A major goal of my research program is to identify the exact molecular nature of the interaction between nitric oxide (NO) and p21ras. Previously, our work suggested that NO may form a nitrosothiol on p21ras and thus regulate function. In collaboration with Dr. Brian T. Chait at Rockefeller University we have identified and quantitated nitrosothiol bonds on p21ras 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 p21ras upon NO addition, we propose to identify which Cys residue is modified. Our p21ras preparation has 5 reduced Cys residues and only one is surface accessible (Cys118). Using either CNBr or trypsin, we will generate peptide fragments of untreated and NO-treated p21ras. These fragments will be analyzed by HPLC-MS. We expect that the peptide fragment containing the nitrosothiol will have a mass of 29 Da (the mass of NO minus that of the substituted hydrogen atom) greater than that of its untreated parent fragment. We will then compare the known sequence of p21ras with the fragments generated by our procedure and thus identify the specific Cys residue modified by NO. Our preliminary data suggest that the formation of this nitrosothiol leads to a conformational change in p21ras. We propose to use ESI-MS to characterize this change through hydrogen-deuterium exchange studies. By allowing exposed protons on native or NO-modified p21ras to exchange with a solvent containing deuterium, we will quantify the rate of exchange using ESI-MS. This will be directly proportional to the amount of protein surface exposed to the solvent. The molecular mass of p21ras is 21,298 Da. With an error of measurement of 0.02%, we will need a change of greater than 4.2 Da upon exchange to yield interpretable results. Based on our previous work in this area and molecular modeling of native p21ras, we expect a change of approximately 300 Da. 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-p21ras interaction we will characterize will likely provide a paradigm by which NO activates other enzymes, such as cyclooxygenase, through nitrosothiol formation.
NIH ExPorter
NIH ExPorter