DOI: 10.5061/dryad.kh18932j2
title: Dual-Cys bacteriophytochromes: intermediates in cyanobacterial phytochrome evolution?

description: 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.
description: 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).
description: # Dual-Cys bacteriophytochromes: intermediates in cyanobacterial
 phytochrome evolution? ## Description of the Data and file structure Data
 are deposited as a gzipped tarball. After extraction, deposited data are
 in three folders: I. Phylogeny II. Mass_Spec III. Absorption_Spectra ####
 I. Phylogeny: Data are all within a single folder, without subdirectories.
 A single phylogeny was inferred for this study, and the deposited files
 are all from this analysis. Three files are deposited: the original
 multiple sequence alignment in CLUSTAL format, the input file for PHYML in
 PHYLIP format after removal of gap-enriched columns, and the final tree
 with transfer bootstrap expectation values in Newick format. The files
 are: DCB_tree.aln (CLUSTAL format) This is the original alignment,
 generated in MAFFT using the E-INS-i algorithm (v7.450). After a header
 indicating the MAFFT version, aligned sequences are presented as
 interleaved blocks. Each block has a segment of each sequence after the
 name of the sequence, with '-' serving as the gap character. As
 an example, here are three arbitrary protein sequences in CLUSTAL format:
 protein MACDEFG--HIKLM prot_2 MGSNEYPGGHLRVW another MSSMEFG--HVQIS
 protein NPQRSTVWY prot_2 PG-KTTLFY another G--MASIYF DCB_tree.phy (Input
 PHYLIP) For phylogenetic inference in PHYML, the CLUSTAL-format alignment
 was converted into the PHYLIP format with the removal of gap-enriched
 positions. The resulting file is now sequential (with one protein sequence
 followed by the next). The header line is also different, indicating the
 number of sequences (3, in the above example) and positions or characters
 (19, in the above case). For the above example, the converted case looks
 like this: 3 19 protein MACDEFGHIKLMNRSTVWY prot_2 MGSNEYPHLRVWPKTTLFY
 another MSSMEFGHVQISGMASIYF (note however that this example is for
 illustrative purposes only and is NOT the actual file deposited).
 DCB_tree.txt (Newick tree) The resulting phylogeny is presented in the
 Newick format, suitable for use with a broad range of tree viewers but not
 well suited for analysis in a text editor or word processor. #### II.
 Mass_spec Data are all within a single folder, without subdirectories.
 Four samples were analyzed using sequential reaction monitoring mass
 spectrometry (SRM-MS). In this technique, the initial ionization of the
 sample is used to select a fraction containing an ion of interest (the Q1
 m/z ratio). This fraction then undergoes a fragmentation step (q2) to
 generate fragment ions (Q3 m/z) for analysis. For this study, the ions
 were peptide fragments generated using proteolytic digestion and then
 separated on a C18 column prior to SRM-MS. Selected peaks on the column
 were analyzed using initial electrospray ionization (Q1 step) followed by
 fragmentation and detection of fragments (Q3). Data from each sample is
 presented in a tab-delimited text file named for that sample: FiDCB_BV.txt
 Wild-type Dual-Cys bacteriophytochrome (DCB) from Fischerella sp. (FiDCB),
 incorporating biliverdin IX-alpha (BV) as a chromophore. FiDCB_C17A_BV.txt
 The C17A variant of FiDCB, in which Cys17 was mutated to Ala, was also
 examined with a BV chromophore. FiDCB_C259I_BV.txt The C259I variant of
 FiDCB, in which Cys259 was mutated to Ile, was also examined with BV.
 FiDCB_WO_Bilin.txt The wild-type protein was examined without a bilin
 chromophore (i.e., apoprotein). In each of these files, the data are
 presented as tab-delimited text with a series of values: * Compound name:
 The assigned initial fragment from the tandem MS analysis. DCB proteins
 can attach to BV through two Cys residues at the same time, so the
 fragments can include discontinuous peptide sequences along with BV. *
 Precursor ion charge state: This is the charge state of the initially
 selected ion in the tandem SRM-MS. * Product ion charge state: This is the
 charge state of the second, detected ion in the tandem SRM-MS. * Fragment
 Ion: Each assigned secondary fragment is indicated. Only discontinuous
 fragments are explicitly named; continuous fragments were instead assigned
 alphanumeric codes based on standard fragmentation schemes. * Light
 Precursor ion Q1 (m/z): Selected m/z value from the initial ionization
 step (Q1). * Light Product ion Q3 (m/z): Observed m/z value after
 collision (Q3). * Collision Energy: The collision energy for that fragment
 at the fragmentation step (q2). * Retention time: Retention time for the
 initial chromatography step. * Area: Integrated peak area from the initial
 chromatography step. #### III. Absorption_Spectra Most work was performed
 with an *E. coli* expression system, but a subset of proteins was also
 examined using expression in the model cyanobacterium *Synechocystis* sp.
 PCC 6803. Data from the spectroscopic characterization of these proteins
 is organized in two folders by expression system. All data files are
 supplied as tab-delimited text with UNIX newlines. Spectra are therefore
 in 2 folders: Synechocystis_expression E_coli_expression
 Synechocystis_expression Within this folder, one file is provided for each
 protein that was studied in this expression system. Each file contains the
 absorption spectra for that protein. The individual files are as follows:
 DrBphP.txt *Deinococcus radiodurans* bacteriophytochrome FiDCB.txt
 *Fischerella* sp. dual-Cys bacteriophytochrome FiTCCP.txt *Fischerella*
 sp. tandem-Cys cyanobacterial phytochrome Le3CP.txt *Leptolyngbya* sp.
 candidate 3-Cys phytochrome NcDCB.txt *Nostoc carneum* dual-Cys
 bacteriophytochrome ToBphP2.txt *Tolypothrix* sp. bacteriophytochrome 2
 ToCph1.txt *Tolypothrix* sp. cyanobacterial phytochrome 1 ToDCB.txt
 *Tolypothrix* sp. dual-Cys bacteriophytochrome ToTCCP.txt *Tolypthrix* sp.
 tandem-Cys cyanobacterial phytochrome The same data are presented for each
 protein, so the organization of each file is identical. Each protein
 expressed in *Synechocystis* was analyzed under native and denaturing
 conditions. Denaturation used acidic guanidinium chloride. For native
 conditions, spectra are presented for the dark-adapted (15Z) state and for
 the photoproduct (15E). For denaturing conditions, the difference spectrum
 between the two photostates is presented. This difference spectrum was
 calculated as (15Z - 15E). Therefore, each file contains the following
 columns: Wavelength (nm) wavelengths for native spectra, in nanometers 15Z
 Absorbance values for the 15Z photostate 15E Absorbance values for the 15E
 photostate d wavelength (nm) wavelengths for denatured difference spectra
 Denatured difference spectra delta-absorbance values (15Z - 15E)
 E_coli_expression Most of the work was done with protein produced in *E.
 coli*, which allowed production at higher levels with higher purity. Data
 are organized into a series of eight folders, each of which has data for
 one set of experiments. The folders are: Native_wildtype_new_proteins
 DCB_single_Cys_removal DCB_second_Cys_addition TCCP_Cys_variants
 BphP_Cph1_Cys_swaps BphP_Cph1_2Cys_variants FiDCB_BV_color_response
 Conversion_timecourses The Conversion_timecourses data are kinetic traces,
 in which data take the form of a series of time/Absorbance pairs, with
 Absorbance reported at a chosen wavelength of interest. All other
 subdirectories contain absorption spectra or difference spectra, in which
 the data take the form of a series of wavelength/Absorbance pairs (as in
 the data from expression in *Synechocystis*). A description of each
 subdirectory follows: (i) Native_wildtype_new_proteins This folder
 contains absorption spectra for previously uncharacterized proteins under
 native conditions. One file is provided for each protein. Each protein is
 designated by a 2-letter code for the host organism (e.g., "Fi"
 = *Fischerella* sp.) followed by the type of protein in all caps: DCB =
 Dual-Cys bacteriophytochrome TCCP = Tandem-Cys cyanobacterial phytochrome
 3CP = candidate 3-Cys cyanobacterial phytochrome Therefore, the files are:
 FiDCB.txt Data for FiDCB (dual-Cys bacteriophytochrome from *Fischerella*
 sp.) FiTCCP.txt Data for TCCP from *Fischerella* sp. Le3CP.txt Data for
 possible 3CP from *Leptolyngbya* sp. NcDCB.txt Data for DCB from *Nostoc
 carneum* ToDCB.txt Data for DCB from *Tolypothrix* sp. ToTCCP.txt Data for
 TCCP from *Tolypothrix* sp. Each of these files is TAB-delimited and has
 the following columns: Wavelength (nm) wavelength for wild-type protein
 with PCB chromophore WT (PCB) 15Z Absorbance for that wavelength in the
 15Z dark-adapted state WT (PCB) 15E Absorbance for the wavelength in the
 15E photoproduct state Wavelength (nm) wavelength for wild-type protein
 with BV chromophore WT (BV) 15Z Absorbance for that wavelength in the 15Z
 dark-adapted state WT (BV) 15E Absorbance for the wavelength in the 15E
 photoproduct state Wavelength (nm) wavelength for the dark-minus-light PCB
 difference spectrum 15Z-15E PCB delta-Absorbance for PCB, as (dark-light)
 or (15Z-15E) Wavelength (nm) wavelength for the dark-minus-light BV
 difference spectrum 15Z-15E BV delta-Absorbance for PCB, as (dark-light)
 or (15Z-15E) (ii) DCB_single_Cys_removal This folder contains spectra for
 variants in DCB proteins in which a single Cys residue has been replaced
 by site-directed substitution mutagenesis. Each file contains data for one
 such variant and is named by the parent protein and the substitution
 (e.g., "FiDCB C17A.txt" contains data for the C17A variant of
 FiDCB, in which Cys17 was replaced with Ala). The individual files are:
 FiDCB C17A.txt FiDCB C259I.txt FiDCB C259L.txt FiDCB C259M.txt NcDCB
 C257I.txt NcDCB C257L.txt NcDCB C257M.txt NcDCB C8A.txt ToDCB C289I.txt
 ToDCB C289L.txt ToDCB C289M.txt ToDCB C40A.txt Each of these files
 contains the absorption spectra for the given protein with BV and PCB
 chromophores in each photostate, except for the C40A variant of ToDCB
 (ToDCB C40A.txt). This variant did not undergo photoconversion with BV, so
 only a 15Z spectrum is provided for this chromophore. Each file is thus of
 the following format: Wavelength (nm) wavelength for the spectra, in
 nanometers BV 15Z Absorbance with BV in the 15Z state BV 15E Absorbance
 with BV in the 15E state (absent in ToCDB C40A) PCB 15Z Absorbance with
 PCB in the 15Z state PCB 15E Absorbance with PCB in the 15E state (iii)
 DCB_second_Cys_addition This folder contains spectra for similar variants
 in which the "second Cys" of TCCP proteins was introduced via
 substitution into different DCB proteins in different contexts. The naming
 convention is the same as in (ii) above; e.g., "FiDCB C17A
 H260C.txt" has data for the C17A H260C variant of FiDCB. The
 individual files are: FiDCB C17A H260C.txt FiDCB H260C.txt NcDCB C8A
 H258C.txt NcDCB H258C.txt ToDCB C40A H290C.txt ToDCB H290C.txt Each file
 has the same format as in (ii), as follows: Wavelength (nm) wavelength for
 the spectra, in nanometers BV 15Z Absorbance with BV in the 15Z state BV
 15E Absorbance with BV in the 15E state PCB 15Z Absorbance with PCB in the
 15Z state PCB 15E Absorbance with PCB in the 15E state (iv)
 TCCP_Cys_variants: This folder contains spectra for variant proteins in
 which similar Cys substitutions were made in TCCP/3CP proteins. This work
 demonstrated that the candidate "3CP" protein is in fact a
 standard TCCP protein, so the folder name reflects that conclusion. The
 file organization matches that used in (ii) and (iii). The individual
 files are: FiTCCP C19A C257H.txt FiTCCP C19A.txt FiTCCP C257H.txt Le3CP
 C19A C260H.txt Le3CP C19A.txt Le3CP C259M C260H.txt Le3CP C260H.txt ToTCCP
 C258H.txt ToTCCP H17C C257M C258H.txt ToTCCP H17C C258H.txt ToTCCP
 H17C.txt Each file is in the following format: Wavelength (nm) wavelength
 for the spectra, in nanometers BV 15Z Absorbance with BV in the 15Z state
 BV 15E Absorbance with BV in the 15E state (absent in some) PCB 15Z
 Absorbance with PCB in the 15Z state PCB 15E Absorbance with PCB in the
 15E state (absent in some) For the C259M C260H variant of Le3CP (Le3CP
 C259M C260H.txt), no photoproduct was formed with PCB and therefore there
 is no column for "15E PCB" included. For the ToTCCP C258H
 variant, no photoproduct was formed with BV and thus there is no column
 for "15E BV" included. For the ToTCCP H17C C257M C258H variant,
 no photoproduct was observed with either chromophore, so only 15Z columns
 are included. (v) BphP_Cph1_Cys_swaps As a comparison to the work
 performed on DCB proteins, similar Cys substitutions were made in
 "reference" proteins belonging to previously recognized types of
 bacterial phytochromes: Cph1 and BphP proteins. Both previously published
 reference proteins (SyCph1 and DrBphP) and newly characterized ones
 (ToBphP1, ToBphP2, and ToCph1) were used for these studies. Each file
 contains the absorption spectra for one protein; wild-type proteins are
 designated by "WT" in the filename, and variants are designated
 by the substitution(s) present in each case along with the name of the
 parent protein (matching above). DrBphP C24A M259C.txt DrBphP M259C.txt
 DrBphP WT.txt SyCph1 L15C E16D C259I.txt SyCph1 L15C E16D.txt SyCph1
 WT.txt ToBphP1 C19A I259C.txt ToBphP1 I259C.txt ToBphP1 WT.txt ToBphP2
 C24A L271C.txt ToBphP2 L271C.txt ToBphP2 WT.txt ToCph1 L15C K16D.txt
 ToCph1 L15C K16D C259L.txt ToCph1 WT.txt Each file is in the following
 format: Wavelength (nm) wavelength for the spectra, in nanometers BV 15Z
 Absorbance with BV in the 15Z state BV 15E Absorbance with BV in the 15E
 state PCB 15Z Absorbance with PCB in the 15Z state PCB 15E Absorbance with
 PCB in the 15E state (absent in one) ToCph1 L15C K16D C259L.txt does not
 contain a column for 15E PCB, because this variant did not exhibit
 formation of the relevant photoproduct. (vi) BphP_Cph1_2Cys_variants: As a
 comparison for the introduction of the TCCP second Cys into DCB proteins,
 similar experiments were again performed with the same panel of BphP and
 Cph1 reference proteins. One file is provided for each of the resulting
 variants, using the same convention in the filenames. Individual files are
 as follows: DrBphP C24A M259C H260C.txt DrBphP M259C H260C.txt SyCph1
 H260C.txt SyCph1 L15C E16D H260C.txt ToBphP1 C19A I259C H260C.txt ToBphP1
 I259C H260C.txt ToBphP2 C24A L271C H272C.txt ToBphP2 L271C H272C.txt
 ToCph1 H260C.txt ToCph1 L15C K16D H260C.txt Each file is in the following
 format: Wavelength (nm) wavelength for the spectra, in nanometers BV 15Z
 Absorbance with BV in the 15Z state BV 15E Absorbance with BV in the 15E
 state (absent in two) PCB 15Z Absorbance with PCB in the 15Z state PCB 15E
 Absorbance with PCB in the 15E state (absent in two) Two variants
 exhibited no photoconversion with either chromophore, so the 15E columns
 are not present in these files (DrBphP C24A M259C H260C.txt and ToBphP2
 C24A L271C H272C.txt). (vii) FiDCB_BV_color_response The color response
 was tested for one DCB protein, FiDCB from *Fischerella* sp. PCC 9605,
 with biliverdin IX-alpha (BV) as a chromophore and with a series of
 different illumination wavelengths. The resulting absorption spectra are
 presented as a series of files, with each file named by the wavelength of
 light used for illumination. Individual files are: 380 nm.txt 405 nm.txt
 460 nm.txt 525 nm.txt 590 nm.txt 620 nm.txt 660 nm.txt 740 nm.txt Each of
 these files has the same columns: wavelength in nm, then 15Z and 15E
 absorption spectra named by the illumination wavelength and the
 configuration. For example, the titles and first data point under 740 nm
 illumination look like this: Wavelength (nm) 740nm 15Z 740nm 15E 250.00
 0.51200 0.52100 (note that tab-delimited text does not line up in the
 README file). (viii) Conversion_timecourses The kinetics of forward
 photoconversion and dark reversion were examined for selected proteins
 (both wild-type and Cys variants) and are deposited as single-wavelength
 Time/Absorbance traces. In this case, each file contains several such
 traces for different proteins to permit easy comparison without having to
 reassemble traces from different files. The filenames thus provide
 approximate "themes" for comparison, along with the direction of
 conversion. In each case, data are presented at both 16 deg. C and 26 deg.
 C. Within each file, the columns are designated as time (in seconds) and
 then by protein and temperature. For example, the first few headers from
 BphP_BV_forward look like this: Time (sec.) FiDCB_PCB_16 FiDCB_PCB_26
 FiDCB_C17A_PCB_16 ... Time (sec.) FiDCB_PCB_16 is the forward conversion
 of FiDCB with PCB at 16 deg. C FiDCB_PCB_26 is the same preparation but at
 26 deg. C FiDCB_C17A_PCB_16 is the C17A variant of FiDCB with PCB at 16
 deg. C (and so on) The individual files are as follows:
 BphP_BV_forward.txt This file includes forward photoconversion traces for
 wild-type FiDCB, for the FdiDCB C259I variant mimicking BphP proteins, and
 for three reference BphP proteins. BphP_BV_reverse.txt This file includes
 thermal relaxation (dark reversion) data for the same proteins. This is
 formally a reverse isomerization reaction relative to the forward
 photoconversion, so the filename reflects that fact. Cph1_PCB_forward.txt
 This file includes forward photoconversion traces for wild-type FiDCB, for
 the FdiDCB C17A variant mimicking Cph1 proteins, and for the reference
 protein SyCph1. Here, the chromophore is phycocyanobilin (PCB).
 Cph1_PCB_reverse.txt This file contains dark reversion data for the same
 proteins. FiDCB_various_forward.txt This file combines the FiDCB data from
 the above "forward" files for easy comparison to each other. No
 reference proteins are included. FiDCB_various_reverse.txt This file is an
 equivalent combination but with dark reversion data from the
 "reverse" files. ## Sharing/Access Information NA