Biophysical Impacts of Mixed Chain Phospholipids on Membrane Structure and Phase Behavior: Insights from Molecular Dynamics and Experimental Approaches

Lipid bilayers are essential for all forms of life and are comprised of hundreds of structurally distinct lipids. Arguably, one of the most important lipid bilayers in nature is the eukaryotic plasma membrane (PM) that forms the external barrier of all living cells. Given the vast diversity of lipid types and structures, a major challenge in membrane biophysics is determining how specific lipid classes contribute to physical properties of the PM. One understudied class of phospholipids consists of lipids where both the chains are saturated but differ in the number of carbons. These mixed chain lipids are thought to alter midplane interactions as compared to their symmetric counterparts. Here, we developed novel methodologies to investigate the fundamental biophysical properties of mixed chain lipids both in vitro and in silico. All-atom molecular dynamics simulations were used to explore the effect of acyl chain mismatch on bilayer properties at an atomic scale. Complementary experimental techniques including FRET, confocal fluorescence microscopy, and cryogenic electron microscopy were used to examine model membrane systems exhibiting phase separation. Simulations reveal that chain mismatch and glycerol backbone positioning significantly influence bilayer properties such as bending modulus, interdigitation, and upward acyl chain snorkeling dynamics. The application of a novel phenomenological model for FRET data enabled precise determination of the miscibility transition temperature and phase behavior of mixed chain lipid systems composed of a high-melting lipid, low melting lipid and cholesterol. Complementary experimental findings further demonstrated that the positional transposition of acyl chains on the glycerol backbone significantly impacts lateral lipid organization in the membrane and other bilayer properties. These findings and methods enhanced our understanding of membrane biophysics, establishing a strong foundation for future studies on the structure, dynamics, and function of complex biomembranes.