| Popis: |
Due to their biological importance and functional diversity, radical S-adenosylmethionine (rSAM) enzymes have become popular targets for electron paramagnetic resonance (EPR) spectroscopic studies. EPR spectroscopy is a powerful tool that allows for the observation of the iron-sulfur clusters as well as paramagnetic reaction intermediates, thus providing insight into their catalytic mechanisms. While the iron-sulfur clusters may be readily observable by EPR spectroscopy in the enzymes' resting states, radical intermediates are often elusive and must be trapped. Here, we describe a protocol for trapping and analyzing the Lys-Trp intermediate of the Lys-Trp-crosslinking rSAM enzyme SuiB, including modified expression and purification steps. This protocol is also intended to serve as a primer for trapping paramagnetic intermediates in other rSAM enzymes for studying by EPR spectroscopy. |
