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.