Photocontrolled reversible self-assembly of dodecamer nitrilase

Qiao Yu,Yong Wang,Shengyun Zhao,Yuhong Ren

doi:10.1186/s40643-017-0167-3

Abstract

BackgroundNaturally photoswitchable proteins act as a powerful tool for the spatial and temporal control of biological processes by inducing the formation of a photodimerizer. In this study, a method for the precise and reversible inducible self-assembly of dodecamer nitrilase in vivo (in Escherichia coli) and in vitro (in a cell-free solution) was developed by means of the photoswitch-improved light-inducible dimer (iLID) system which could induce protein–protein dimerization.ResultsNitrilase was fused with the photoswitch protein AsLOV2-SsrA to achieve the photocontrolled self-assembly of dodecamer nitrilase. The fusion protein self-assembled into a supramolecular assembly when illuminated at 470 nm. Scanning electron microscopy showed that the assembly formed a circular sheet structure. Self-assembly was also induced by light in E. coli. Dynamic light scattering and turbidity assay experiments showed that the assemblies formed within a few seconds under 470-nm light and completely disassembled within 5 min in the dark. Assembly and disassembly could be maintained for at least five cycles. Both in vitro and in vivo, the assemblies retained 90% of the initial activity of nitrilase and could be reused at least four times in vitro with 90% activity.ConclusionsAn efficient method was developed for the photocontrolled assembly and disassembly of dodecamer nitrilase and for scaffold-free reversible self-assembly of multiple oligomeric enzymes in vivo and in vitro, providing new ideas and methods for immobilization of enzyme without carrier.

Highlights

Photoswitchable proteins act as a powerful tool for the spatial and temporal control of biological processes by inducing the formation of a photodimerizer
Several strategies have been developed to utilize LOV2 domain isolated from Avena sativa (AsLOV2) as a photoswitch, including using tunable light-inducible dimerization tags to control the interaction between the AsLOV2 domain and an engineered PDZ domain, and using the improved light-inducible dimer system to control the location of transmembrane proteins (Guntas et al 2014)
BNAS retained about 97% of the specific activity of nitrilase from Burkholderia cenocepacia J2315 (BCNIT) (Fig. 2b), indicating that the protein fused with AsLOV2-SsrA did not suffer any drastic structural change in vitro

Summary

Introduction

Photoswitchable proteins act as a powerful tool for the spatial and temporal control of biological processes by inducing the formation of a photodimerizer. A method for the precise and reversible inducible self-assembly of dodecamer nitrilase in vivo (in Escherichia coli) and in vitro (in a cell-free solution) was developed by means of the photoswitch-improved light-inducible dimer (iLID) system which could induce protein– protein dimerization. Protein assembly has been extensively studied (King and Lai 2013; Yu et al 2015), and represents a useful tool for biology and bioengineering processes owing to its potential applications in protein material development nanobiotechnology, and multienzyme biocatalysis (Matsumoto et al 2016; Smith et al 2011; Fairman and Åkerfeldt 2005; Holmes 2002). Several strategies have been developed to utilize AsLOV2 as a photoswitch, including using tunable light-inducible dimerization tags to control the interaction between the AsLOV2 domain and an engineered PDZ domain, and using the improved light-inducible dimer (iLID) system to control the location of transmembrane proteins (Guntas et al 2014). The iLID presents several advantages, such as possessing strictly monomeric components, being expressed in Escherichia coli, and having a broad dynamic range, a highly tunable affinity, and a fast reversion rate in the dark (Guntas et al 2014)

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