Data from: Quantifying a lightinduced energetic change in bacteriorhodopsin by force spectroscopy
Data files
Jan 27, 2024 version files 129.98 MB

Fig2B.csv

Fig2C.csv

Fig3A.csv

Fig3B.csv

Fig4A.csv

Fig4B.csv

Fig4C.csv

Fig5B.csv

Fig5C.csv

FigS10.csv

FigS11A.csv

FigS11B.csv

FigS12B.csv

FigS12C.csv

FigS12D.csv

FigS1A.csv

FigS1B.csv

FigS2A.csv

FigS2B.csv

FigS2C.csv

FigS3B.csv

FigS4.csv

FigS6A.csv

FigS7A_trace1.csv

FigS7A_trace2.csv

FigS7A_trace3.csv

FigS9A.csv

FigS9B.csv

README.md
Abstract
Ligandinduced 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 atomicforcemicroscopy–based singlemolecule force spectroscopy. Precisely timed light pulses triggered the bR photocycle while we measured the equilibrium unfolding and refolding of the terminal 8aminoacid region of bR’s Ghelix. These dynamics changed when the EFhelix 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 lightinduced destabilization of 3.4 ± 0.3 kcal/mol, lasting 38 ± 3 ms. The kinetics and pHdependence of this destabilization were consistent with prior measurements of bR’s open phase. The frequency of lightinduced 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 longerlived putative misfolded state. Through this work, we establish a general singlemolecule force spectroscopy approach for measuring ligandinduced energetics and lifetimes in membrane proteins.
README: Dataset Overview:
This repository contains raw data files for the figures presented in "Quantifying a lightinduced 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 timeseries data)
Lc_4_1508_18: Contour length in meters (example timeseries data)
hist_bins: Bin centers in meters of histogrammed contourlength 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: xaxis of freeenergy landscape, corresponding to contour lengths in meters
groundFEL_4_1508_18: yaxis of freeenergy landscape, corresponding to free energy of ground/closed state in kcal/mol
excitedFEL_4_1508_18: yaxis of freeenergy 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 timeseries data)
Lc: Contour length in meters (example timeseries data)
hist_bins: Bin centers in meters of histogrammed contourlength 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: xaxis of freeenergy landscape, corresponding to contour lengths in meters
groundFEL_10_83_18: yaxis of freeenergy landscape, corresponding to free energy of ground/closed state in kcal/mol
excitedFEL_10_83_18: yaxis of freeenergy 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: xaxis of destabilizing events at pH 7.8 (s)
fraction_destab_ph78: Corresponding fraction surviving to a given time
time_destab_ph95: xaxis of destabilizing events at pH 9.5 (s)
fraction_destab_ph95: Corresponding fraction surviving to a given time
time_misfold_ph78: xaxis of misfolding events at pH 7.8 (s)
fraction_misfold_ph78: Corresponding fraction surviving to a given time
time_misfold_ph95: xaxis of misfolding events at pH 9.5 (s)
fraction_misfold_ph95: Corresponding fraction surviving to a given time
time_Perrino_ph8: xaxis 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: xaxis of experimental data (s)
fraction_data: Corresponding fraction surviving to a given time
time_fit: xaxis for the doubleexponential 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 lightinduced 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 200us light pulse, in m
contact_400us_1, contact_400us_2, contact_400us_3: Three examples of apparent deflection after 400us 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 200us 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 dataacquisition 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 inverseBoltzmann 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 twostate force value at which measurement was made for 01 transition (N)
ddG_01: Corresponding measured freeenergy difference (kcal/mol)
F_12: Average twostate force value at which measurement was made for 12 transition (N)
ddG_12: Corresponding measured freeenergy difference (kcal/mol)
Corresponding Figure: Fig. S4 in the SI.
FigS6A.csv
Description: Apparent free energy stabilization of misfolded state I_G^MF in lightactivated 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 timeseries data (s)
Lc_segment: Contour length of timeseries data (m)
hist_bins: Bin centers of contourlength 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 timeseries 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 doubleexponential 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 timeseries data (s)
Lc_segment: Contourlength axis of timeseries data (m)
hist_bins: Bin centers of contourlength histogram (m)
Hist: Normalized histogram
In FigS11B.csv
Lc_time: Time axis of timeseries data (s)
Lc_segment: Contourlength axis of timeseries data (m)
hist_bins: Bin centers of contourlength 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.