The final outcome of complement activation on cells is the formation of the membrane attack complex (MAC). This membrane channel structure is fashion by the sequential fusion of five plasma proteins C5b through C9. There are two questions which I am addressing in this grant: what is the structure of these components, and how do they assemble to form transmembrane channels. Addressing the issue of structure, all these proteins have been sequenced; therefore, other levels of structural organization need to be investigated with emphasis on forming relationships among the different levels. One of the aims include deciphering the disulfide bridging patterns of C6 and C9. Also as a result of the successful crystallization of human complement component C5, X-ray diffraction analysis will be used to decipher the entire three dimensional architecture of this protein. Since C5 belongs to a family of proteins including C3, none of which have been delineated in terms of their tertiary structure, the successful decipherment of the three dimensional structure of C5 will provide insight into the folding of an entire family of Human Immunity Proteins. Immuno-electron microscopy and image reconstructions of single particles of poly (C9) will be employed to obtain the most detailed picture to date of the ultrastructure of this assembly. Molecular chimeras of C9 and perforin (a lytic protein from T-lymphocytes) will be studied to identify both structural and functional aspects of segments of these proteins that are involved in tubule formation, lysis and C5b-89 interaction. In addition recombinant perforin will be characterized using a wide spectrum of techniques including circular dichroism spectroscopy, electron microscopy and protein modification. Although all the proposed work is basic science, it is my view that t knowledge obtained ultimately will have health relatedness. The lytic components of the immune system have been implicated in disorders such as multiple sclerosis, Guillain-Barre', and myasthenia gravis where damage to membranes occurs. To develop methods to deal with such medical problems requires a through understanding of the molecules of the immune system that are responsible for membrane disruption.
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