Investigations of Metalloproteins by Purification and Unlabeled Nitrogen NMR

Abstract

The first half of this study concerns the fungal oxidoreductase Chloroperoxidase, and the possibility of its modification towards a model system for the newly-discovered class of enzymes, Aromatic Peroxidases. CPO was synthesized in transgenic bacteria first as the wild-type protein, then again after directed mutagenesis, with the goal of incorporating the heme prosthetic group into the apoprotein and regenerating function. Unfortunately, despite promising extant results in the literature, every attempt ultimately failed at the reconstitution step. The second half of this study concerns \(^{14}\)N NMR spectroscopy. While this nucleus is usually dismissed outright as useless, an initial survey of the field suggested that it may be of more use than previously thought. While the end goal was to perform unlabeled nitrogen NMR experiments on proteins, and that particular tangent failed under the conditions available, a great deal of promising data was gathered.\(^{14}\)N NMR experiments successfully probed a number of organic small molecules and peptides, and in particular a binding isotherm for interactions between lysine's amine nitrogens and hydrogen was developed. These findings served to validate \(^{14}\)N NMR as a worthwhile paradigm, and highlighted a number of paths for further study.