Class II molecules of the Major Histocompatibility Complex (MHC) are central to antigen presentation in T cell-dependent immune responses. The lateral and rotational dynamics and membrane distribution of Class II molecules have been implicated in signal transduction and antigen presentation. The cytoplasmic domains of Class II molecule alpha- and beta-chains are critical for normal functioning of these molecules and are major determinants of their membrane motions. In collaboration with Dartmouth Medical School, we will examine the effects of mutations within Class II molecules on their membrane dynamics and biological function. B cells expressing mutant Class II molecules with truncated and substituted cytoplasmic domains, as well as Class II molecules with mutations in the contact residues of the Class II (alpha- beta2) dimer, will be examined using biophysical methods to determine if specific Class II molecular structural features and domains have roles in modulating molecular dynamics and intermolecular associations. These phenomena have been implicated in regulating Class II functions including signal transduction in the B cell and antigen presentation to T cells.
Specific aims i nclude: l) Time-resolved phosphorescence anisotropy, polarized fluorescence depletion and fluorescence photobleaching recovery studies on cytoplasmic mutants of Class II to determine which amino acid residues regulate Class II molecular rotational and lateral motions. An additional aspect of this aim is to examine Class II molecules with mutations affecting signalling. The effect of mutations on molecular motions will be correlated with signalling defects and correlated with nearest neighbor analysis of protein-protein interactions which contribute and control Class II's membrane motions and physiology; 2) Lateral and rotational diffusion studies of wild type and mutant Class II molecules in the presence or absence of immunogenic peptides. These studies will examine the ability of peptides to regulate Class II aggregation in the presence or absence of cytoplasmic domain structures of Class II molecules; and 3) Examination of the effect of mutations in the contact amino acids between alpha-beta molecules in the Class II (alpha-beta)2 dimer. Class II molecular aggregation appears required for efficient signal transduction and antigen presentation. Studies in this aim will examine whether changes in dimer-forming ability, evaluated as rotational and energy transfer changes measurements, correlate with the efficacy of antigen presentation.
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