Both soluble proteins and detergent-solubilized integral membrane proteins can be tethered to the surface of inorganic substrates via bifunctional organic chain molecules possessing a headgroup specific for the substrate surface and an endgroup specific for one or more residues exposed on the protein's surface. Such tethering can provide a single, vectorially-oriented monolayer of the fully-functional protein on the surface of the substrate which is ideal for correlated structural and functional studies, especially if the solid substrate has been suitably tailored by Molecular Beam Epitaxy for x-ray (or neutron) diffraction studies. We have most recently employed sulfhydryl-endgroups for covalently tethering yeast cytochrome c, in which the heme iron atom was isomorphously replaced by cobalt, to the surface of Ge/Si multilayer substrates; the so-tethered cytochrome c monolayer was then used as a template to electrostatically bind detergent-solubilized mitochondrial cytochome aa3 oxidase to thereby form a vectorially-oriented monolayer of the bimolecular cytochrome c-cytochrome oxidase complex on the substrate's surface. Resonance x-ray diffraction effects on the meridional kinematical x-ray diffraction from the cytochrome c heme cobalt atom and from the cytochrome oxidase heme a and heme a3 iron atoms have been detected, utilizing a position-sensitive proportional counter, with good signal-to-noise over a sufficiently wide range of photon momentum transfer Qz. The on-going analysis of these resonance effects on the meridional diffraction will permit an unambiguous determination of the locations of the resonant atoms within the already determined nonresonant profile structure of the bimolecular complex within the tethered monolayer. Such structural studies are critical to our understanding of the actual structure of this bimolecular complex and the role of this structure in intermolecular electron transfer phenomena, including the possible electrogenic nature of the intermolecular electron transfer in the generation of transmonolayer electrochemical potentials.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR001633-14
Application #
5223467
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
14
Fiscal Year
1996
Total Cost
Indirect Cost
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