Abstract
Free volume within lipid bilayers plays a crucial role in determining membrane properties, such as fluidity, permeability, and domain organization, which in turn modulate the functions of membrane proteins and cellular processes. The presence of hydrophobic molecules within bilayers reciprocally influences free volume, and previous studies have shown that solidification of such hydrophobic molecules induces exclusion from the bilayer interior, leading to a reduction in bilayer thickness. Here, we investigate the inclusion and exclusion behaviors of hydrocarbon intercalants by quantifying bilayer thickness. The excluded volume effect expelled the long-chain compound squalane, whereas smaller intercalants, n-decane, p-xylene, and tetralin, remained incorporated, increasing bilayer thickness. In contrast, crystallizable intercalants, naphthalene and durene, were excluded at concentrations likely to promote aggregation. This exclusion led to a depletion force, resulting in bilayer thinning. Understanding of free volume modulation through membrane composition will not only advance fundamental insights into biological membranes but also guide the further development of synthetic membrane systems.