Vectorially-Oriented Monolayers of Cytochrome Oxidase:  Fabrication and Profile Structures

Abstract

Vectorially-oriented monolayers of detergent-solubilized bovine heart cytochrome c oxidase have been formed by self-assembly from solution and Langmuir-Blodgett (LB) deposition. Both quartz and Ge/Si multilayer substrates, the latter fabricated by molecular beam epitaxy, were alkylated with an amineterminated alkylsiloxane monolayer prior to introduction to the protein. For the self-assembled protein monolayers, the amine end group surface provided for primarily electrostatic interactions with the protein, thereby encouraging a nearly unidirectional vectorial 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 along the axis normal to the substrate plane to a spatial resolution of 10 Å. These profiles are consistent with the three-dimensional structure of the protein, obtained from electron microscopy. Patterson function analysis of meridional X-ray diffraction from the LB-deposited monolayers has shown the profile structure of the so-deposited protein monolayers to be qualitatively similar to that obtained via selfassembly from solution, thereby suggesting that the LB-deposited monolayers are similarly vectoriallyoriented. Optical spectroscopy using quartz substrates has also indicated that the LB monolayers tend to be more densely packed than their self-assembled counterparts. Optical linear dichroism has confirmed that the planes of the oxidase's two heme groups and, hence, the molecule's long axis are more perpendicular to the monolayer plane in the LB case than for the self-assembled monolayers, consistent with the profile length of the molecule along the axis normal to the monolayer plane. Such densely packed, vectoriallyoriented monolayers in a fully hydrated state now provide a unique opportunity to perform directly correlated structural-functional studies on this membrane protein.

Heme Structure and Orientation in Single Monolayers of Cytochrome c on Polar and Nonpolar Soft Surfaces

“…All substrates were cleaned and coated with the organic self-assembled monolayer (SAM) compounds following the general procedure of Sagiv (1980) with modifications that have been described previously (Xu et al, 1993;Edwards et al, 1997). Nonpolar surfaces were formed by a 6:1 ratio of dodecyltrichlorosilane (methyl-endgroup surface) and 11-trichlorosilylundecyl thioacetate (protected sulfhydryl-endgroup surface); while polar, but uncharged, surfaces were produced using a 6:1 ratio of trichlorosilylacetoxyundecane (protected hydroxyl-endgroup surface) and 11-trichlorosilylundecyl thioacetate (protected sulfhydryl-endgroup surface).…”

“…While other research groups have focused on measurements of the functional aspects of such (or closely related, e.g., Langmuir-Blodgett) monolayer systems that were not structurally characterized (Song et al, 1993;Cullinson et al, 1994;Owaku et al, 1995;Jiang et al, 1996;Guo et al, 1996), we have focused instead on developing the physical techniques essential to determining the key structural features of the proteins within such vectorially oriented single monolayers. To date, this work has included both nonresonance and resonance x-ray diffraction (Pachence and Blasie, 1987;Pachence et al, 1989), optical linear dichroism (Pachence et al, 1990), and x-ray interferometry/holography (Blasie et al, 1992;Chupa et al, 1994;Edwards et al, 1997Edwards et al, , 1998.…”

Polarized XAS on vectorially oriented single monolayers of cytochrome c

“…Langmuir-Blodgett) monolayer systems that were not structurally characterized (Song et al, 1993;Cullinson et al, 1994;Owaku et al, 1995;Jiang et al, 1996;Guo et al, 1996), we have focused instead on developing the physical techniques essential to determining the key structural features of the proteins within such vectorially oriented single monolayers. To date, this work has included resonance x-ray diffraction (Pachence et al, 1989), optical linear dichroism (Pachence et al, 1990), and x-ray interferometry/holography (Blasie et al, 1992;Chupa et ai., 1994;Edwards et al, 1997 andEdwards et al, 1998). We have further demonstrated the potential of applying polarized XAFS to such systems in order to obtain information on both the orientation of the protein with respect to the substrate and the detailed structure around the metal site(s) of the protein (Zhang et al, 1997).…”

Vectorially Oriented Monolayers of the Cytochrome c/Cytochrome Oxidase Bimolecular Complex

“…After another rinsing in 1 mM TRIS buffer, the cytochrome c monolayers were ready for study before subsequent incubation in the second protein, cytochrome c oxidase. This integral membrane protein was isolated and purified from beef heart mitochondria using further modifications of the method of Yonetani et al (Okunuki et al, 1958;Yonetani et al, 1960) as described elsewhere (Edwards et al, 1997). To produce the bimolecular complex monolayer samples, the cytochrome c monolayers were incubated for 3-4 h in solutions of approximately 6 M cytochrome oxidase in 0.1% n-dodecyl ␤-D-maltoside (Sigma) in 1 mM KPO 4 (Sigma), pH 7.4.…”

“…To date, this work has concerned the determination of the so-called profile structures of such monolayers uniquely and to a resolution of ϳ7 Å by x-ray interferometry/holography (Blasie et al, 1992;Chupa et al, 1994) which is essential to the verification of the monolayer assembly process including the protein vectorial orientation, the positions of the protein metal redox centers within this profile structure by resonance x-ray diffraction to within Ϯ1-3 Å (Pachence et al, 1989), and the orientations of the redox centers within the monolayer structure by polarized x-ray spectroscopy (Zhang et al, 1997) and optical linear dichroism (Pachence et al, 1990). These techniques have recently been successfully applied to vectorially oriented monolayers of both cytochrome c covalently tethered to thiol SAM endgroups (Chupa et al, 1994) and detergent-solubilized cytochrome oxidase electrostatically tethered to amine SAM endgroups (Edwards et al, 1997). Cyclic voltammetry studies performed by others on similar, but structurally uncharacterized, supposedly monolayer, systems containing cytochrome c (Collinson at al., 1992) and cytochrome oxidase (Cullinson et al, 1994) nevertheless indicate that these membrane protein systems are fully functional.…”