Bacterial Phytochromes, Cyanobacteriochromes and Allophycocyanins as a Source of Near-Infrared Fluorescent Probes

Abstract

Bacterial photoreceptors absorb light energy and transform it into intracellular signals that regulate metabolism. Bacterial phytochrome photoreceptors (BphPs), some cyanobacteriochromes (CBCRs) and allophycocyanins (APCs) possess the near-infrared (NIR) absorbance spectra that make them promising molecular templates to design NIR fluorescent proteins (FPs) and biosensors for studies in mammalian cells and whole animals. Here, we review structures, photochemical properties and molecular functions of several families of bacterial photoreceptors. We next analyze molecular evolution approaches to develop NIR FPs and biosensors. We then discuss phenotypes of current BphP-based NIR FPs and compare them with FPs derived from CBCRs and APCs. Lastly, we overview imaging applications of NIR FPs in live cells and in vivo. Our review provides guidelines for selection of existing NIR FPs, as well as engineering approaches to develop NIR FPs from the novel natural templates such as CBCRs.

Keywords: allophycocyanin; bacterial photoreceptor; cyanobacteriochrome; near-infrared fluorescent protein; phytochrome; tetrapyrrole.

Figure 1

Chromophores and structures of selected bacterial photoreceptors. Linear tetrapyrrole chromophores are: (A) biliverdin (BV); (B) phycocyanobilin (PCB); (C) phycoviolobilin (PVB); and (D) phycoerythrobilin (PEB); (E) Crystal structure of BphP XccBphP from Xanthomonas campestris. PAS domain is shown in green, GAF in cyan, PHY in magenta and HisK in brown (PDB ID: 5AKP); (F) Crystal structure of CBCR AnPixJg2 from Anabaena sp. PCC 7120 in red-light-absorbing state (PDB ID: 3W2Z); (G) Crystal structure of CBCR TePixJg2 from Thermosynechococcus elongatus in green-light-absorbing state (PDB ID: 3VV4); (H) Crystal structure of APC B from Synechocystis PCC 6803 (PDB ID: 4PO5). BV and PCB chromophores in panels E–H are shown as spheres.

Figure 2

Structural properties of cyanobacteriochromes (CBCRs): (A) topology diagram of CBCR GAF domain; and (B) sequence alignment of chromophore-binding pockets in CBCRs. Representative CBCRs of the red/green subfamily (red), NpR3784 subfamily (black), DXCF subfamily (blue), green/red subfamily (green), and far-red/orange subfamily (purple) are included. Key CBCR residues are highlighted: Asp-motif is in green; β1 Phe, β2 Phe, helix Phe and “gate” Trp are in yellow; chromophore-binding Cys is in red; and conserved residue following chromophore-binding Cys is in blue.

Figure 3

Molecular evolution steps, methods and techniques employed to develop near-infrared fluorescent proteins (NIR FPs). Left column lists typical molecular evolution steps, such as gene template mutagenesis, construction of library of mutants, methods of high-throughput screening, and characterization of advanced variants of NIR FP in vitro and in cells. Specific conditions, techniques and studied parameters of mutant for each molecular evolution step are indicated in the right column.

Figure 4

Applications of near-infrared fluorescent proteins. (A) Image of iRFP713 transgenic newborn mice and their wild-type non-fluorescent littermates (adapted from [26], copyright 2014 by the Japanese Association for Laboratory Animal Science); (B) Impaired lymphatic drainage due to metastatic spread. Lymphatic channels are shown in green and the iRFP713-labeled tumor is shown in red (adapted from [138], copyright 2014; available under the Creative Commons Attribution license); (C) Odyssey LI-COR scan of iRFP expressing 3T3 fibroblasts 16 days post transfection with cMyc/Ha-RasG12V. Fluorescent signal is shown in pseudocolor (adapted from [139], copyright 2014; available under the Creative Commons Attribution license); (D) Photoacoustic microscopy image of multiple layers of RpBphP1-expressing cells (right). Depth is shown in pseudocolor (adapted from [140]); (E) Detection of the bJun-bFos interaction in living mice expressing two split-iRFP713 constructs. Fluorescence pseudocolor images of tumor xenografts expressing bJun-iRN and iRC-bFos fusion proteins. Images for the constructs split at positions 97 (left) and 123 (right) of iRFP713 are shown (adapted from [141], copyright 2015 by Elsevier B.V.); (F) Drosophila brain cells co-expressing Histone-2B-GFP and the iCasper protease reporter. Apoptotic cells are shown in red (adapted from [142]); (G) Fluorescence lifetime images of cells expressing the mKate2-DEVD-iRFP713 reporter before (left) and after (right) induction of the apoptosis (adapted from [143], copyright 2016 by BioTechniques); (H) Stimulus-induced degradation of the NIR IκBα reporter, upon activation of the NF-κB pathway (adapted from [35]); (I) NIR Fucci cell cycle reporter in cells at different time points during cell cycle progression (adapted from [35]).