# Experimental measurement of binding energy, selectivity and allostery using fluctuation theorems

## TL;DR

This paper introduces a fluctuation theorem-based experimental method using single-molecule force spectroscopy to measure binding energies, selectivity, and allostery in biological molecules, especially in systems where traditional thermodynamic assumptions fail.

## Contribution

It develops a model-independent approach to study ligand binding and allostery, extending fluctuation theorems beyond simple folding reactions.

## Key findings

- Successful measurement of binding energies and selectivity in nucleic acids and proteins.
- Demonstration of the method's applicability to non-dilute, complex biological systems.
- Bridging thermodynamics of small systems with chemical equilibrium laws.

## Abstract

Thermodynamic bulk measurements of binding reactions critically rely on the validity of the law of mass action and the assumption of a dilute solution. Yet important biological systems such as allosteric ligand-receptor binding, macromolecular crowding, or misfolded molecules may not follow this fundamental law and require a particular reaction model. Here we introduce a fluctuation theorem for ligand binding and an experimental approach using single-molecule force-spectroscopy to determine binding energies, selectivity and allostery of nucleic acids, proteins and peptides in a model-independent fashion. This work extends the use of fluctuation theorems beyond unimolecular folding reactions, bridging the thermodynamics of small systems and the basic laws of chemical equilibrium.