Engineered heme peroxidases as genetically encoded probes with diverse applications in cell biology

Abstract

Proteins perform myriad functions inside cells. In vitro studies of purified proteins yield important information about their function, but it is an important pursuit in cell biology to study proteins within the native context of intact cells, since their physiological functions might not be recapitulated in any other environment. The most basic question, the location of a protein within the cell, is difficult to answer, since most proteins do not possess a spectroscopic handle that causes them to stand out above all the other proteins and biomolecules in the cell. Fluorescent proteins such as GFP have been revolutionary in allowing specific proteins to be tracked within living cells, but the resolution of fluorescence microscopy (~200 nm) is often inadequate for determining precise subcellular localization. Electron microscopy (EM), on the other hand, offers far superior resolution (<10 nm), but no genetic tag equivalent to GFP has been developed for EM. Horseradish peroxidase (HRP), a multifunctional reporter enzyme widely used in bioanalytical assays, has been utilized for EM because of its ability to generate electron-dense deposits. In addition, HRP produces extremely sensitive chemiluminescent, colorimetric, and fluorescent readouts and has applications for promiscuous protein labeling and detection of hydrogen peroxide. However, the failure of HRP to become active in most cellular compartments has hampered its adoption as a genetic tag. This thesis describes our engineering of genetically encoded peroxidase probes with diverse applications in cell biology. We describe the development of enhanced ascorbate peroxidase (APEX), a HRP-like tag that works in all cellular compartments. We present our applications of this tag for EM imaging, live-cell proteomic mapping, and intracellular detection of hydrogen peroxide. We also discuss the discovery and characterization of APEX2, a greatly improved variant of APEX. Finally, we describe the development of a split HRP protein complementation assay using a combination of rational design and directed evolution, followed by our application of split HRP for detection of neurological synapses.