Quantifying a light-induced energetic change in bacteriorhodopsin by force spectroscopy.

Area of Science:

• Biophysics
• Structural Biology
• Membrane Protein Dynamics

Background:

• Ligand-induced conformational changes are crucial for membrane protein function.
• Bacteriorhodopsin (bR) uses a photocycle to pump protons, involving complex protein motions.
• Quantifying the energetics underlying these motions has been challenging.

Purpose of the Study:

• To measure the intramolecular energetics of conformational changes in bR on the millisecond timescale.
• To apply atomic-force-microscopy-based single-molecule force spectroscopy to membrane proteins.
• To investigate light-induced energetic shifts during the bR photocycle.

Main Methods:

• Utilized single-molecule force spectroscopy with atomic force microscopy.
• Applied precisely timed light pulses to trigger the bR photocycle.
• Measured equilibrium unfolding/refolding of the bR G-helix terminal region.

Main Results:

• Observed a light-induced destabilization of 3.4 ± 0.3 kcal/mol, lasting 38 ± 3 ms in ~60% of trials.
• Destabilization kinetics and pH-dependence align with bR's open phase.
• A longer-lived misfolded state was unexpectedly stabilized in ~40% of trials.

Conclusions:

• Established a novel single-molecule force spectroscopy approach for measuring ligand-induced energetics in membrane proteins.
• Demonstrated the ability to quantify millisecond-timescale energetic changes in protein conformational dynamics.
• Provided new insights into the energetic landscape of the bacteriorhodopsin photocycle.