Our project entitled """"""""Molecular Immunology of MHC-Restricted T Cell Responses"""""""" aims at understanding the initial events of T cell activation. In order to do so, we will attempt the structure determination of a number of molecules retained in the TCR complex such as CD8ab and CD3 ge and de. Protein engineering will be used to produce single chain CD8-MHC molecules to facilitate crystallization of the CD8-MHC and CD8ab/TCRp-MHC complexes. In addition new crystallization techniques will be attempted for CD8 and CD3 molecules with the usage of viral display. The platform of the N-w virus and CPMV virus will be used for these studies. Also, a large number of Fab-CD3, and Fab- CD8ab complexes will be produced and used for crystallization.
The second aim i s to characterize the lateral interactions of CD3 dimers with T cell receptors by surface plasmon resonance and FRET using monomers and tetramers of the CD3 dimers and purified TCR molecules. Similar studies will be carried out with the pre-TCR and gd TCR. The first two aims will converge and help us into our attempt at reconstituting artificial TCRC in membranes. Observation of these structures will be done by single molecule microscopy in supported bilayers. The association of the different subunits of the TCRC will be studied in supported membranes by total internal reflection microscopy. Recombinant molecules will be labeled in vitro with fluorophores and reattached to membranes through chelating lipids. Interactions between individual components will be measured and compared to affinity numbers in solution. Behavior of reconstituted TCRC will be observed upon ligation with appropriate p-MHC complexes. These dynamic studies will be complemented by attempts at 2-dimensional crystallization of the TCRC. Finally, studies of the pre-TCR and the characterization of its ligand will aim at determining its structure and the structure of its ligand. Recombinant pre-TCR monomers and tetramers will be used to isolate a ligand using advanced proteomics approaches. Expression cloning will be attempted if biochemical techniques fail. Confirmation of ligand identity and binding properties will be studied by using transfection, expression of soluble recombinant forms of the molecule and surface plasmon resonance.
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