Dataset DOI: 10.5061/dryad.qz612jmxd

Sequences used in the study for phylogeny and alignment and data generated in this study.

Rhodopsin, G-protein DNA sequences, and reference sequences for Neogonodactylus oerstedii were retrieved from NCBI. 

Dark absorption spectra, action spectra, and G-protein coupled activity were generated in this study.

Files and variables

File: Data_S1.xlsx

Description: Sequences used in this study for expression and phylogeny. 

Tab 1: Full_rhodopsin_sequences

Column A: Abbreviation – abbreviation used for the rhodopsin in this study.

Column B: Other_names – other names or identifiers associated with the rhodopsin.

Column C: Length_AA – protein length in amino acids.

Column D: DNA_ID – database identifier for the corresponding DNA sequence. GenBank https://www.ncbi.nlm.nih.gov/genbank/

Column E: Protein_ID – database identifier for the corresponding protein sequence in Genbank https://www.ncbi.nlm.nih.gov/genbank/

Column F: DNA_sequence_raw_from_GenBank – nucleotide sequence as obtained from https://www.ncbi.nlm.nih.gov/genbank/

Column G: Amino_acid_sequence_GenBank – amino-acid sequence corresponding to GenBank. https://www.ncbi.nlm.nih.gov/genbank/

Tab 2: Partial_rhodopsin_sequences excluded from study

Partial rhodopsin sequences retrieved from GenBank. Columns have the same definitions as in Full_rhodopsin_sequences.

Tab 3: Reference_rhodopsin_sequences

Reference rhodopsin protein sequences used for sequence comparisons.

Column A: Gene – gene/protein name.
Column B: Organism – source organism.
Column C: PDB_ID – Protein Data Bank identifier, where applicable.
Column D: Amino_acid_sequence – amino acid sequence.

Tab 4: Gprotein_sequences

G-protein sequences used in the study.

Column A: Gene – gene/protein name.
Column B: Organism – source organism.
Column C: Source – source of the sequence.
Column D: Length – protein length.
Column E: Amino_acid_sequence – amino acid sequence.

File: Data_S2.xlsx

Description: Contains dark-state absorption spectra for the indicated rhodopsins.

Worksheets in order:

LWS_R3, NOL1, NOL2, NOL6, NOL7, NOL8, NOL9, NOL12, NOL14, NOL15, NOL16, NOL17, NOL19, NOL20, NOM5, and NOM6.

The worksheets contain the following variables:

Column A: Wavelength (nm) – wavelength of the measurement in nanometers.
Column B: Wavenumber (µm^-1) – reciprocal wavelength expressed as wavenumber in µm^-1.
Column C: Fit Datapoints – data points used for spectral fitting, where applicable.
Column D: Averaged norm. absorbance – averaged normalized absorbance.
Column E: SEMs – standard errors of the mean for the normalized absorbance, where available.

Normalization is dimensionless. Absorbance values are reported as normalized values rather than absolute extinction coefficients.

File: Data_S3.xlsx

Description: Contains wavelength-dependent light-induced luminescence measurements for the indicated rhodopsins, including measurements for activating light stimulus (on) and, where available, after deactivating light stimulus followed by an activation light stimulus (off).

Worksheets:

NOL12_on, NOL13_on, NOL13_off, NOL14_on, NOL14_off, NOL16_on, NOL17_on, NOL19_on, NOM5_on, NOM5_off, NOM6_on, and NOM6_off.

The worksheets contain the following variables:

Column A: Wavelength (nm) – wavelength of the measurement in nanometers.
Column B: Wavenumber (µm^-1) – reciprocal wavelength expressed as wavenumber in µm^-1.
Column C: Fit Datapoints – data points used for spectral fitting, where applicable.
Column D: Averaged norm. luminescence – averaged normalized luminescence.
Column E: SEMs – standard errors of the mean for the normalized luminescence.
Normalized luminescence values are dimensionless.

Action spectra were measured using the Aequorin Gq-assay. Light stimulations were delivered using a Polychrome V (TILL Photonics) in combination with neutral density filters to keep an equal photon flux across wavelengths.

Wavelength-dependent aequorin luminescent activation/deactivation responses. Activation is induced by a 1s light pulse of fixed wavelengths (1x10¹⁶ photons cm^-2 s^-1,n=3 experiments). Deactivation is assayed by two consecutive 1s light pulses, one with the fixed wavelength leading to maximal activation followed by a second light pulse of varying wavelength.

File: Data_S4.xlsx

Description: Contains light-intensity titration measurements used to assess rhodopsin activity in the presence of different G-protein subtypes, together with normalized datasets and fitted dose-response parameters.

The rhodopsin-specific worksheets include:

GqX_GsX NOL12, GqX_GsX NOL13, GqX_GsX NOL14, GqX_GsX NOL16, GqX_GsX NOL17, GqX_GsX NOL19, GqX_GsX NOM5, GqX_GsX NOM6

Variables:

• Light stimulus (Log10 Photons/cm²/s) – logarithm (base 10) of the incident photon flux in photons cm^{-2 s}-1.
• Columns named <condition>_<replicate> – individual experimental measurements for the indicated rhodopsin/G-protein condition and replicate.

Examples of Gq conditions include noGqX, Gqs, Gqi, Gqo, Gqz, Gq, Gq12, Gq13, Gq15, Gq14, and GqNO. Gs-family conditions include noGsX, Gs, Gsi, Gso, Gst, Gsz, Gsq, Gs12, Gs13, and Gs15, as applicable to the experiment.

The suffix _1, _2, etc. identifies biological/experimental replicates within a condition.

Worksheets beginning with GqX_norm contain normalized light-intensity titration data for NOL12, NOL13, NOL14, NOL16, NOL17, and NOL19.

Normalization was performed within the corresponding experimental dataset; normalized response values are dimensionless.

GqX_GsX_Statistical_parameters and GqX_norm_parameters contain parameters obtained from light-intensity response curve fitting.

Variables:

Column A: Condition – experimental rhodopsin/G-protein condition.
Column B: Bottom – fitted lower asymptote.
Column C: Top (Emax) – fitted upper asymptote/maximal response.
Column D: LogEC50 – base-10 logarithm of the EC50 expressed in photon flux units.
Column E: EC50 – fitted half-maximal effective photon flux.
Column F: Span – difference between fitted top and bottom values.
Column G: R2 – coefficient of determination for the fitted curve.
Column H: Sy.x – standard deviation of the residuals from the fitted curve.
Column I: n (Y) – number of response observations included in the fit.
Column J: GsX Selectivity (%) or GqX selectivity (%) – calculated selectivity metric, where applicable.

Luminescence was measured for 1s every 60s. Pre-illumination baseline was recorded for 5 cycles before recording was paused and the plate ejected. Light-dependent GsX responses were acquired using light-titration curves. Each well was exposed once to a single light intensity pulse of the same wavelength, starting from the highest intensity and gradually decreasing in 0.5 log10 steps to capture the peak response, especially small signals from low intensities.

As NOL opsins did not signal efficiently through GsX proteins, GqX chimeric proteins were employed instead, following the same principle. When LogEC50 is unstable, the Emax is set manually to 0 to avoid aberrant GqX selectivity values. 

File: Data_S5.xlsx

Description: Luminescence response coupling to human G-proteins Gs, Gi and Gq.

Worksheets:
Gs, Gs_NOL14, Gs_NOMS, Gi, Gi_NOL14, Gi_NOL20, Gi_NOMS, Gq, Gq_NOL12,L16,L17,L19, Gq_NOL13, Gq_NOL14_L20, Gq_NOMS

Variables in these worksheets generally include:

• Time (min) or Time (s) – time after the start of the assay, in minutes or seconds as indicated by the worksheet.
• no opsin_<replicate> – measurements from the no-opsin control.
• <rhodopsin>_<replicate> – measurements from the indicated rhodopsin condition.

The exact rhodopsin conditions and number of replicates vary among worksheets.

Worksheets ending in _parameters contain statistical comparisons corresponding to the associated time-course dataset.

Depending on the analysis, variables include:

Column A: Analysis_Comparison – comparison performed.
Column B: Test_Applied – statistical test used.
Column C: n1– sample sizes for the two compared groups.
Column D: n2– sample sizes for the two compared groups.
Column E: Mean_1– group 1 mean.
Column F: SEM_1– standard errors of group 1 mean.
Column G: Mean_2 – group 2 mean.
Column H: SEM_2– standard errors of group 2 mean.
Column I: Mean_Diff – difference between group means.
Column J: SEM_Diff – standard error of the difference.
Column K: t_stat – test statistic for analyses using a t-test.
Column L: df – degrees of freedom.
Column M: p_value – p-value, where applicable.
Column N: Tail – directionality of the statistical test, where applicable.
Column O: Significance – significance classification, where applicable. Non-significance is marked as "ns".

The Gq_NOMS_parameters worksheet uses median-based statistics and therefore contains Median_1, Median_2, Median_Diff_HL, and U_stat rather than the corresponding mean/t-test variables.

Luminescence was measured from a single well in a plate reader (Infinite 200Pro, Tecan) with an integration time of 1s every 0.5s for Gq assays, every 60s for Gs and with an integration time of 300ms every 0.5s for Gi assays. Dark luminescent baseline was measured for 5 cycles until the measurement was paused and the plate ejected for illumination. 

For Gq assays, one well was subjected to a 1s illumination using a pE-4000 (coolLED Ltd.) and measured. For Gs assays, luminescence was measured for all wells before ejection. 1s light pulse was applied subsequently to each well before the measurement was resumed. 

For Gi assays, 2µM forskolin (Sigma-Aldrich) was added to each well to elevate cAMP levels and recording was resumed for 20-30min and illuminated as before. In the 3 assays, luminescent responses were compared to cells transfected only with the corresponding luminescent reporter, subjected to the same illumination conditions as the opsin. 

File: Data_S6.xlsx

Description: Activity measurements for comparison of cloning strategies.

In order to establish which cloning strategy is best for the expression and activity study of the rhodopsins in the study, a luminescence measurement was done comparing different constructs. Comparison of relative aequorin luminescent levels of the most effective (highest signal) constructs for each opsin following a 1s activation. Titration is defined as light luminescence response at different light intensities. 

Worksheets:

LWS_R3 titration, NOL1 titration, NOL2 titration, NOL6 titration, NOL7 titration, NOL12 titration, NOL13 titration, NOL14 titration, NOL16 titration, NOL17 titration, NOL19 titration, NOM5 titration, and NOM6 titration.

Each titration worksheet contains:

Column A: Light stimulus (Log10 Photons/cm²/s) – logarithm (base 10) of the incident photon flux in photons cm^{-2 s}-1.
Column B and onward: <construct>_<replicate> – response measurement for the indicated rhodopsin construct and replicate.

Construct names identify the rhodopsin and experimental construct:

• Lucyrho-... – rhodopsin-containing Lucy-Rho N-terminal tag for trafficking and expression.
• T2A-SiriusFP – reference/control construct containing SiriusFP.
• mScarlet-ER – mScarlet-tagged construct targeted to the endoplasmic reticulum with fluorescent tag.
• IRES-mCitrine – construct containing an IRES-linked mCitrine reporter.
• Δ17 – construct lacking the indicated N-terminal 17 amino acids.
• Ntrunc16AA – construct with the indicated N-terminal 16-amino-acid truncation.
• 1D4 – construct containing the 1D4 epitope/tag in the C-terminal end.

The replicate suffix (_1, _2, etc.) identifies individual experimental replicates.

Data_S6.xlsx also contains the following worksheets:

LWS_R3 titration_norm, NOL1 titration_norm, NOL2 titration_norm, NOL6 titration_norm, NOL7 titration_norm, NOL12 titration_norm, NOL13 titration_norm, NOL14 titration_norm, NOL16 titration_norm, NOL17 titration_norm, NOL19 titration_norm, NOM5 titration_norm, NOM6 titration_norm

These contain normalized versions of the corresponding titration measurements. Normalized response values are dimensionless.

NOL8,L9,L15,L20_traces contains time-resolved traces for the indicated rhodopsins.

Column A: Time (s) – time in seconds.
Column B onward, <construct>_<replicate> – measured response for the indicated construct and replicate.

NOLs-NOMs_titrations_parameters contains fitted parameters for the rhodopsin titration curves.

Same columns as the Data_S4 parameter sheets: Condition, Bottom, Top (Emax), LogEC50, EC50, Span, R2, Sy.x, n (Y), with the same definitions and units. Condition is the construct name with the replicate suffix removed.

Variables:

Column A: Condition – rhodopsin/construct condition.
Column B: Bottom – fitted lower asymptote.
Column C: Top (Emax) – fitted upper asymptote/maximal response.
Column D: LogEC50 – base-10 logarithm of EC50.
Column E: EC50 – fitted half-maximal effective photon flux.
Column F: Span – difference between fitted top and bottom values.
Column G: R2 – coefficient of determination.
Column H: Sy.x – standard deviation of the residuals from the fitted curve.
Column I: n (Y) – number of response observations included in the fit.

Missing data and data conventions

• NA denotes a condition that was not measured.
• N/A indicates that the value is not applicable to the corresponding measurement or condition.
• Blank cells are used where no measurement was performed (abortive measurement due to unresponsive sample).
• In all raw and normalized measurement sheets, numeric zeroes are actual recorded/calculated zero values and should not be interpreted as missing data.
• In statistical parameters of Data_S4.xlsx when the LogEC50 is unstable, the Emax is manually set to 0 to avoid aberrant GqX selectivity values percentage.
• Replicate numbers identify separate measurements within the same experimental condition.
• The different levels of significance are indicated by * for p < 0.05, ** for p < 0.01, *** if p < 0.001, and **** if p < 0.0001.
• Normalized measurements are dimensionless unless otherwise stated.

Software and analysis

The Excel workbooks are intended to be readable using standard spreadsheet software capable of opening .xlsx files.

Curve-fitting and statistical-analysis results are provided directly in the parameter worksheets so that the deposited dataset contains the numerical results used for the reported analyses. The original experimental measurements are retained in the corresponding data worksheets.

Access information

Other publicly accessible locations of the data:

• GenBank
• Uniprot
• PDB Protein Data Bank

Data was derived from the following sources:

• https://www.ncbi.nlm.nih.gov/genbank/
• https://www.uniprot.org/
• https://www.rcsb.org/