The aim of this application is to pursue the structural study of proteins--especially membrane proteins--by high resolution electron microscopy and image processing of electron micrographs. The first part of the proposal deals with studies of membrane proteins which have been prepared in two-dimensionally ordered arrays (two-dimensional crystals) and emphasizes the determination of the three-dimensional structure of cytochrome c oxidase at higher resolution (ca. 10-15 angstroms and in a more natural environment than previously. This will be accomplished by recording electron micrographs of specimens which have been frozen in thin layers of vitreous ice and maintained at near liquid nitrogen temperatures. Specific labeling of functionally important sites will be accomplished with antibody preparations and with undecagold cluster compounds which have been derivatized to render them selectively reactive to certain amino acid side-chains. A second part of the proposal deals with the examination of the surface structures of membranes by freeze-drying and high resolution heavy-atom shadowing (both unidirectional and rotary). Particles contrasted by shadowing membrane surfaces will reveal to a resolution approaching 20 angstroms: 1) How far membrane proteins protrude from the bilayer surface. 2) The oligomeric state of membrane proteins in phospholipid bilayers. 3) Locations of binding sites of small soluble proteins on membrane proteins (e.g. the high affinity cytochrome c binding site on cytochrome oxidase). 4) Whether there are associations between different membrane proteins, e.g. respiratory chains in mitochondrial membranes. This approach will initially be applied to """"""""artficial"""""""" membranes, reconstituted proteo-liposomes, in order to characterize the structures of purified membrane proteins and any structural changes in response to changes in functional state such as changes in oxidation state, membrane potential etc. It will eventually be extended to studies of membrane surface structures of natural membranes such as the inner mitochondrial membrane.
| Valpuesta, J M; Henderson, R; Frey, T G (1990) Electron cryo-microscopic analysis of crystalline cytochrome oxidase. J Mol Biol 214:237-51 |
