Cardiolipin (CL) is a complex phospholipid that is specifically found in mitochondria. Owing to the association of the CL levels with mitochondrial physiopathology such as in Parkinson’s disease, we study the molecular effect of CL on membrane organization using model Langmuir monolayer, fluorescence microscopy, and x-ray reflectivity. We find that the liquid-expanded phase in membranes increases with increasing CL concentration, indicating an increase in the elasticity of the mixed membrane. The Gibbs excess free energy of mixing indicates that the binary monolayer composed of CL and DPPC is most thermodynamically stable at ΦCL = 10 mol %, and the stability is enhanced when the surface pressure is increased. Additionally, when ΦCL is small, the expansion of the membrane with increasing CL content was slower at higher surface pressure. These abnormal results are indicative of a folding structure being present before a collapsing structure, which was confirmed by using fluorescence microscopy and was characterized by using x-ray reflectivity with the electron density profile along the membrane’s surface normal.
