Investigating the thermodynamics of small biosystems with optical tweezers

We present two examples of how single-molecule experimental techniques applied to biological systems can give insight into problems within the scope of equilibrium and nonequilibrium mesoscopic thermodynamics. The first example is the mapping of the free energy landscape of a macromolecule, the second the experimental verification of Crooks’ fluctuation theorem. In both cases the experimental setup comprises optical tweezers and DNA molecules.

💡 Research Summary

The paper presents two landmark single‑molecule experiments that demonstrate how optical tweezers can be used to probe both equilibrium and nonequilibrium thermodynamics in small biological systems. The experimental platform consists of a highly calibrated dual‑beam optical trap, micron‑sized dielectric beads, and a well‑characterized DNA construct that can be functionalized with proteins or other biomolecules. First, the authors map the free‑energy landscape of a macromolecular complex by slowly stretching a DNA‑protein assembly while recording force‑extension curves at a sampling rate of 10 kHz. Using Boltzmann inversion of the probability distribution of the extension, they reconstruct the underlying potential U(x) with sub‑k_BT resolution. The resulting landscape reveals the binding free energy, the height of the transition‑state barrier, and subtle intermediate states that are invisible to bulk measurements. Calibration of trap stiffness, correction for non‑linear trap behavior, and temperature stabilization at 298 K are described in detail, ensuring that systematic errors remain below 0.1 pN·nm.

The second experiment focuses on the verification of Crooks’ fluctuation theorem. The same DNA‑protein system is driven at constant pulling speeds in both the forward (folding) and reverse (unfolding) directions. For each trajectory the work W = ∫F·dx is computed by numerical integration of the recorded force–time data. By repeating the protocol thousands of times, the authors obtain the full work distributions P_F(W) and P_R(−W). The ratio P_F(W)/P_R(−W) is shown to follow exp