Cholesterol microcrystals and cochleate cylinders: attachment of pyolysin oligomers and domain 4

Using an established organic solvent injection procedure for the preparation of aqueous cholesterol microcrystal suspensions, it has now been shown that a new, hollow, cylindrical, tightly-coiled, multi-bilayer form of cholesterol can be generated, termed the cochleate cylinder. Cholesterol cochleate cylinders are formed in larger numbers at intermediate temperatures (40-75°C) but are not formed at 100°C. The structure of the cholesterol microcrystals and cochleate cylinders is shown in negatively stained electron micrographs. Oligomerization and attachment of pyolysin to cholesterol microcrystals and cochleate cylinders is shown, as is the attachment of the pyolysin "cholesterol-binding" domain 4 (D4) fragment. The bound D4 domain forms a linear array on the two planar surfaces and edges of the cholesterol microcrystals and a quasi helical array on the surface of the cochleate cylinders. Little evidence has been obtained to support the possibility that interaction or hetero-oligomerization can occur between intact pyolysin and the pyolysin D4 fragment on the surface of cholesterol microcrystals. Using immobilized cholesterol crystals attached to a carbon support film, single-sided linear labelling of the cholesterol surface with pyolysin D4 has been achieved, which correlates well with the images from the microcrystal suspensions and our earlier data using non-cytolytic streptolysin O mutants.