Chain Packing in the Inverted Hexagonal Phase of Phospholipids: A Study by X-ray Anomalous Diffraction on Bromine-labeled Chains

Abstract

Although lipid phases are routinely studied by X-ray diffraction, construction of their unit cell structures from the diffraction data is difficult except for the lamellar phases. This is due to the well-known phase problem of X-ray diffraction. Here we successfully applied the multiwavelength anomalous dispersion (MAD) method to solve the phase problem for an inverted hexagonal phase of a phospholipid with brominated chains. Although the principle of the MAD method for all systems is the same, we found that for lipid structures it is necessary to use a procedure of analysis significantly different from that used for protein crystals. The inverted hexagonal phase has been used to study the chain packing in a hydrophobic interstice where three monolayers meet. Hydrophobic interstices are of great interest, because they occur in the intermediate states of membrane fusion. It is generally believed that chain packing in such a region is energy costly. Consequently, it has been speculated that the inverted lipid tube is likely to deviate from a circular shape, and the chain density distribution might be nonuniform. The bromine distribution obtained from the MAD analysis provides the information for the chain packing in the hexagonal unit cell. The intensity of the bromine distribution is undulated around the unit cell. The analysis shows that the lipid chains pack the hexagonal unit cell at constant volume per chain, with no detectable effect from a high-energy interstitial region.