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

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.