Dual-Cys bacteriophytochromes: intermediates in cyanobacterial phytochrome evolution?

This work (Yang et al., "Dual-Cys bacteriophytochromes: intermediates in cyanobacterial phytochrome evolution?" in FEBS. J.) involves the characterization of new cyanobacterial phytochrome photoreceptor proteins and engineering chromophore preferences via Cysteine mutagenesis both in those proteins and in known reference proteins. Proteins were characterized using absorption and mass spectrometry, phylogenetic analysis, and X-ray crystallography. The crystal structure is available through the RCSB PDB (PDB ID: 9JRY). All other data are in this deposit.

Methods
Three types of data are deposited: absorption spectroscopy, mass spectrometry, and phylogeny.

Absorption data were collected at 16°C or 26°C using a Shimadzu UV-1900 spectrophotometer equipped with a TCC-100 temperature controller. Data were exported as text files.

Selected reaction monitoring mass spectrometry (SRM-MS) analysis was conducted using a 1290 Infinity Ⅱ Bio 2D-LC System (Agilent Technologies, USA) connected with a 6495D Triple Quadrupole mass spectrometer (Agilent Technologies, USA) with a Jet-stream electrospray source. The separation was performed on a ZORBAX Eclipse Plus C18 column (ID: 959759-902, length: 150 mm, pore size: 95 Å, particle size: 1.8 μm, column temperature 40℃) (Agilent Technologies, USA) using Mobile Phase A [0.1% (vol/vol) formic acid in water] and Mobile Phase B [0.1% (vol/vol) formic acid in acetonitrile]. The peptides derived from protein digestion were separated using a 30-minute gradient. The gradient started at 3% Mobile Phase B for 4 min, then increased to 25% for 22 min, 40% for 24 min, and 60% for 25 min. Finally, the system was equilibrated to 3% for 28 min. The separation was performed at a flow rate of 0.4 mL/min. The samples, with a concentration of 0.48 mg/mL, were introduced into the column by injection with 5 μL. SRM-MS Data were analyzed using Skyline.

For phylogenetic analysis, a multiple sequence alignment of 175 sequences and 995 characters was constructed in MAFFT v7.450 (command-line settings --genafpair --maxiterate 16 --clustalout –reorder). After gap removal (5% cutoff), 477 characters remained. The resulting file was used to infer a maximum likelihood phylogeny using PhyML-3.1 (command-line settings -m WAG -d aa -s SPR -a e -c 4 -v e -o tlr). Support was evaluated using the Shimodaira–Hasegawa approximate likelihood test as implemented in PhyML (SH-aLRT).