STRUCTURE - ACTIVITY STUDIES OF COMPLEMENT PROTEIN C9

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The ninth component of complement (C9) is responsible for the cytotoxic action of complement. It is, therefore, an important component of the immune system. C9 is a 70 Kda glycoproteins which in order to be effective must change from a stably folded, water soluble protein into an intrinsic membrane protein. The nature of this transformation must be understood to appreciate how this protein functions. To gain such information a thorough investigation of the structure C9 in its native water soluble form and in its membrane bound form will be initiated. Information static structures will be obtained by surface mapping, EPR and fluorescence spectroscopy, electron microscopy and 3-D image reconstruction, and small angle neutron scattering. The kinetics of structural changes as they occur when the protein enters a membrane will be followed by EPR and fluorescence spectroscopy. All techniques will benefit from labeling of the protein with specific probes. In the past specific labeling was limited by the small number of sites available on a protein. However, this problem can now be circumvented to some extend by site-directed mutagenesis and the use of newer spectroscopic techniques of sufficient sensitivity to examine small amounts of mutant proteins. The approach is to employ genetically engineered labeling sites in the C9 sequence for the attachment of labels. The specific aims for the next grant period are: 1. To identify the peptide region necessary for hemolytic function by generation of C9 mutants with free cysteines for site-specific attachment of labels, and of hybrid molecules composed of segments from lytic (human) and non-lytic (horse) C9 molecules. 2. To determine the presence (or absence) of transmembrane regions in membrane-bound C9 by mapping of the surfaces of native C9 and membrane- bound C9 with immunologic, enzymatic and spectroscopic methods. 3. To identify contact sites between C8 and C9 and to measure the kinetics of C9 refolding and insertion into membranes. 4. To generate a low resolution structure of membrane-bound C9 by 3- dimensional image reconstruction and by small angle neutron scattering combined with contrast variation procedures.