DOI: 10.5061/dryad.rn8pk0pp7
title: Data for: Circular dichroism spectroscopy reveals multiple phytochrome photoproducts in equilibrium

description: This work involves characterization of the model cyanobacterial
 phytochrome photoreceptor Cph1 from Synechocystis sp. PCC6803, using
 characterization of different truncations and variant proteins constructed
 using site-directed mutagenesis. Wild-type Cph1 photoconverts between two
 photostates: a red-absorbing, dark-adapted Pr state in which the
 phycocyanobilin (PCB) chromophore is in the 15Z configuration, and a
 far-red-absorbing Pfr photoproduct in which PCB is instead in the 15E
 configuration. Proteins were characterized using absorption and circular
 dichroism spectroscopy. Absorption spectra were taken under native and
 denaturing conditions. Absorption and circular dichroism (CD) spectra were
 also taken at different pH values. All variants were examined after
 recombinant co-expression with enzymes for the synthesis of PCB in E.
 coli. One additional variant was also examined after expression and
 purification without chromophore biosynthesis, followed by in vitro
 assembly with PCB.
description: Absorption spectra were acquired using Cary 50 or Cary 60
 spectrophotometers. CD spectra were acquired using an
 Applied Photophysics Chirascan circular dichroism spectrometer.
description: # Data for: Circular dichroism spectroscopy reveals multiple phytochrome
 photoproducts in equilibrium ## Description of the Data and file structure
 Data are deposited as a gzipped tarball containing a series of
 tab-delimited text files with Unix newlines. After extraction, the
 deposited data are in six folders: I. Standard_native_absorption II.
 Standard_CD III. In_vitro_assembly IV. Estimated_photoisomerization V.
 Analysis_of_CD VI. Analysis_of_pH_effects ### I.
 Standard\_native\_absorption: This folder contains absorption spectra for
 purified proteins after co-expression with PCB biosynthesis enzymes,
 measured under standard conditions (pH 7.8). Two wild-type sequences were
 examined: a truncation having the first 322 N-terminal amino acids (N322),
 and another having the first 514 amino acids (N514). The N514 construct
 was also used to construct a series of variant proteins via site-directed
 mutagenesis. Absorption spectra for each of these proteins are included in
 this folder, for a total of 22 proteins. Spectra for each protein are
 included in a file named for that protein; thus,
 "N514_F178A.txt" contains absorption spectra for the F178A
 variant in the N514 construct, whereas "N322_wildtype.txt"
 contains absorption spectra for the wild-type N322 sequence. Each file
 contains a wavelength column and two spectra, demonstrating
 photoconversion of each protein between two photostates. In the case of
 N514_Y203A.txt, these spectra are labeled "before R" and
 "after R (higher 15E)". For this protein, "before R"
 means "before red light illumination," and "after R (higher
 15E)" means "after red light illumination" (red light
 resulted in the formation of higher levels of the 15E isomer of the PCB
 chromophore). In this case, the 15E chromophore could not be efficiently
 reverted to 15Z using far-red light or incubation in darkness, so data are
 presented in this form. In the other cases, cleaner 15Z and 15E
 populations could be obtained. In those cases, the spectra are labeled as
 "15Z' (equivalent to the Pr form of wild-type protein) and
 "15E" (equivalent to the Pfr form). However, many variants did
 not exhibit Pfr formation but did exhibit formation of the 15E chromophore
 isomer. Absorption spectra have been baseline-corrected. The individual
 files in this folder are: N322_wildtype.txt N514_wildtype.txt
 N514_F178A.txt N514_F178I.txt N514_Y198F.txt N514_L201A.txt N514_H202A.txt
 N514_Y203A.txt N514_Y203F.txt N514_S206A.txt N514_D207N.txt N514_I208L.txt
 N514_I208V.txt N514_M267A.txt N514_H290A.txt N514_E480A.txt N514_R441A.txt
 N514_W450A.txt N514_S474A.txt N514_S474C.txt N514_F475A.txt N514_W478A.txt
 ### II. Standard\_CD: This folder contains CD spectra for the samples
 examined in the "Standard_native_absorption" folder, except for
 F178A, which gave poor results. Each file contains a wavelength column and
 two spectra, which have been zeroed and buffer-subtracted. The Y203A
 variant (file N514_Y203A_coexpression_CD.txt) is described as "net
 before light" and "net after light" to reflect the poor
 conversion from 15E to 15Z. All other samples are described as
 "15Z" and "15E" (matching the absorption spectra).
 Individual files are: N322_wildtype_CD.txt N514_wildtype_CD.txt
 N514_F178I_CD.txt N514_Y198F_CD.txt N514_L201A_CD.txt N514_H202A_CD.txt
 N514_Y203A_coexpression_CD.txt N514_Y203F_CD.txt N514_S206A_CD.txt
 N514_D207N_CD.txt N514_I208L_CD.txt N514_I208V_CD.txt N514_M267A_CD.txt
 N514_H290A_CD.txt N514_R441A_CD.txt N514_W450A_CD.txt N514_S474A_CD.txt
 N514_S474C_CD.txt N514_F475A_CD.txt N514_W478A_CD.txt N514_E480A_CD.txt
 ### III. In\_vitro\_assembly: The Y203A variant was also purified in the
 absence of chromophore biosynthesis (apoprotein) for in vitro assembly
 with PCB. This assay allowed examination of the 15Z state without the
 presence of a 15E population. Three files are presented: 1.
 Y203A_assembly_with_PCB.txt This file contains absorption spectra under
 native conditions for free PCB ("free PCB" column), for scaled
 PCB (dilution-corrected free PCB spectrum, "scaled PCB” column), and
 for holoprotein generated after 2 hours of in vitro assembly ("after
 120' in vitro assembly" column). 2.
 Y203A_assembled_photoreaction.txt This file contains native absorption
 spectra for holoprotein in the initial 15Z state generated by in vitro
 assembly ("pre red light") and after photoconversion to generate
 a mix of 15Z and 15E species ("post red light"). Spectra are
 baseline-subtracted. 3. Y203A_assembly_with_PCB_CD.txt This file contains
 buffer-subtracted CD spectra for holoprotein in the two photostates, with
 "net before red" being the 15Z form before illumination and
 "net after red" being at photoequilibrium. ### IV.
 Estimated\_photoisomerization: Selected samples were also examined using a
 denaturation assay to assess the chromophore composition at
 photoequilibrium. In this assay, 0.1 ml of protein solution was added to 1
 ml of 6M guanidinium chloride/1% HCl (v/v). Under these conditions, the
 15E form can be converted to 15Z by white light. The cyanobacteriochrome
 RcaE can be effectively converted to either configuration in the native
 state using red or green light, permitting calculation of a standard curve
 that can then be used to estimate chromophore configuration at
 photoequilibrium. This folder contains such a standard curve, a series of
 files with assignment of photoconversion using that standard curve, and a
 file compiling the resulting values of estimated photoconversion classed
 into Type 1 constructs (exhibiting formation of the 15E far-red-absorbing
 Pfr state, as in wild-type N514 and full-length protein) and Type 2
 variants (exhibiting formation of an alternate 15E photoproduct). Each
 protein is represented by two files, one containing the denaturation data
 (such as "denatured_N514_D207N.txt") and one containing the
 estimate of photoconversion (such as
 "estimated_15E_N514_D207N.txt"). Each file of denatured data has
 buffer-subtracted spectra for the 15Z and 15E forms of that protein, where
 the "15E" form is at photoequilibrium and the 15Z form is
 generated from that by illuminating the denatured sample with white light.
 Each estimate of photoconversion contains the scaled spectrum for that
 protein along with the reference curves used to make the estimate.
 Proteins are thus represented by the following files:
 denatured_N322_wildtype.txt estimated_15E_N322_wildtype.txt
 denatured_N514_D207N.txt estimated_15E_N514_D207N.txt
 denatured_N514_E480A.txt estimated_15E_N514_E480A.txt
 denatured_N514_F178A.txt estimated_15E_N514_F178A.txt
 denatured_N514_F178I.txt estimated_15E_N514_F178I.txt
 denatured_N514_F475A.txt estimated_15E_N514_F475A.txt
 denatured_N514_I208L.txt estimated_15E_N514_I208L.txt
 denatured_N514_I208V.txt estimated_15E_N514_I208V.txt
 denatured_N514_L201A.txt estimated_15E_N514_L201A.txt
 denatured_N514_R441A.txt estimated_15E_N514_R441A.txt
 denatured_N514_S474A.txt estimated_15E_N514_S474A.txt
 denatured_N514_S474C.txt estimated_15E_N514_S474C.txt
 denatured_N514_W450A.txt estimated_15E_N514_W450A.txt
 denatured_N514_W478A.txt estimated_15E_N514_W478A.txt
 denatured_N514_Y203F.txt estimated_15E_N514_Y203F.txt
 denatured_N514_wildtype.txt estimated_15E_N514_wildtype.txt In addition,
 this directory includes two other files related to this analysis. The file
 "denatured_standard_curve.txt" has reference curves for
 denatured PCB from 100% 15E to 100% 15Z, in 5% increments. The file
 "photoconversion_by_type.txt" contains the resulting estimates,
 sorted into two categories. Type 1 proteins exhibit Pfr formation, whereas
 type 2 proteins instead undergo photoisomerization to the 15E state but do
 not form the far-red-absorbing Pfr photoproduct. ### V. Analysis\_of\_CD:
 This folder contains several files analyzing the CD spectra measured in
 this work. Four files are provided. 1. Gaussian_decomposition_of_N514.txt
 This file contains the Soret region (330-500 nm) of the 15E Pfr CD
 spectrum for wild-type Cph1-N514. That region was fit to three Gaussian
 functions, which are also included in this file along with the sum of the
 Gaussians and the residuals for the fit. 2.
 comparison_R441A_I208L_F475A.txt This file contains a comparison of two
 type 1 variants (R441A and I208L) along with one type 2 variant (475A).
 Spectra for all three in the 15E state are included. The file also
 contains the difference spectrum for the two type 1 variants, calculated
 as R441A - I208L. That difference spectrum is also present after scaling
 (multiplication by 5.0) to facilitate comparison to the F475A spectrum. 3.
 peak_wavelengths_and_CD_intensity_ratios.txt This file includes absolute
 values of relative rotational strength (measured CD divided by the
 corresponding absorbance) for the two chromophore peaks (S0-S1 and S0-S2
 transitions) in selected protein spectra, along with the ratio of those
 absolute values. The absolute value function allows a straightforward
 comparison of type 1 and type 2 cases, even though the transitions are not
 of the same sign, and the data are separated into type 1 and type 2 cases.
 Additionally, 15E S0-S1 peak wavelengths are provided for each case, along
 with equivalent data for wild-type Cph1-N514 at pH 6 and pH 9. 4.
 relative_rotational_strength_by_type.txt This file has relative rotational
 strengths for the 15E S0-S1 transitions (long-wavelength transitions) of
 type 1 and type 2 proteins. Absolute values are also provided for type 2
 proteins. As a further comparison, relative rotational strengths are
 provided for the 15E photoproduct states of selected red/green
 cyanobacteriochromes, distant Cph1 relatives that also use a PCB
 chromophore, and of green/teal cyanobacteriochromes that instead use a
 phycoviolobilin chromopore. ### VI. Analysis\_of\_pH\_effects This folder
 contains spectra recorded for three proteins at different pH values. The
 three proteins are wild-type N322 and N514, along with the S474C variant.
 The files are as follows: 1. N322_wildtype_15Z_Absorbance.txt
 Buffer-subtracted absorption spectra are shown for 15Z Cph1-N322 at
 selected pH values. 2. N322_wildtype_15Z_pH6_photoconversion.txt
 Absorption spectra are shown for 15Z Cph1-N322 at pH 6 before (column
 "15Z") and after ("15Z photoproduct") illumination
 with red light, along with a (pre-post) difference spectrum to show
 photoconversion at pH 6. 3. N322_wildtype_15Z_pH9_photoconversion.txt
 Absorption spectra are shown for 15Z Cph1-N322 at pH 9 before (column
 "15Z") and after ("15Z photoproduct") illumination
 with red light, along with a (pre-post) difference spectrum to show
 photoconversion at pH 9. 4. N322_wildtype_15E_Absorbance.txt
 Buffer-subtracted absorption spectra are shown for 15E Cph1-N322 at pH 6
 and pH 9 after subtraction of residual 15Z signal at each pH. 5.
 N322_wildtype_15E_pH6_photoconversion.txt Absorption spectra are shown for
 15E Cph1-N322 at pH 6 before (column "15E") and after ("15E
 photoproduct") illumination with red light, along with a (pre-post)
 difference spectrum to show photoconversion at pH 6. 6.
 N322_wildtype_15E_pH9_photoconversion.txt Absorption spectra are shown for
 15E Cph1-N322 at pH 9 before (column "15E") and after ("15E
 photoproduct") illumination with red light, along with a (pre-post)
 difference spectrum to show photoconversion at pH 9. 7.
 N322_wildtype_15E_pH_titration.txt Absorption values at 678 nm are
 reported at different pH values for 15E wild-type Cph1-N322 after buffer
 subtraction, subtraction of signals from residual 15Z protein, and
 renormalization. 8. N514_wildtype_15Z_Absorbance.txt Buffer-subtracted
 absorption spectra are shown for 15Z Cph1-N514 at selected pH values. 9.
 N514_wildtype_15Z_pH6_photoconversion.txt Absorption spectra are shown for
 15Z Cph1-N514 at pH 6 before (column "15Z") and after
 ("15E") illumination with red light, along with a (pre-post)
 difference spectrum to show photoconversion at pH 6. 10.
 N514_wildtype_15Z_pH9_photoconversion.txt Absorption spectra are shown for
 15Z Cph1-N514 at pH 9 before (column "15Z") and after ("15Z
 photoproduct") illumination with red light, along with a (pre-post)
 difference spectrum to show photoconversion at pH 9. 11.
 N514_wildtype_15E_Absorbance.txt Buffer-subtracted absorption spectra are
 shown for 15E Cph1-N514 at pH 6 and pH 9 after subtraction of residual 15Z
 signal at each pH. 12. N514_wildtype_15E_pH6_photoconversion.txt
 Absorption spectra are shown for 15E Cph1-N514 at pH 6 before (column
 "15E") and after ("15E photoproduct") illumination
 with red light, along with a (pre-post) difference spectrum to show
 photoconversion at pH 6. 13. N514_wildtype_15E_pH6_DarkReversion.txt
 Absorbance values at 704 nm (reporting the 15E Pfr state) and 662 nm
 (reporting the 15Z Pr state) are reported at different timepoints to allow
 assessment of photoproduct stability at pH 6. 14.
 N514_wildtype_15E_pH9_photoconversion.txt Absorption spectra are shown for
 15E Cph1-N514 at pH 9 before (column "15E") and after ("15E
 photoproduct") illumination with red light, along with a (pre-post)
 difference spectrum to show photoconversion at pH 9. 15.
 N514_wildtype_15E_pH_titration.txt Absorption values at 704 nm are
 reported at different pH values for 15E wild-type Cph1-N514 after buffer
 subtraction, subtraction of signals from residual 15Z protein, and
 renormalization. 16. N514_wildtype_CD_pH6.txt Wild-type Cph1-N514 was
 concentrated 10-fold and then diluted 10-fold into pH 6 buffer to permit
 measurement of CD spectra in each photostate at pH 6. Spectra are reported
 for the 15Z and 15E photostates. 17. N514_wildtype_CD_pH9.txt Wild-type
 Cph1-N514 was concentrated 10-fold and then diluted 10-fold into pH 9
 buffer to permit measurement of CD spectra in each photostate at pH 9.
 Spectra are reported for the 15Z and 15E photostates. 18.
 N514_S474C_15E_Absorbance.txt Denaturation analysis of the S474C variant
 demonstrated that photoconversion was essentially complete, allowing
 analysis of pH effects without correction for residual 15Z species.
 Buffer-subtracted absorption spectra are shown for 15E S474C at selected
 pH values. 19. N514_S474C_15E_pH6_photoconversion.txt Absorption spectra
 are shown for 15E S474C at pH 6 before (column "15E") and after
 ("15E photoproduct") illumination with red light, along with a
 (pre-post) difference spectrum to show photoconversion at pH 6. 20.
 N514_S474C_15E_pH_titration.txt Absorption values at 704 nm are reported
 at different pH values for 15E S474C after buffer subtraction and
 renormalization. 21. peak_wavelengths_and_intensity_ratios.txt Peak
 wavelengths and intensities are reported for absorption spectra at pH 6
 and pH 9, along with the intensity ratio for the two peaks (S1:S2 ratio).
 ## Sharing/Access Information NA ## Version changes **2-July-2025: **This
 version adds data in one file and notes that addition in the internal
 README.txt file. Data were added to the
 "relative_rotational_strength_by_type.txt" file in the
 "Analysis_of_CD" subdirectory to provide an additional
 comparison. The data added are for the photoproduct states of green/teal
 cyanobacteriochromes, extending the previous analysis that included only
 data for red/green cyanobacteriochrome photoproducts.