Photochemical and biochemical characterization of GUN4 reactions with protoporphyrin IX and phycocyanobilin
Data files
May 05, 2026 version files 414.04 KB
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GUN4_spectroscopy_photochemistry.tar.gz
408.47 KB
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README.md
5.57 KB
Abstract
These data include characterization of the reactions of GUN4 protein with bound tetrapyrroles, using GUN4 from the model green alga Chlamydomonas. In almost all oxygenic photosynthetic organisms, GUN4 functions as a fourth "accessory" subunit of magnesium chelatase (MgCh), the enzyme carrying out the committed step in chlorophyll biosynthesis. GUN4 is able to bind a wide range of tetrapyrroles, including the protoporphyrin IX (PPIX) substrate and magnesium protoporphyrin IX (MgPPIX) product of the MgCh reaction. GUN4 can also bind linear tetrapyrroles (bilins) such as phycocyanobilin (PCB), and past work has shown that the GUN4•PCB complex stimulates MgCh activity by an order of magnitude in vitro and that both GUN4 and bilin are required for maintenance of the catalytic subunit of MgCh (CHLH1) in vivo. The current dataset examines the behavior of the GUN4/PCB/PPIX system both in vivo and in vitro. In vitro measurements used Chlamydomonas GUN4 protein purified after recombinant expression in Escherichia coli. Four sub-directories span a range of data types, all presented as tab-delimited text files. In addition to the overall README for this dataset, detailed information about the measurements and reported values are included in a README text file in each directory.
Dataset DOI: 10.5061/dryad.47d7wm3vq
Description of the data and file structure
Four sub-directories span a range of data types, all presented as tab-delimited text files. Detailed information about the measurements and reported values are included in a README text file in each directory. The four sub-directories are as follows:
1. in_vivo_tetrapyrrole_protein_levels
This sub-directory includes in vivo measurements of tetrapyrrole metabolites and protein levels in Chlamydomonas cells.
PPIX and MgPPIX levels are reported for wild-type cells (using the 4A+ background), gun4 cells, hmox1 cells, and gun4 hmox1 cells. The different mutations are loss-of-function for GUN4 protein, for the plastid heme oxygenate HMOX1 required for PCB biosynthesis in Chlamydomonas, or for both.
Protein levels were measured as a function of time after dark-to-light transition, comparing GUN4 and CHLH1 degradation to that of the stable CHLI1 subunit of MgCh and to that of the known light-labile photosystem protein D1.
Chlorophyll levels are reported for Chlamydomonas cells with wild-type GUN4 protein (4A+ wild-type), no GUN4 protein (gun4 mutant), or with the wild-type GUN4 allele replaced by mutant alleles carrying W189A or N219G substitutions.
2. oxidation_of_Trp_residues
This sub-directory includes measurements of oxidation of Trp residues both in vivo and in vitro for GUN4 protein, examining a known consequence of PPIX binding in other proteins. Tandem LC-MS was used to map specific sites of post-translational modification.
In vitro measurements are reported for four conditions: GUN4 + PPIX in darkness, GUN4 + PPIX under near-ultraviolet (nUVA) light, GUN4 + PPIX + PCB in darkness, and GUN4 + PPIX + PCB under nUVA light.
In vivo measurements are reported for two strains, both of which use a Strep-tagged GUN4 allele in combination with a gun4 mutant strain. This approach allows facile purification of GUN4 protein from Chlamydomonas cells and unambiguous identification of the resulting peptides. This allele was used in either wild-type HMOX1 cells or in hmox1 cells lacking bilin biosynthesis.
3. fluorescence_of_Trp_residues
This sub-directory includes in vitro measurements of fluorescence from GUN4 tryptophan (Trp) residues. Both static fluorescence and time-resolved fluorescence lifetime measurements are included, along with a summary table of the resulting parameters.
Static Trp emission spectra are reported for GUN4 protein as purified, after supplementation with PCB, after supplementation with PCB and subsequent treatment with violet light, after supplementation with both PCB and PPIX, and after supplementation with both ligands and violet illumination.
Static PPIX emission spectra are also reported for direct excitation of PPIX (using 405 nm to excite the strong porphyrin Soret band) and after excitation of Trp at 280 nm, to assess possible energy transfer between the two chromophores.
Fluorescence lifetime data are reported for GUN4 protein as purified or with the same combination of ligands and illumination conditions used in the static measurements. As for other proteins, Trp fluorescence decay in GUN4 was well described by a three-exponential fit, and the results of that analysis are included for each condition.
4. ligand_photochemistry
This subdirectory includes characterization of the in vitro photochemical reactions of GUN4 protein, PCB, and PPIX. Wild-type GUN4 protein, the N219G variant, and the Y197Q variant were examined using absorption and fluorescence spectroscopy. Photochemical degradation of free PCB was assessed using absorption spectroscopy under identical conditions.
Each of the three proteins was characterized using absorption and fluorescence spectroscopy after initial purification from E. coli cells engineered to synthesize PCB. These measurements assess the initial levels of bound PCB and PPIX. Each protein was then supplemented with additional PCB, with binding assessed by absorption spectroscopy prior to assessment of photochemistry under violet light. In parallel, samples were supplemented with PPIX as well as PCB with similar characterization and photochemistry. In all cases, the progress of photochemistry was assessed as a 30 minute time course with spectra taken every 3 minutes. For wild-type proteins, aliquots were withdrawn after 0, 9, and 30 minutes and diluted into acidic guanidinium chloride to permit characterization in the absence of spectral tuning effects from GUN4 protein.
Files and variables
File: GUN4_spectroscopy_photochemistry.tar.gz
Description: This file contains the four sub-directories listed in the overall README file.
- in_vivo_tetrapyrrole_protein_levels
- oxidation_of_Trp_residues
- fluorescence_of_Trp_residues
- ligand_photochemistry
Additional information for each directory is provided in a README file for that directory. All files are deposited as tab-delimited text files.
Code/software
All files are deposited as tab-delimited text files. Spectroscopic data and protein levels were processed using Kaleidagraph; metabolite levels and mass spectrometry data were processed using Excel. Mass spectrometry data are deposited with a mix of text and floating-point values in some cases, meaning that software packages that infer contents from only the first few lines will not work properly.
Tetrapyrrole levels were measured using published procedures, with initial solvent extraction and subsequent separation using high-performance liquid chromatography (HPLC). Protein levels were measured using Western blotting with subsequent quantitation by densitometry. Trp oxidation used tandem LC-MS, and in vivo measurements used a Strep-tagged allele of GUN4 to facilitate isolation from Chlamydomonas cells. Complementation of the gun4 allele by the Strep-tagged allele was confirmed using growth assays with and without acetate under a range of light conditions that included high light stress and exposure to near-UV, both conditions that are associated with growth defects in gun4 cells. Static fluorescence measurements were taken on a QM-6/2005SE fluorimeter at room temperature (Photon Technology International 814 Series). Fluorescence lifetime measurements were taken using an EasyLife lifetime fluorescence spectrometer (PTI) with a 280 nm LED source, using a 320 nm long-pass Schott glass filter on the emission channel for fluorescence measurements and neutral density filters for measuring the instrument response function (IRF). Dilute solutions of Ludox TM-50 or of Percoll were used to measure the IRF. Absorption spectra were taken at 25 °C using a Cary 60 spectrophotometer. Spectra were acquired every 2 nm over 250-900 nm, with an integration time of 0.125 s per wavelength (equaling 10 flashes at 80 Hz), and photochemical illumination used a high-intensity LED (THORLABs M405L4) from a distance of approximately 1 cm at room temperature in an unsealed cuvette. For absorption spectra under denaturing conditions, 100 µl aliquots were withdrawn at selected times and added to 1 ml of 6 M guanidinium chloride/1 % HCl (v/v).