A major goal of our research program is to identify the exact molecular nature of the interaction between nitric oxide (NO) and p2l'. Previously, our work suggested that NO may form a nitrosothiol on p2l"""""""" and thus regulate function. In collaboration with Dr. Brian T. Chait at Rockefeller University we have identified and quantitated nitrosothiol bonds on p2l'"""""""" using ESI-MS. We expanded this study to characterize and examine the chemical nature of nitrosothiols and developed methodology to make ESI-MS a useful tool for investigators studying NO biology. These studies are the first reported use of ESI-MS in this context and may help investigators better understand the consequences of NO-protein interactions. These results were reported in a paper in J. BioL Chem. Having found that a single nitrosothiol is formed on p2l' upon NO addition, we set about to identify which Cys residue is modified. Our p2l's preparation has 5 reduced Cys residues although only one is surface accessible (Cys 118). Using CNBr we generated peptide fragments of untreated and NO-treated p2l'. These fragments were analyzed by HPLC-MS. The peptide fragment containing the nitrosothiol has a mass of 29 Da (the mass of NO minus that of the substituted hydrogen atom) greater than that of its untreated parent fragment. The specific residue that was modified turned out to be CySl 18. A report descring this result was published in Nature 381 (1996) 379 and a more complete description appeared in J. Biol. Chem. 272, 4323-4326, 1997. Our data suggest that the formation of this nitrosothiol leads to a conformational change in p2 I'"""""""". We propose to use NMR to characterize this conformational change. The results derived from these studies will provide a critical understanding of the molecular nature by which NO initiates a signaling cascade from the cell membrane to the nucleus. Furthermore, the NO-p2l... interaction we will characterize will likely provide a paradigm by which NO activates other enzymes, such as cyclooxygenase, through nitrosothiol formation.
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