Protein sensor design for the quantification of heme concentration in cells

Due to the potentially toxic nature and hydrophobicity of the heme metalloporphyrin, the necessity for this prosthetic group to be trafficked across cell membranes suggests the existence of highly controlled delivery mechanisms. Furthermore, accumulating evidence on the signaling and regulatory properties of heme is indicative of a much wider spectrum of roles for this ubiquitous cofactor than traditionally thought. New insights into the biological significance of the labile heme pool are required in order to elucidate heme roles within the cell metabolism and homeostasis. As a contribution to this grand scheme, the aim of the project is to design and build a sensor for the quantification of labile heme concentration in cells. This will involve the construction of fluorescently-labelled proteins which can bind heme under a range of heme concentrations and conditions. Moreover, the optical properties of the fusion heme-binding proteins will be exploited for imaging purposes, trying to track heme into cells.

Recombinant proteins will be mainly expressed in E. Coli making use of properly engineered plasmid vectors. Purification and characterization will involve standard molecular biology procedures. The quantification of the heme-sensor binding and the imaging studies will require the use spectroscopy/imaging techniques such as FLIM and FLIM-FRET.

The project is under the supervision of Prof. Emma Raven at the University of Bristol and will involve the collaboration of Dr. Andrew Hudson (Leicester University) and the Wolfson Biomaging center (Bristol).