Protein nucleation and crystallization by homologous protein thin film template.

A new method of protein nucleation and crystallization based on Langmuir-Blodgett technology is here utilized for the template stimulation of crystal growth of so far non-crystallized proteins. Microcrystals (60-120 microm) of bovine cytochrome P450scc and human protein kinase CKII alpha subunit were obtained with use of the homologous protein thin film template by vapor diffusion modified hanging drop method. The induction of microcrystals nucleation by the thin template confirms in the two different important classes of proteins, until now never crystallized, the positive stimulatory influence for crystal formation of protein thin film template, which was observed in an earlier study with a model system (chicken egg white lysozyme) as an unexpected acceleration and enhancement in the crystal growth.

Direct visualization of a cardiolipin-dependent cytochrome P450scc-induced vesicle aggregation.

Cytochrome P450scc can be reconstituted successfully into large unilamellar phospholipid vesicles by a combined octylglucoside dialysis/adsorption method. Freeze-fracture electron microscopy was used to analyze the morphology, distribution, and protein topology of the cytochrome P450scc vesicles in dependence on lipid composition. Particles were observed only in close contact to the vesicle surface, probably representing tightly associated cytochrome P450scc at the outer vesicle surface. In cytochrome P450scc vesicles similar in lipid composition to the inner membrane of bovine mitochondria direct evidence by freeze-fracturing was found for a specific cytochrome P450scc-induced aggregation of the vesicles. The vesicle aggregation critically depends on the content of the specific mitochondrial membrane constituent cardiolipin. The aggregation and thus the intervesicular contacts were observed to be inhibited by both addition of anti-cytochrome P450scc IgG and adrenodoxin. Enzymatic reduction of cytochrome P450scc in the liposomal membrane by its electron transfer partners completely indicates an asymmetrical localization in/at the outer side of the bilayer membrane. It is suggested that vesiculation of the inner mitochondrial membrane may be a consequence of the characteristic cardiolipin-dependent cytochrome P450scc membrane topology: the cardiolipin binding, peripheral, non-bilayer-spanning integration as an oligomer in the outer leaflet of the membrane may play a role in the dynamics of formation and dissociation of intramitochondrial vesicles with a functional importance for steroidogenesis.

Is cytochrome P-450scc a transmembrane protein?

The topology of cytochrome P-450scc in the inner mitochondrial membrane of adrenal cortex has been investigated using monospecific antibodies to cytochrome P-450scc and its fragments F1 (Ile1-Arg250), F2 (Asn257-Ala481) and F3 (Asn257-Arg399). Antibodies to F1 and F2 were shown to effectively bind to the matrix and cytosolic sides of the inner membrane. Antibodies to F3 specifically interacted only with the matrix side of the membrane. These data are consistent with a model of molecular organization which shows that cytochrome P-450scc is a transmembrane protein, both N- and C-terminal sequences of the cytochrome being able to span the membrane.