# Data from: Quantifying a light-induced energetic change in bacteriorhodopsin by force spectroscopy

## Cite this dataset

Jacobson, David; Perkins, Thomas (2024). Data from: Quantifying a light-induced energetic change in bacteriorhodopsin by force spectroscopy [Dataset]. Dryad. https://doi.org/10.5061/dryad.z08kprrm1

## Abstract

Ligand-induced conformational changes are critical to the function of many membrane proteins and arise from numerous intramolecular interactions. In the photocycle of the model membrane protein bacteriorhodopsin (bR), absorption of a photon by retinal triggers a conformational cascade that results in pumping a proton across the cell membrane. While decades of spectroscopy and structural studies have probed this photocycle in intricate detail, changes in intramolecular energetics that underlie protein motions have remained elusive to experimental quantification. Here, we measured these energetics on the millisecond time scale using atomic-force-microscopy–based single-molecule force spectroscopy. Precisely timed light pulses triggered the bR photocycle while we measured the equilibrium unfolding and refolding of the terminal 8-amino-acid region of bR’s G-helix. These dynamics changed when the EF-helix pair moved ~9 Å away from this end of the G helix during the “open” portion of bR’s photocycle. In ~60% of the data, we observed abrupt light-induced destabilization of 3.4 ± 0.3 kcal/mol, lasting 38 ± 3 ms. The kinetics and pH-dependence of this destabilization were consistent with prior measurements of bR’s open phase. The frequency of light-induced destabilization increased with the duration of illumination and was dramatically reduced in the triple mutant (D96G/F171C/F219L) thought to trap bR in its open phase. In the other ~40% of the data, photoexcitation unexpectedly stabilized a longer-lived putative misfolded state. Through this work, we establish a general single-molecule force spectroscopy approach for measuring ligand-induced energetics and lifetimes in membrane proteins.

## README: Dataset Overview:

This repository contains raw data files for the figures presented in "Quantifying a light-induced energetic change in bacteriorhodopsin by force spectroscopy" (PNAS, 2024) by David R. Jacobson and Thomas T. Perkins, including figures from the SI Appendix.

**File Format:**

All data files are in CSV format.

Data are comma delimited.

Units are in SI, except for energies, which are in kcal/mol.

## Data Files and Descriptions:

### Fig2B.csv, Fig2C.csv

```
Description: Data from photoactivation experiments of single bR molecules.
Variables:
In Fig2B.csv
time_index: Time in seconds
Lc_4_1508_18, Lc_18_1054_22, Lc_24_887_112: Contour lengths in meters for three different molecules (destabilizing behavior)
In Fig2C.csv
time_index: Time in seconds
Lc_10_83_58, Lc_11_446_96, Lc_20_814_32: Contour lengths in meters for three different molecules (misfolding behavior)
Corresponding Figure: Fig. 2B and 2C in the paper.
```

### Fig3A.csv, Fig3B.csv

```
Description: Data showing Lc vs. time during a photoactivation event, histogram of Lc in both phases, and calculated energy landscape
Variables:
In Fig3A.csv
time_index: Time in seconds (example time-series data)
Lc_4_1508_18: Contour length in meters (example time-series data)
hist_bins: Bin centers in meters of histogrammed contour-length data
groundHist_4_1508_18: Normalized histogram bin occupancy for ground/closed state
excitedHist_4_1508_18: Normalized histogram bin occupancy for photoexcited/open state
In Fig3B.csv
FEL_Lc: x-axis of free-energy landscape, corresponding to contour lengths in meters
groundFEL_4_1508_18: y-axis of free-energy landscape, corresponding to free energy of ground/closed state in kcal/mol
excitedFEL_4_1508_18: y-axis of free-energy landscape, corresponding to free energy of photoexcited/open state in kcal/mol
Corresponding Figure: Fig. 3A and 3B in the paper.
```

### Fig4A.csv, Fig4B.csv, Fig4C.csv

```
Description: Characterization of misfolding behavior in bR.
Variables:
In Fig4A.csv
time_index: Time in seconds (example time-series data)
Lc: Contour length in meters (example time-series data)
hist_bins: Bin centers in meters of histogrammed contour-length data
groundHist_10_83_18: Normalized histogram bin occupancy for ground/closed state
excitedHist_10_83_18: Normalized histogram bin occupancy for photoexcited/open state
In Fig4B.csv
hist_bins: Bin centers of histogrammed I_G^MF positions, expressed as a percentage between established states
positions_hist: Corresponding bin occupancies (absolute number recorded)
In Fig4C.csv
hist_bins: x-axis of free-energy landscape, corresponding to contour lengths in meters
groundFEL_10_83_18: y-axis of free-energy landscape, corresponding to free energy of ground/closed state in kcal/mol
excitedFEL_10_83_18: y-axis of free-energy landscape, corresponding to free energy of photoexcited/open state in kcal/mol
Corresponding Figure: Fig. 4A, 4B, and 4C in the paper.
```

### Fig5B.csv, Fig5C.csv

```
Description: Kinetics of destabilization and misfolding events at different pH levels.
Variables:
In Fig5B.csv
time_destab_ph78: x-axis of destabilizing events at pH 7.8 (s)
fraction_destab_ph78: Corresponding fraction surviving to a given time
time_destab_ph95: x-axis of destabilizing events at pH 9.5 (s)
fraction_destab_ph95: Corresponding fraction surviving to a given time
time_misfold_ph78: x-axis of misfolding events at pH 7.8 (s)
fraction_misfold_ph78: Corresponding fraction surviving to a given time
time_misfold_ph95: x-axis of misfolding events at pH 9.5 (s)
fraction_misfold_ph95: Corresponding fraction surviving to a given time
time_Perrino_ph8: x-axis of events from Perrino et al. (determined from reported time constant at pH 8) (s)
fraction_Perrino_ph8: Corresponding fraction surviving to a given time
In Fig5C.csv
time_data: x-axis of experimental data (s)
fraction_data: Corresponding fraction surviving to a given time
time_fit: x-axis for the double-exponential fit (s)
fraction_fit: Corresponding fraction surviving to a given time, as predicted from fit parameters
Corresponding Figure: Fig. 5B and 5C in the paper.
```

### FigS1A.csv, FigS1B.csv

```
Description: Data on light-induced movement of surface in the absence of bR.
Variables:
In FigS1A.csv
time_index: Common x (time) axis for all records, in s
contact_200us_1, contact_200us_2, contact_200us_3: Three examples of apparent deflection after 200-us light pulse, in m
contact_400us_1, contact_400us_2, contact_400us_3: Three examples of apparent deflection after 400-us light pulse, in m
In FigS1B.csv
time_index: Common x (time) axis for all records, in s
noncontact_200us_1, noncontact_200us_2, noncontact_200us_3: Three examples of apparent deflection after 200-us light pulse, in m
Corresponding Figure: Fig. S1A and S1B in the SI.
```

### FigS2A.csv, FigS2B.csv, FigS2C.csv

```
Description: Data acquisition protocol details.
Variables:
In FigS2A.csv
time_index: Time in seconds
Z_height: Cantilever Z height during the data-acquisition protocol, in m
In FigS2B.csv
time_index: Time in seconds
F_force: Force measured based on cantilever deflection during the protocol, in N
In FigS2C.csv
force_time: x (time) axis of zoomed force data
force: Corresponding force, in N
force_var_time: x (time) axis of variance calculated from zoomed force
force_var: Corresponding variance, in N^2
Corresponding Figure: Fig. S2A, S2B, and S2C in the SI.
```

### FigS3B.csv

```
Description: Fitting and analysis details of inverse-Boltzmann method of energetic analysis.
Variables:
closed_data_bins: Bin centers for ground/closed state corresponding to contour length (m)
closed_data: Normalized histogram bin occupancy from experimental data
closed_fit_bins: Bin centers for ground/closed state corresponding to contour length (m)
closed_fit1, closed_fit2, closed_fit3, closed_fit4: Normalized histogram bin occupancy for each Gaussian fit to ground/closed state (which are added to give the total fit)
open_data_bins: Bin centers for photoexcited/open state corresponding to contour length (m)
open_data: Normalized histogram bin occupancy from experimental data
open_fit_bins: Bin centers for photoexcited/open state corresponding to contour length (m)
open_fit1, open_fit2, open_fit3, open_fit4: Normalized histogram bin occupancy for each Gaussian fit to photoexcited/open state (which are added to give the total fit)
Corresponding Figure: Fig. S3B in the SI.
```

### FigS4.csv

```
Description: Scatter in ΔΔG_open values from individual molecules.
Variables:
F_01: Average two-state force value at which measurement was made for 0-1 transition (N)
ddG_01: Corresponding measured free-energy difference (kcal/mol)
F_12: Average two-state force value at which measurement was made for 1-2 transition (N)
ddG_12: Corresponding measured free-energy difference (kcal/mol)
Corresponding Figure: Fig. S4 in the SI.
```

### FigS6A.csv

```
Description: Apparent free energy stabilization of misfolded state I_G^MF in light-activated phase.
Variables:
force: Force in pN.
ddG: Corresponding ddG_misfold* (kcal/mol)
Corresponding Figure: Fig. S6A in the SI.
```

### FigS7A_trace1.csv, FigS7A_trace2.csv, FigS7A_trace3.csv

```
Description: Misfolding behavior in equilibrium data without photoactivation, three examples.
Variables:
In each file:
Lc_time: Time axis of time-series data (s)
Lc_segment: Contour length of time-series data (m)
hist_bins: Bin centers of contour-length histogram (m)
ground_hist: Normalized histogram of ground/closed state
excited_hist: Normalized histogram of photoexcited/open state
Corresponding Figure: Fig. S7A in the SI.
```

### FigS9A.csv, FigS9B.csv

```
Description: Misfolding during reduced force surrounding light pulse.
Variables:
In Fig9A.csv
time_index: Time in seconds
Z_smth: Cantilever Z position (m)
In Fig9B.csv
time_index: Time in seconds
force: Force axis of time-series data (N)
hist_bins: Bin centers of force histogram (N)
closed_hist: Normalized histogram of ground/closed state
misfold_hist: Normalized histogram of photoexcited/misfolded state
Corresponding Figure: Fig. S9A and S9B in the SI.
```

### FigS10.csv

```
Description: Photoexcited state lifetime at elevated pH (9.5).
Variables:
time_data: x (time) axis of experimental data (s)
fraction_data: Corresponding fraction surviving to a given time
time_fit: x (time) axis of double-exponential fit to data (s)
fraction_fit: Corresponding fraction surviving to a given time
Corresponding Figure: Fig. S10 in the SI.
```

### FigS11A.csv, FigS11B.csv

```
Description: Data from D96G/F171C/F219L triple mutant under light activation.
Variables:
In FigS11A.csv
Lc_time: Time axis of time-series data (s)
Lc_segment: Contour-length axis of time-series data (m)
hist_bins: Bin centers of contour-length histogram (m)
Hist: Normalized histogram
In FigS11B.csv
Lc_time: Time axis of time-series data (s)
Lc_segment: Contour-length axis of time-series data (m)
hist_bins: Bin centers of contour-length histogram (m)
closed_hist: Normalized histogram of ground/closed state
misfold_hist: Normalized histogram of photoexcited/misfolded state
Note: Some Lc_segment data during light pulse were redacted (marked NaN) due to bleedthrough into the cantilever deflection channel.
Corresponding Figure: Fig. S11A and S11B in the SI.
```

### FigS12B.csv, FigS12C.csv, FigS12D.csv

```
Description: Mechanical properties of a modified AFM cantilever.
Variables:
In FigS12B.csv
tau: Time axis of Allen deviation (s)
AD: Corresponding Allen deviation (N)
In FigS12C.csv
time_index: Time axis of autocorrelation (s)
autocorr: Corresonding autocorrelation (normalized to unity at low time)
In FigS12D.csv
force_time: Time axis of experimental data (s)
force: Force axis of experimental data (N)
fit_time: Time axis of exponential fit to data (s)
force_fit: Force axis of exponential fit to data (N)
Corresponding Figure: Fig. S12B, S12C, and S12D in the SI.
```

## Additional Notes:

```
The data from Fig. S2D are a subset of those saved in FigS2B.csv.
Fig. S3A contains the same data as Fig3A.csv.
Fig. S6B contains the same data as Fig4C.csv.
Fig. S7B contains the same data as Fig4A.csv.
```

## Funding

National Science Foundation, Award: MCB-2139572

National Science Foundation, Award: PHY-2317149

National Institute of General Medical Sciences, Award: K99GM140439

National Institute of Standards and Technology