| Chemical footprinting of proteins using the hydroxyl radical as a probe is a well characterized method for investigating protein-ligand interfaces. One current method, in which the radicals are formed by millisecond irradiation with synchrotron light, can only be performed at the National Synchrotron Light Sources. Other methods generate hydroxyl radicals on seconds or greater timescales, but long oxidation times may compromise the results. We developed a 248-nm laser method, called Fast Photochemical Oxidation of Proteins (FPOP), to cleave homolytically H2O2 in 17 nanoseconds. The solution contains a scavenger to quench the radicals in one microsecond. FPOP probes more residues than other methods, ensuring that more surface area is probed, and at a sufficient speed that the protein cannot unfold as a result of the oxidations. With apomyoglobin as a model, we confirmed that the F-helix is exposed to solvent and that the ligand binding pocket is closed in the absence of the heme ligand. With myoglobin, the F-helix is conformationally constrained, and the H-helix acts as a hinge for the ligand binding site, decreasing the solvent accessibility of leucine 137 when the heme is bound. A modified continuous beam footprinting method showed that apomyoglobin does sample the open conformation when the radicals are present for 30 seconds. This supports our hypothesis that FPOP at the microsecond timescale captures a snapshot of the protein in solution. During the course of these investigations, we unexpectedly found that nearly 100% single oxidation of apomyoglobin occurs when the protein and 100-mM H2O2 were incubated at -80°C for two hours. The phenomenon depends on time, temperature, and [H2O 2]. While incubation at -15°C produced nearly 100% double oxidation, minimal oxidation occurs at 4 and 22°C, demonstrating that the reaction is promoted in the ice lattice, an effect not previously reported in the literature. We also found that the oxidant can diffuse over the surface of the protein, but not through the bulk media. We conclude the thesis with some perspective on the FPOP method and its applicability. |
