Investigating molecular effects on membrane structure, dynamics and function

Biological membranes are heterogeneous structures with complex electrostatic profiles arising from lipids, sterols, membrane proteins, and water molecules. We investigated the effect of cholesterol and its derivative 6-ketocholestanol (6-kc) on membrane electrostatics by directly measuring the dipole electric field (F [arrow above F] [subscript d] ) within lipid bilayers containing cholesterol or 6-kc at concentrations of 0−40 mol% through the vibrational Stark effect (VSE). We found that adding low concentrations of cholesterol, up to ∼10 mol %, increases F [arrow above F] [subscript d], while adding more cholesterol up to 40 mol% lowers F [arrow above F] [subscript d]. In contrast, we measured a monotonic increase in F [arrow above F] [subscript d] as 6-kc concentration increased. We proposed that this membrane electric field is affected by multiple factors: the polarity of the sterol molecules, the reorientation of the phospholipid dipole due to sterol, and the impact of the sterol on hydrogen bonding with surface water. We used molecular dynamics simulations to examine the distribution of phospholipids, sterol, and helix in bilayers containing these sterols. At low concentrations, we observed clustering of sterols near the vibrational probe whereas at high concentrations, we observed spatial correlation between the positions of the sterol molecules. This work demonstrated how a one-atom difference in a sterol changes the physicochemical and electric field properties of the bilayer. Additionally, we set out to understand how a small molecule interacts with the lipid bilayer differently based on its charge. Our laboratory had previously reported that tryptophan permeated through a phosphatidylcholine lipid bilayer membrane at a faster rate when it was positively charged (Trp+) than when negatively charged (Trp−), which corresponded to a lower potential of mean force (PMF) barrier determined through simulations. In the work described here, we demonstrated that Trp+ partitions into the lipid bilayer membrane to a greater degree than Trp− by interacting with the ester linkage of a phosphatidylcholine lipid, where it is stabilized by the electron withdrawing glycerol functional group. These results are in agreement with tryptophan’s known role as an anchor for transmembrane proteins, though the tendency for binding of a positively charged tryptophan is surprising. We discussed the implications of our results on the mechanisms of unassisted permeation and penetration of small molecules within and across lipid bilayer membranes based on molecular charge, shape, and molecular interactions within the bilayer structure.