VECTORIALLY-ORIENTED MONOLAYERS OF CYTOCHROME-OXIDASE - FABRICATION AND PROFILE STRUCTURES

Vectorially-oriented monolayers of detergent-solubilized bovine heart cytochrome c oxidase have been formed by self-assembly from solution a nd Langmuir-Blodgett (LB) deposition. Both quartz and Ge/Si multilayer substrates, the latter fabricated by molecular beam epitaxy, were alk ylated with an amine-terminated alkylsiloxane monolayer prior to intro duction to the protein. For the self-assembled protein monolayers, the amine end group surface provided for primarily electrostatic intel ac tions with the protein, thereby encouraging a nearly unidirectional ve ctorial orientation of the so-adsorbed integral membrane protein. This was demonstrated by the analysis of meridional X-ray diffraction data from the monolayers so-adsorbed onto the Ge/Si multilayer substrates, which directly provided electron density profiles of the protein alon g the axis normal to the substrate plane to a spatial resolution of 10 Angstrom. These profiles are consistent with the three-dimensional st ructure of the protein, obtained from electron microscopy. Patterson f unction analysis of meridional X-ray diffraction from the LB-deposited monolayers has shown the profile structure of the so-deposited protei n monolayers to be qualitatively similar to that obtained via self-ass embly from solution, thereby suggesting that the LB-deposited monolaye rs are similarly vectorially-oriented. Optical spectroscopy using quar tz substrates has also indicated that the LB monolayers tend to be mor e densely packed than their self-assembled counterparts. Optical linea r dichroism has confirmed that the planes of the oxidase's two heme gr oups and, hence, the molecule's long axis are more perpendicular to th e monolayer plane in the LB case than for the self-assembled monolayer s, consistent with the profile length of the molecule along the axis n ormal to the monolayer plane. Such densely packed, vectorially-oriente d monolayers in a fully hydrated state now provide a unique opportunit y to perform directly correlated structural-functional studies on this membrane protein.