Class I molecules are heterotrimers, based on considerable evidence that the integrity of the class I H chain is dependent upon it being assembled with Beta 2m and bound by a peptide ligand. In the endoplasmic reticulum both Beta 2m and peptide are known to be critical factors in the de novo folding of the class I ligand binding site, although their separate roles are poorly defined. Furthermore, association with Beta 2m and peptide is critical for maintaining the integrity of the class I ligand binding site at the cell surface. Surface class I H chains are capable of exchanging Beta 2m and/or peptide, however, the mechanism and inter-relationship of these two processes is unknown. The experiments proposed here will resolve several outstanding questions concerning the precise structural and functional implications of peptide and Beta 2m interactions with class I H chains. The structure of non-peptide bound class I molecules will be characterized, using a mAb specific for empty molecules. This mAb will be used to trap and stabilize empty class I molecules for crystallographic resolution. Using both immunochemical and crystallographic approaches, we will also determine how differences in peptide sequence influence the conformation of the ligand binding site. Furthermore, using a peptide library we will determine whether the same peptides bind to class I in the presence or absence of Beta 2m. Finally, we will define the interrelationship of peptide and Beta 2m exchange by surface class I molecules, and determine whether these two processes are cooperative or antagonistic.
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