Single-molecule identification of the entropy/enthalpy contributions in macromolecule crowding induced by Carboxylated chitosan

Macromolecular crowding effects frequently drive phase separation and have a significant impact on a series of biochemical processes in the cell. Owing to the significant differences in the physical mechanisms of the macromolecular crowding effects induced by different macromolecules, revealing the underlying mechanisms is crucial. From this perspective, in this study, an innovative method combining high-resolution magnetic tweezers and a statistical analysis based on the van’t Hoff relation was applied to systematically study the kinetics and thermodynamics of the folding–unfolding transition for an individual DNA hairpin in a crowded environment induced by carboxylated chitosan(CC). More specifically, from the magnetic tweezer-based experiments, the nonlinear dependence of the critical force of the DNA hairpin on the CC concentration in experimental buffer with a fixed pH value was identified. It was found that the critical force of the DNA hairpin first decreased and then increased with increasing CC concentration. Zeta potential measurement indicated that the Zeta potential of the experimental buffer was sharply reduced with increasing CC concentration from 0.05% to 0.4%. However, increasing the CC concentration above 0.4% did not result in obvious Zeta-potential variation, suggesting that entropy contributions in macromolecular crowding effects must dominate over the enthalpy contributions above this value. The van’t Hoff relation was established based on a series of single-molecule experiments performed at a constant stretching force within a temperature range of 275 K to 305 K, and the thermodynamics results suggested that the total free energy change in a 0.8% CC solution, opposite to that in a 0.2% CC solution, is negative. The proposed method provides a viable pathway to quantify the contributions of entropy and enthalpy to macromolecular crowding effects.