Research Interests: The Lagarias Laboratory

Biliprotein Engineering: Directed Evolution of Phytochromes

The spectroscopic and photophysical properties of linear tetrapyrroles (bilins) are strongly influenced by their chemical environment (Figure 4). In aqueous solution, bilins adopt cyclic porphyrin-like conformations that strongly absorb light in the near-ultraviolet region and mainly dissipate absorbed light energy by radiationless processes. Upon association with proteins such as the phycobiliprotein antennae of blue-green, red and cryptomonad algae or the phytochrome photoreceptors of plants, cyanobacteria and many non-photosynthetic microorganisms, bilins assume more extended conformations, which significantly increases their visible light absorption and alters the pathways used for de-excitation. Adapted for efficient transfer of excitation energy to membrane-bound photosynthetic reaction centers, phycobiliproteins are intensely fluorescent with fluorescence quantum yields (ΦF) near unity. Phytochromes, by contrast, are poorly fluorescent biliproteins with ΦF < 0.005 due to efficient C15Z-to-E double bond isomerization of their bilin prosthetic group (link to Figure 3 in Photochrome Photochemistry).

Figure 4. Biliproteins tune the conformation, spectra and photophysical properties of bilins. Bilins in solution adopt porphyrin-like conformations that are also seen in bilin complexes in insecticyanin, and myoglobin as well as those in the enzymes heme oxygenase and phycocyanobilin:ferredoxin oxidoreductase and phycoerythrobilin synthase. This contrasts with the more extended conformations of the bilin prosthetic groups of the intensely fluorescent phycobiliproteins phycocyanin and allophycocyanin and the phytochromes DrBphP from Deinococcus radiodurans, RpBphP3 from Rhodopseudomonas palustris, PaBphP  from Pseudomonas aeruginosa and Cph1 from Synechocystis sp. PCC 6803.



Figure 5. The Cph1-derived phytofluor (left) is intensely fluorescent while Pr and Pfr forms of wild-type Cph1 is not.

The unique spectral characteristics of phytochromes in the red (R) and near infrared (NIR) region as well as their ability to autocatalytically assemble with their bilin chromophores make them ideal subjects for engineering new R/NIR fluorescent reporters and photoswitches. Fluorescent phytochromes with unique fluorescence excitation/emission spectra that could be used with commonly available laser-based confocal microscopes and flow cytometers are highly desireable targets. Unlike the self-assembling Green Fluorescent Protein (GFP) and its relatives, fluorescent phytochrome engineering for cell-based applications requires production of two components - an apoprotein and a bilin precursor. Since bilins can passively diffuse across membranes, unnatural bilin precurors can be used to tune the spectroscopic properties of phytochromes. This was the basis for the development of the first phytofluor, a phytochrome-based yellow-orange fluorescent biliprotein that can be produced in live cells (Figure 5).

 

 

 

 


Figure 6. Expression of holophytochrome in bacteria. E. coli strain LMG194 engineered to express apoCph1Δ and a bilin biosynthetic pathway consisting of a heme oxygenase and a ferredoxin-dependent bilin reductase, PcyA (PCB) or HY2 (PΦB) are shown here after co-induction and cell pelleting by centrifugation.

To complement chromophore-based modification approaches, the technique of directed evolution was used to introduce random protein modifications to alter phytochrome's photophysical properties. Initial studies utilized a dual plasmid expression system developed earlier in our lab - a system that is capable of producing holophytochrome within live bacterial cells (Gambetta & Lagarias, 2001). With this approach, one plasmid produces a cyanobacterial phytochrome Cph1Δ apoprotein while the other plasmid encodes the enzymes heme oxygenase and PcyA, needed for the conversion of endogenous heme to the chromophore precursor phycocyanobilin (PCB). E. coli cells expressing the two plasmids are brilliantly blue-colored due to production of holophytochrome (Figure 6).

In our first directed evolution study, we introduced a library of mutations into a truncated cyanobacterial phytochrome 1 (Cph1Δ) using the technique of error-prone polymerase chain reaction (PCR) amplification (Figure 7). We expected a range of phenotypes to be represented in this library, including mutant proteins that could no longer assemble with bilin, others with shifted spectra, and possibly others with enhanced fluorescence. For absorbance screens, we used a Kairos MacroDIS imaging spectrophotometer system to identify optically shifted Cph1Δ mutants on agar plates, while fluorescence-activated cell sorting (FACS) was used to identify fluorescent Cph1Δ mutants.

Figure 7. Directed evolution of the cyanobacterial phytochrome 1. Four libraries of Cph1Δ mutants were constructed by error-prone PCR amplification of the PAS, GAF and PHY domains or all three domains together. After subcloning the library into an expression plasmid, the mutant libraries were introduced into a bilin-producing E. coli strain (mutations indicated by asterisks). Co-expression yields holophytochromes within cells that were screened by an absorbance-based method or sorted by FACS to identify optically-shifted and fluorescent cell lines (Fischer & Lagarias, 2004).

FACS screening was performed using a red laser (647 nm excitation) and a red cutoff filter (> 660 nm). A population of roughly 50 million cells was sorted, identifying 18 fluorescent colonies. After re-isolation of plasmid DNA from these clones and transformation into a new strain of bacteria, one red-fluorescent Cph1Δ named phytofluor red 1 (PR1) was recovered. DNA sequence analysis of PR1 identified three amino acid mutations only one of which, i.e. Tyr176His, was found to be responsible for the gain-of-function red fluorescence. Photographs of WT and PR1 are shown in Figure 8.

Figure 8. Solutions of wild-type (WT) and PR1 mutant of Cph1Δ photographed under white light (left) and UV light (right) revealing the intense red fluorescence of PR1, not seen for WT.