Mimicking the inner mitochondrial membrane with curved supported lipid bilayers: A neutron reflectometry study

Cardiolipin (CL) is a phospholipid with an unusual molecular structure exhibiting four acyl chains bound to a polar headgroup. CL is found in curved regions within biological membranes, such as the poles of cylindrically shaped bacteria and the cristae in mitochondria. Like bacterial cells, mitochondria are characterised by two cell membranes. CL is exclusively found in the inner membrane of mitochondria (IMM), and it is vital for the proper functioning of this organelle. Together with CL, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the main lipid components of the IMM. Interestingly, most of the CL in human tissues exhibits C18 acyl chains and alterations of its molecular structure are related to severe diseases. Here we investigated supported lipid bilayers (SLBs) composed of PC, PE and CL as model systems mimicking the IMM. In our experiments we used tetra-oleyl cardiolipin (TOCL), which exhibits four C18 acyl chains and is therefore close to the CL normally present in the IMM, and tetra-myristoyl cardiolipin (TMCL), which exhibits considerably shorter acyl chains, i.e. C14. All samples were investigated both in the presence and in the absence of Ca2+ ions. The structure of the produced samples was characterised by neutron reflectometry (NR). Our data indicate that all samples with TMCL were organised as a regular SLB with a small impact of TMCL on the bilayer structure. On the other hand, at a TOCL concentration above 10% mol and upon injection of Ca2+, we observed a large structural rearrangement of the initially formed SLB compatible with the formation of curved bilayer regions that protrude towards the bulk solvent. To the best of our knowledge, this is the first example where specular NR and off-specular scattering revealed buckled SLBs. This experimental evidence indicates the crucial role of CL acyl chain composition in favouring the proper folding of the IMM.