Orange fluorescent proteins constructed from cyanobacteriochromes chromophorylated with phycoerythrobilin

Abstract

Cyanobacteriochromes are a structurally and spectrally highly diverse class of phytochrome-related photosensory biliproteins. They contain one or more GAF domains that bind phycocyanobilin (PCB) autocatalytically; some of these proteins are also capable of further modifying PCB to phycoviolobilin or rubins. We tested the chromophorylation with the non-photochromic phycoerythrobilin (PEB) of 16 cyanobacteriochrome GAFs from Nostoc sp. PCC 7120, of Slr1393 from Synechocystis sp. PCC 6803, and of Tlr0911 from Thermosynechococcus elongatus BP-1. Nine GAFs could be autocatalytically chromophorylated in vivo/in E. coli with PEB, resulting in highly fluorescent biliproteins with brightness comparable to that of fluorescent proteins like GFP. In several GAFs, PEB was concomitantly converted to phycourobilin (PUB) during binding. This not only shifted the spectra, but also increased the Stokes shift.The chromophorylated GAFs could be oligomerized further by attaching a GCN4 leucine zipper domain, thereby enhancing the absorbance and fluorescence of the complexes. The presence of both PEB and PUB makes these oligomeric GAF-"bundles" interesting models for energy transfer akin to the antenna complexes found in cyanobacterial phycobilisomes. The thermal and photochemical stability and their strong brightness make these constructs promising orange fluorescent biomarkers.

Genomic Survey and Biochemical Analysis of Recombinant Candidate Cyanobacteriochromes Reveals Enrichment for Near UV/Violet Sensors in the Halotolerant and Alkaliphilic Cyanobacterium Microcoleus IPPAS B353

“…However, CBCRs lacking signaling domains other than GAF(s), such as FdIflA (50), or containing multiple cystathione ␤-synthase domains, such as TeSesA (51), were not identified in this genome. Like the majority of CBCRs, the natural chromophore of these Phys and CBCR proteins is very likely to be PCB, rather than BV (18) or phycoerythrobilin (52). The Microcoleus genome harbors only PCB biosynthesis genes, which include two heme oxygenases (ho1, mbr4200, and mbr2519) and a phycocyanobilin: ferredoxin oxidoreductase (pcyA, mbr2212).…”

Natural Photoreceptors as a Source of Fluorescent Proteins, Biosensors, and Optogenetic Tools

“…This can be achieved by preventing light-induced chromophore isomerization or by incorporating heterologous chromophores, which cannot isomerize. The latter approach was demonstrated by substituting PCB with phycoerythrobilin (PEB), which has saturated bonds between the C- and D-rings (35). As a result, FPs with quantum yield of up to 0.72 from the cyanobacterial phytochrome Cph1 and 0.55 for CBCRs were obtained.…”

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

“…It is considered that CBCRs initially bind PCB as a chromophore, but some dual-Cys CBCRs can then isomerize PCB into phycoviolobilin chromophore [ 18 , 39 , 40 ]. In heterologous expression systems, some CBCRs can also autocatalytically incorporate phytochromobilin and phycoerythrobilin [ 40 , 41 , 42 , 43 ]. Furthermore, several CBCRs can covalently incorporate as chromophore not only PCB but also BV [ 42 , 44 , 45 , 46 ].…”

“…Photochemically inert red/green CBCR Npf2164g5, found in Nostoc punctiforme , was unable to photoconvert; instead, it exhibited red fluorescence with a quantum yield of 10–15% [ 75 ]. Incorporation of PEB chromophore, which carries saturated bridge between pyrrole rings C and D and cannot isomerize [ 108 ], resulted in five-fold higher quantum yield of CBCR, Slr1393g3, as compared to its PCB chromophorilated form [ 43 , 84 ]. Suppression of CBCR photoisomerization can be achieved by mutation of the key amino acid residues, including those within the Asp-motif, conserved residue following chromophore-binding Cys, “gate” Trp residue and conserved Phe ( Figure 2 B).…”