Isotopic exchange of amide hydrogens in proteins has been an important tool for identifying which parts of a protein participate directly in conformational changes. For example, hydrogen exchange has been used to determine that the allosteric changes in hemoglobin are centered on the C-terminal segment of the beta- chain. Likewise, the specific surface of cytochrome c that binds to a monoclonal antibody has been determined by hydrogen exchange. Despite the apparent utility of hydrogen exchange as a sensitive probe for investigating conformational changes of proteins, it has been applied to only a few problems because of limitations inherent to current methods -- NMR or tritium exchange -- for detecting hydrogen exchange. We propose to develop methods based on directly-coupled HPLC fast atom bombardment mass spectrometry (HPLC FABMS) to detect and quantify hydrogen exchange in proteins. Amide hydrogens in proteins will be selectively exchanged with deuterium by an established exchange-in/exchange-out procedure. The deuterium content of specific segments of the protein will be determined by proteolytically fragmenting the protein into peptides, whose molecular weights will be determined by HPLC FABMS. The deuterium content of very short segments of the protein, including specific amide sites, will be determined by subdigestion or MS/MS. In addition to facilitating accurate determination of hydrogen exchange rates in large and complex proteins that cannot be studied by NMR, HPLC FABMS offers the advantages of higher speed and sensitivity, as well as the possibility of providing new types of information. Directly-coupled HPLC FABMS will be used to determine rates of hydrogen exchange in a variety of challenging protein structure/function problems. The site at which Band 3 protein, an enzyme inhibitor, binds to aldolase and glyceraldehyde-3-phosphate dehydrogenase will be determined from changes in the rates of hydrogen exchange found in the enzymes. Hydrogen exchange methods will also be used to investigate conformation changes in lens proteins that may be related to cataractogenesis. In addition, the noncovalent binding of carbohydrates to proteins will be investigated by determining the rates of hydrogen exchange in free and complexed L-arabinose-binding protein.
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