Correlated x-ray diffraction and freeze-fracture studies on membrane model systems. Perturbations induced by freeze-fracture preparative procedures

Abstract

Lipid-water and protein-lipid-water phases have been examined by X-ray methods before and after freezing. Frozen samples have been subsequently fractured and replicated, thus permitting an evaluation of the nature of structural perturbations in samples examined by freeze-fracture electron microscopy. Important results are summarized: (1) Freezing low water content (approx. less than 25%) phases causes perturbations in the packing of hydrocarbon chains. The results suggest that freezing liquied paraffin chains produces a condensed "glass-like" packing. (2) Additional perturbations occur in high water content samples. After freezing, much smaller lamellar repeat distances, intense ice reflections, and extensive perturbation of fracture faces are consistant with the expulsion of water from between lamellae. Presence of glycerol generally relieves these perturbations but in some cases introduces additional lattice disorder. (3) Surprisingly, cooling by a stream of cold N2 gas (-140 degrees C) produces qualitatively the same results as rapid cooling in liquid Freon-22 (-160 degrees C). (4) Complex perturbations occur in phases containing integral membrane proteins. Interesting results have been obtained with cytochrome b5-lecithin lamellar associations which display both smooth and rough fracture faces without clearly defined particles.