The binding of antigenic peptides by class I major/histocompatibility complex (MHC) molecules is a crucial step in the immune response. Our long-term objectives are to understand how a given class I molecule can selectively interact with a large number of antigens, and how peptides contribute to class I assembly, conformation and stability. A thorough understanding of peptide-MHC interactions will surely help guide the design of peptide vaccines and high-affinity ligands for MHC molecules associated with autoimmune disease. We have recently succeeded in the isolation of soluble, empty (peptide-free) class I molecules (mouse H-2Kb) from recombinant Drosophila cells. Subsequently, we have established direct-peptide binding assay system in vitro using these molecules. Based on these studies, we propose the following research plans.
The specific aims and methods for achieving the goals are (1) to quantitatively analyze the interactions between class I molecules and natural peptides (octamers for Kb molecules). Using purified, soluble Kb molecules and [3H]-labelled octapeptides, the kinetic and equilibrium parameters for peptide binding will be determined by gel filtration, equilibriium dialysis and fluorescence spectroscopy; (2) to define the distinctive chemical and structural motifs of peptides required for selective binding to class I molecules. Using natural peptide analogs with single or multiple amino-acid substitutions, the affinities of these peptides for Kb molecules will be determined to elucidate peptide-binding motifs. Also, randomly synthesized octapeptides will be used to select high-affinity peptides; (3) to characterize the changes in conformation and stability of class I molecules upon peptide binding. The stabilities of the soluble forms of empty and peptide-bound Kb molecules will be determined by monitoring circular dichroism (CD) spectra as a function of temperature. The conformational changes of the class I heavy chains upon peptide binding as well as beta2m association will be also assessed by measuring CD spectra.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29AI031965-02
Application #
3455980
Study Section
Allergy and Immunology Study Section (ALY)
Project Start
1992-07-01
Project End
1993-08-31
Budget Start
1993-05-01
Budget End
1993-08-31
Support Year
2
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037