The T cell receptor (TCR) is a multi-subunit complex that mediates recognition of peptide antigens complexed to MHC molecules. Owing to the fact that the individual components of the receptor complex are transmembrane molecules and therefore, not amenable to study in solution, the structural basis of this recognition is ill-defined. Recently I have devised a general method to facilitate association of soluble TCR alpha and beta subunits through the use of leucine zipper sequences that permit only heterodimer formation. In the present proposal, a detailed analysis of TCR-peptide/MHC interaction will be performed utilizing TCRs specific for a well-characterized vesicular stomatitis virus (VSV) octapeptide in the context of the Kb MHC class I molecule. First soluble TCRs will be engineered for secretion in eukaryotic cells (Lec328l CHO) which synthesize homogeneous glycans and whose glycoproteins can be readily deglycosylated using Endo-H. Material will be provided for x-ray crystallography in the form of the """"""""apoprotein"""""""" by itself as well as complexed with homogeneously VSV-loaded Kb molecules that have been previously shown to defract to high resolution by x-ray crystallography. The sTCRs will also be complexed with various Fab fragments of anti-TCR mAbs directed against variable region, constant region and clonotypic determinants. Second, mutations in TCR residues as well as Kb a helical residues will be created by site-directed mutagenesis and, in conjunction with peptide variants at the p1, p4 and p6 position of the VSV octapeptide thought to be TCR contacts, used in functional studies to probe the basis of TCR-peptide MHC recognition. Comparison of different TCRs recognizing the same VSV-8/Kb complex will be made. In addition, altered peptide ligands (APL) of several of these TCRs will be identified by functional criteria and complexed with Kb for structural studies. The latter will offer considerable insight into peptide/MHC complexes which are stimulatory or antagonistic of T cell activation. Third, biophysical analysis of TCR/VSV-Kb interaction will be performed using plasmon resonance. The affinity of several TCRs for VSV/Kb as well as APL/Kb will be characterized. Correlations between monomeric receptor affinity for Kb with variant peptides and the processes of thymic selection using TCR transgenic animals in the Rag-2-/- background will be investigated.
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