Abstract
Heme, or iron protoporphyrin IX, has been traditionally understood to bind exclusively as a cofactor to proteins. However, regulatory roles have since been uncovered, for example, in circadian rhythms, gas sensing, gene expression, and immunity. As a result, the need to uncover more regulatory heme proteins has become apparent. Many methods for studying heme binding to proteins are available, including traditional techniques (such as mutational analysis and measurements of heme binding affinity) and novel approaches (machine learning and proteomic analyses). However, little is known about how heme is made available to bind to these proteins. Knowledge of the bioavailability and the oxidation state of heme in the cell is required to understand the physiological context in which these proteins bind heme.To this aim, we have adopted a dual approach, the first of which is an investigation into the use of bioinformatics to predict heme binding. To do this, we have employed the ligand binding prediction tool, ProFunc, in combination with AlphaFold. The predicted binding sites were assessed and compared to existing crystal structures (with or without heme bound) and with suspected binding residues from the literature. This work is the start of an exploration into the techniques available for predicting heme binding and demonstrates their practical uses and limitations. The second part of the approach is the creation of a genetically encoded redox-sensitive heme sensor for deployment in live cells. The sensor is a recombinant fusion protein of the mKate2 fluorescent protein and a variant of the heme-binding protein, myoglobin, and the first steps of its development are described in this thesis. This sensor will provide much-needed understanding into the significance of redox state in heme binding and cellular regulation and be an important step forward for the heme community.
Date of Award | 6 Dec 2022 |
---|---|
Original language | English |
Awarding Institution |
|
Supervisor | Emma Raven (Supervisor) & Avinash J Patil (Supervisor) |