wayne A Johnston,Selvakumar Edwardraja,Patricia Walden,Hui-Wang Ai,Jacobus P J Ungerer,Shayli Varasteh Moradi,Rinky D Parakra,Jonathan J Phillips

doi:10.1038/s41467-022-28425-2

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.

Highlights

Allostery enables proteins to interconvert different biochemical signals and form complex metabolic and signaling networks
We demonstrate that the developed biosensors can be engineered to recognize both proteins and small molecules, and can be used to for detection in biological samples with sensitivities exceeding the requirements for practical utilization
We propose that this approach may be applied to other protein scaffolds, thereby representing a path to standardized approach to construction of artificial allosteric switches

Summary

Introduction

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. We test an approach, where entropically driven local unfolded inactive reporter domain can be structured and activated by ligand-induced non-covalent intramolecular cyclisation. We propose that this approach may be applied to other protein scaffolds, thereby representing a path to standardized approach to construction of artificial allosteric switches

Methods

Results

Conclusion