Minimal domain of bacterial phytochrome required for chromophore binding and fluorescence.

Konstantin A Rumyantsev,Vladislav V Verkhusha,Alexander V Emelyanov,Konstantin K Turoverov,Natalia I Zakharova,Daria M Shcherbakova

doi:10.1038/srep18348

Abstract

Fluorescent proteins (FP) are used to study various biological processes. Recently, a series of near-infrared (NIR) FPs based on bacterial phytochromes was developed. Finding ways to improve NIR FPs is becoming progressively important. By applying rational design and molecular evolution we have engineered R. palustris bacterial phytochrome into a single-domain NIR FP of 19.6 kDa, termed GAF-FP, which is 2-fold and 1.4-fold smaller than bacterial phytochrome-based NIR FPs and GFP-like proteins, respectively. Engineering of GAF-FP involved a substitution of 15% of its amino acids and a deletion of the knot structure. GAF-FP covalently binds two tetrapyrrole chromophores, biliverdin (BV) and phycocyanobilin (PCB). With the BV chromophore GAF-FP absorbs at 635 nm and fluoresces at 670 nm. With the PCB chromophore GAF-FP becomes blue-shifted and absorbs at 625 nm and fluoresces at 657 nm. The GAF-FP structure has a high tolerance to small peptide insertions. The small size of GAF-FP and its additional absorbance band in the violet range has allowed for designing a chimeric protein with Renilla luciferase. The chimera exhibits efficient non-radiative energy transfer from luciferase to GAF-FP, resulting in NIR bioluminescence. This study opens the way for engineering of small NIR FPs and NIR luciferases from bacterial phytochromes.

Highlights

A mixture of mutated genes was electroporated into Escherichia coli strain LMG194 host cells (Life Technologies/Invitrogen) containing the pWA23h plasmid[8] facilitating biliverdin synthesis via co-expression of heme oxygenase
The LMG194 cells were grown overnight at 37 °C in RM minimal medium supplemented with ampicillin and kanamycin
Bacterial cells were washed with phosphate-buffered saline (PBS) and diluted with PBS to an optical density of 0.03 at 600 nm

Summary

Introduction

For the anaerobic protein maturation assay the LMG194 bacterial cells containing pBAD/HisB-GAF-FP and either pWA23h or pPL-PCB plasmids were grown on LB/ampicillin/kanamycin Petri dishes supplemented with 0.02% rhamnose overnight at 37 °C. After analysis Petri dishes were transferred to 4 °C for additional 48 h incubation in the presence of oxygen. During this time only already synthetized FP will be able to bind chromophore, while the synthesis of new protein will be significantly slowed-down. The dishes were analyzed again, level of fluorescence after incubation at 4 °C was considered to be 100%. All quantitative measurements of fluorescence signal for bacteria were performed using the ImageJ software[3]

Methods

Results

Conclusion