Harnessing phytochrome's glowing potential

Abstract

Directed evolution of a cyanobacterial phytochrome was undertaken to elucidate the structural basis of its light sensory activity by remodeling the chemical environment of its linear tetrapyrrole prosthetic group. In addition to identifying a small region of the apoprotein critical for maintaining phytochrome's native spectroscopic properties, our studies revealed a tyrosine-to-histidine mutation that transformed phytochrome into an intensely red fluorescent biliprotein. This tyrosine is conserved in all members of the phytochrome superfamily, implicating direct participation in the primary photoprocess of phytochromes. Fluorescent phytochrome mutants also hold great promise to expand the present repertoire of genetically encoded fluorescent proteins into the near infrared.

Fig. 1.

Structure and photointerconversions of the phytochrome chromophore. (A) The proposed structure of the Pr and Pfr chromophores of plant (R₁₈, vinyl; P, propionate) and cyanobacterial (R₁₈, ethyl; P, propionate) phytochromes are based on vibrational spectroscopy and semiempirical vibrational energy calculations (35, 36). The Pr to Pfr conversion is thought to involve large motions: a Z-to-E configurational isomerization of the C15 double bond (a) followed by rotation around the C14–C15 bond (b), whereas the reverse reaction appears to be a concerted configurational and conformational inversion (c). (B) Ground and excited state potential energies of the Pr to Lumi-R photointerconversion as a function of the C15 dihedral angle are based on the trans-to-cis photoisomerization processes of stilbene (37). In phytochromes, light excitation energy (hv) is mainly dissipated on the picosecond time scale by photoproduct formation (k_p) involving the formation of a twisted C15 double bond, electronically excited intermediate (I*) that is gated by a thermal activation energy barrier (E_a) (7). This excited state intermediate decays radiationlessly into Pr (C15Z-isomer) and Lumi-R (C15E-isomer) in an ≈5:1 ratio. Lumi-R thermally converts to Pfr via a series of spectroscopically distinct intermediates in which the syn-to-anti conformational change occurs (data not shown). Lumi-R to Pr photoconversion is envisaged to proceed through the same intermediate (I*). It is proposed that the PR-1 Y176H mutation identified in this work leads to a larger thermal activation energy barrier (E_a), thereby greatly decreasing the rate of photoproduct formation (k_p) and leading to enhanced fluorescence (k_f).

Fig. 2.

Directed evolution of the photosensory domains of Cph1Δ.(A) Phytochrome Cph1 consists of the three related PAS, GAF, and PHY photosensory domains terminated with the histidine kinase domain (HKD) module. The truncated Cph1Δ photosensory construct lacks the HKD domain. The conserved cysteine site of bilin attachment at position 259 is shown in black. Four mutagenesis libraries were generated from either single domains or a combination of the entire photosensory domain (PGP). These libraries were coexpressed in E. coli with a bilin biosynthetic plasmid for the production of holoCph1Δ mutants in vivo. Holoproteins were analyzed for alterations in absorbance (ABS) or fluorescence (FACS). (B) The position and residue change of the 21 mutants having single amino acid substitutions identified from the PHY library absorbance screen. The alleles are colored with respect to phenotypic class: black, WT; purple, mutant class I; red, mutant class II; blue, mutant class III; teal, mutant class IV. The pink bar designates the conserved HisG insert that is present in the PHY domain of all know phytochromes.

Fig. 3.

Properties of the red fluorescent phytochrome mutant PR-1. (A) Purified WT Cph1Δ and PR-1 PCB adducts imaged in white (Left) or UV(Right) light. (B) Comparative flow cytometry analysis of PR-1 and WT Cph1Δ in PCB-producing cell lines by using the 635-nm red diode laser as an excitation source. (C) The three amino acid substitutions identified in the original PR-1 mutant are shown in red. (D) Colony fluoroimaging assay of WT Cph1Δ, PR-1 and the six possible single and double amino acid substitution combinations as PCB adducts produced in E. coli. The membrane was imaged under UV light to observe fluorescence.

Fig. 4.

Spectroscopic properties of the PCB adducts of WT Cph1Δ and PR-1 (Y176H). (A) Phytochrome difference absorption spectra for WT Cph1Δ (dashed line) and PR-1 Y176H single mutant (solid line). (B) Corrected fluorescence excitation (solid line) and emission (dotted line) spectra for the PR-1 Y176H single mutant. (C) Pr absorption spectra for WT Cph1Δ (dashed line) and PR-1 Y176H single mutant (solid line).

Fig. 5.

Tyrosine gating hypothesis for Pr excited state decay. Tyrosine 176 is proposed to act as a molecule gate to photoisomerization of the C15 double bond. Upon photoexcitation to Pr*, simultaneous torsional rotation of the C15 double bond (a) and the tyrosine phenol ring (a′) must occur to generate the proposed excited state intermediate (I*). This intermediate vibrationally relaxes to ground state Lumi-R (Lr) or Pr via radiationless pathways labeled b and c, respectively. The activation barrier for Pr* to I* interconversion, i.e., E_a (WT), is proposed to be greater for the Y176H mutant because of H-bonding of the histidine imidazole side chain that would inhibit C15 photoisomerization (shown at Right). The increase in E_a leads to an increased lifetime and enhanced fluorescence from Pr*.