@article{13401,
  keywords = {Multidisciplinary},
  author = {David Jacobson and Thomas Perkins},
  title = {Quantifying a light-induced energetic change in bacteriorhodopsin by force spectroscopy},
  abstract = {<jats:p>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.</jats:p>},
  year = {2024},
  journal = {Proceedings of the National Academy of Sciences},
  volume = {121},
  pages = {e2313818121},
  month = {2024-02},
  publisher = {Proceedings of the National Academy of Sciences},
  issn = {0027-8424, 1091-6490},
  doi = {10.1073/pnas.2313818121},
}