| Construction and characterization of protein assemblies on solid substrates have a wide range of potential applications in basic biological research and biotechnological development in medicine and industry. Heme-containing electron transfer proteins, cytochrome b5 and cytochrome c, provide excellent subjects for developing synthetic methods for assembling proteins into thin films. Due to their inherent robustness and well understood physical properties, both of these cytochromes serve as model proteins for studying recognition, structure and stability at the solid/liquid interface.; Orientation of cytochrome b5, either on gold-supported alkanethiol monolayers or maleimide terminated lipid bilayers on mica, was achieved through covalent attachment of a genetically engineered surface cysteine. Monolayers of cyt b5 on gold were then used to look at orientation and ionic strength dependence of complex formation with the complementary protein, cytochrome c, from solution. It was shown that cyt c preferentially binds to cyt b5 when the heme edge of the latter is accessible from solution and that the predominant, but not unique, force controlling the affinity is electrostatic in nature. Surface forces measurements between bilayer supported monolayers of each of the cytochromes, using the surface forces apparatus, probed the interactive force between the two proteins as a function of distance. Orientation of the cyt b5, relative to cyt c, affected both the overall adhesion and long range forces observed.; In order for protein assemblies to serve in medical or industrial capacities, they must be stable in variable and often non-native environments. Determination of structural stability and functionality, in terms of electroactivity, of covalently immobilized cytochrome c was performed in various harsh conditions. Organic, chemical and thermal denaturants altered the properties of cyt c in the heterogeneous environment but much of their overall integrity was maintained.; Scanning probe microscopies, particularly atomic force microscopy and scanning tunneling microscopy, offer structural biologists new and powerful methods for understanding biointerfacial systems. Both STM and AFM provide information about the structure of protein monolayers and these techniques, including their current limitations, are discussed. |
