Determination of the Separate Lipid and Protein Profile Structures Derived from the Total Membrane Profile Structure of Isolated Sarcoplasmic Reticulum Via X-Ray and Neutron Diffraction
Sarcoplasmic reticulum (SR) membranes were prepared to contain biosynthetically deuterated SR phospholipids utilizing specific and general phospholipid exchange proteins (PLEP). Functional measurements and freeze fracture on SR dispersions and x-ray diffraction of hydrated oriented membrane multilayers revealed that the exchanged SR membranes were very similar to unexchanged SR membranes. Low resolution (28-Å) neutron diffraction studies utilizing SR membranes exchanged with either protonated or perdeuterated SR phospholipids allowed direct determination of the lipid profile within the isolated SR membrane at two different unit cell repeat distances. These lipid profile structures were found to be highly asymmetric regarding the conformation of the fatty acid chain extents and compositional distribution of phospholipid molecules in the inner vs. outer monolayer of the SR membrane bilayer. The relatively high resolution (11-Å) electron-density profile from x-ray diffraction was decomposed by utilizing the asymmetry in the number of phospholipid molecules residing in the inner vs. outer monolayer of the SR lipid bilayer as obtained from the neutron diffraction study. This model refinement analysis of the corresponding electron density profile was found to be dependent upon the direct results obtained via the neutron diffraction study. The asymmetry in the isolated SR profile structures was the result of a localization of a greater portion of the calcium pump protein (and correspondingly less lipid) in the extravesicular half of the SR membrane in contrast to the intravesicular half, and the cylindrically averaged static conformational shape and molecular dimensions of the calcium pump protein as it resides in the isolated SR membrane were obtained. To our knowledge, this represents the first direct determination of a lipid bilayer profile structure within an isolated membrane system.