Previous experiments suggest a dynamic model for class II intracellular trafficking, which is regulated by conformational alternatives in the peptide binding domain. The focus of the experiments in this proposal are to test this model and explore the role of hydrogen bonds between class II and its bound peptide. The investigator will: 1. Delineate the contribution of hydrogen bonds to class II peptide binding. Loss of a single hydrogen bond between class II and peptide has been shown to have dramatic consequences on peptide binding and intracellular trafficking. The consequences of hydrogen bond loss at other sites will be explored, using PCR mutagenesis coupled with peptide binding and cell biology studies. 2. Determine the specificity of CLIP to the function of Ii in class II biochemistry and function. The Ii-derived CLIP segment is critical for assembly of Ii with the class II molecule. In the experiments in this Aim, they will determine whether the amino acid sequence of CLIP contributes unique aspects to the fate of class II, and whether the affinity of the """"""""CLIP"""""""" peptide for class II affects the biological consequences of Ii. 3. Determine whether peptide occupancy changes the intracellular fate of 81m and 82m. In this Aim, the investigator will evaluate whether peptides that have stable peptide binding to mutant class II molecules can change their fate within cells. Ii constructs with high affinity peptides in the """"""""CLIP"""""""" segment of Ii will be introduced into the mutant cells and consequences on their intracellular fate will be evaluated. 4. Evaluate the role of hydrogen bonds to the interaction of class II molecules with DM and on intracellular trafficking of class II molecules. A model for DM function is that it stabilizes an alternate conformational state of the class II molecule that favors peptide dissociation. The investigator will test whether this involves de-stabilization of hydrogen bonds between class II and peptide. Mutant molecules with altered potential for hydrogen bonds will be tested for altered interactions with DM and post-endosomal sorting. These experiments will provide insight into the basic molecular mechanism of DM function and may reveal influences of DM on the intracellular targeting of normal class II molecules that relate to peptide occupancy.
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