Class II MHC proteins are a b heterodimers (M.W ~ 66,000). The peptide is streched in the binding site forming a network of hydrogen bonds with the MHC protein. Some of the peptide side chains interact with deep pockets in the binding site. Strong interaction of the peptide with the binding site is likely to be physiologicaly important, it allows the complex to presist and increase its immunogenicity. To get new insight on the structure of the MHC II: I-Ak-HEL 48-62 we probed the dynamics of the peptide backbone binding by hydrogen exchange kinetics in combination with LC-MS/MS. A soluble form of I-Ak protein was made with a covalently attached HEL 48-62. After proteolysis the complex is occupied by a unique peptide. The MHC is completely deutrated and H/D back exchange is initiated by diluting the solution in H2O at pH 7.4 for varying times. The exchange is stopped by lowering the pH to 2.75. The peptide deuterium content and distribution was investigated by rapid LC-MS/MS analysis. A new data processing algorithm and a dedicated computer program were developed to deconvulute the deuterium content from other isotopic contributions and calculate, from time-dependent product-ion spectra of the deuterium-enriched peptide, the rate constants for exchange at most of the amide linkages of the bound peptide. The amount of exchange is related to the extent of protection afforded the antige nic peptide in the MHC and is remarkably consistent with what is seen in the crystal structure.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR000954-22
Application #
6118601
Study Section
Project Start
1998-08-01
Project End
1999-07-31
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
22
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Washington University
Department
Type
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
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