DOI: 10.5061/dryad.z08kprrm1
title: Data from: Quantifying a light-induced energetic change in bacteriorhodopsin by force spectroscopy

description: 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.
description: # 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. ```