Reaction Cycle of Operating Pump Protein Studied with Single-Molecule Spectroscopy

Area of Science:

• Biophysics
• Physical Chemistry
• Molecular Biology

Background:

• Biological processes often depend on nonequilibrium dynamics for sustained function.
• Understanding protein conformational transitions and entropy production is crucial for complex reaction cycles.

Purpose of the Study:

• To investigate the temperature dependence of conformational transitions in bacteriorhodopsin during proton pumping.
• To quantify flux, affinity, enthalpy, and entropy production in specific reaction cycle portions.

Main Methods:

• Utilized multidimensional single-molecule fluorescence lifetime correlation spectroscopy.
• Measured forward and reverse conformational transitions of bacteriorhodopsin.
• Analyzed temperature-dependent changes in reaction cycle thermodynamics.

Main Results:

• Observed a decrease in the affinity of irreversible conformational transitions with increasing temperature.
• Found that increasing temperature diminishes both flux and entropy production.
• Demonstrated that transition affinity's temperature dependence follows the Gibbs-Helmholtz relation.

Conclusions:

• The Gibbs-Helmholtz relation allows experimental extraction of transition enthalpy (ΔHtrans).
• Temperature negatively impacts the thermodynamic driving force of these protein transitions.
• Findings provide insights into the energy transduction mechanisms in biological systems.