Fluorescence Imaging-Based High-Throughput Screening of Fast- and Slow-Cycling LOV Proteins

Fuun Kawano,Moritoshi Sato,Yuki Aono,Hideyuki Suzuki,Andrew J Roe

doi:10.1371/journal.pone.0082693

Abstract

Light-oxygen-voltage (LOV) domains function as blue light-inducible molecular switches. The photosensory LOV domains derived from plants and fungi have provided an indispensable tool for optogenetics. Here we develop a high-throughput screening system to efficiently improve switch-off kinetics of LOV domains. The present system is based on fluorescence imaging of thermal reversion of a flavin cofactor bound to LOV domains. We conducted multi site-directed random mutagenesis of seven amino acid residues surrounding the flavin cofactor of the second LOV domain derived from Avena sativa phototropin 1 (AsLOV2). The gene library was introduced into Escherichia coli cells. Then thermal reversion of AsLOV2 variants, respectively expressed in different bacterial colonies on agar plate, was imaged with a stereoscopic fluorescence microscope. Based on the mutagenesis and imaging-based screening, we isolated 12 different variants showing substantially faster thermal reversion kinetics than wild-type AsLOV2. Among them, AsLOV2-V416T exhibited thermal reversion with a time constant of 2.6 s, 21-fold faster than wild-type AsLOV2. With a slight modification of the present approach, we also have efficiently isolated 8 different decelerated variants, represented by AsLOV2-V416L that exhibited thermal reversion with a time constant of 4.3×103 s (78-fold slower than wild-type AsLOV2). The present approach based on fluorescence imaging of the thermal reversion of the flavin cofactor is generally applicable to a variety of blue light-inducible molecular switches and may provide a new opportunity for the development of molecular tools for emerging optogenetics.

Highlights

Blue light photoreceptors and photosensory domains derived from plants and fungi have been tested as light-inducible molecular switches or photoswitches to optogenetically control specific molecular processes in living cells, such as cellular signaling processes and gene expression (Fig. 1A) [1]
Fluorescence imaging of thermal reversion of AsLOV2 expressed in bacterial colonies AsLOV2 is a photosensory domain that binds with flavin mononucleotide (FMN) as a cofactor [18]
In the process of experimental design of the present multi site-directed mutagenesis of AsLOV2, we focused on Val416, Thr418, Asn425, Ile427, Ile466, Phe494 and Leu496

Summary

Introduction

Blue light photoreceptors and photosensory domains derived from plants and fungi have been tested as light-inducible molecular switches or photoswitches to optogenetically control specific molecular processes in living cells, such as cellular signaling processes and gene expression (Fig. 1A) [1]. These natural photoswitches suffer from their slow switch-off kinetics [2,3,4,5,6] that prevents them from accurately controlling spatiotemporal activities of cellular proteins [7]. Thermally controlled reversion of the flavin cofactor in the dark condition requires minutes to hours depending on species of LOV domains [2,3,4,6,9], indicating that the thermal reversion is the rate-determining step for the photocycle (Fig. 1B, right to left)

Methods

Results

Conclusion