A longstanding hypothesis proposes that cholesterol in membranes partitions into two distinct populations: a sequestered pool stabilized by lipid interactions, and a more dynamic pool of “free” cholesterol. In this study, we investigate whether hydrogen-bonding populations can provide molecular evidence for such partitioning. Membranes composed of a ternary mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), palmitoyl sphingomyelin (PSM), and cholesterol were chosen in order to systematically vary cholesterol content while maintaining a single liquid phase. We applied a spectral-decomposition method to 2D-IR measurements of these membranes, which resolved six distinct hydrogen-bonding states of PSM ranging from non-bonded to multiply bonded configurations involving the amide and carbonyl groups. The same series of membranes were then simulated using molecular dynamics to provide atomistic resolution of hydrogen-bond populations, lifetimes, and bonding motifs to understand how cholesterol alters the hydrogen-bonding environment of PSM at the membrane-water interface. By tracking how these populations of the hydrogen-bonding states shift as cholesterol increases, we can assess whether the cholesterol-PSM hydrogen-bonding populations continue to scale proportionally or plateau beyond a threshold, consistent with the emergence of a non-sequestered pool. This integrated MD-spectroscopy approach provides molecular-level insight into the cholesterol-PSM interactions and allows us to assess how cholesterol content shapes both interfacial hydrogen bonding and membrane organization.
Scott et al. (Sun,) studied this question.