Expression, Purification and Molecular Characterisation of Sideroflexin Proteins
Members of the sideroflexin family are poorly characterised membrane proteins of the mitochondrial inner membrane. Understanding their function is now a high priority, as patients have been identified who carry mutations in human sideroflexin 4, causing a severe mitochondrial, neurological and haematological disease phenotype. The work in this thesis aims to describe the molecular properties of the sideroflexin proteins in order to elucidate their function. In the first part, yeast and the five human sideroflexin proteins were successfully expressed in mitochondria of Saccharomyces cerevisiae and purified to homogeneity. In the absence of a functional assay, thermostability assays and size exclusion chromatography were used to show that the purified proteins were folded, and therefore amenable to further biophysical and functional analyses. Next, the effect of lipids on the stability of human sideroflexin 5 was investigated, demonstrating that cardiolipin is an important stabilising factor. Using these stabilising conditions, it was subsequently established that the protein is dimeric in detergent solution. Based on the principle that interactions with compounds increase the stability of proteins, a compound library was screened in thermostability assays. The results show that human sideroflexin 5 binds zinc ions, which was confirmed subsequently by native mass spectrometry and elemental mass spectrometry. Following this result, the groundwork was laid to find the zinc binding site by combining scanning alanine mutagenesis with thermostability assays. Furthermore, all five human paralogues and yeast sideroflexin were successfully expressed in Lactococcus lactis for zinc binding or transport studies to establish the functional role of the sideroflexins. Finally, in an unexpected way, haem was found to be associated with concentrated human sideroflexin 5, opening up the possibility that sideroflexins could be involved in haem metabolism, which would explain the haematological disease phenotype well. This notion was explored further by Förster resonance energy transfer experiments and binding equilibrium assays using hemin, but hemin intercalates into detergent micelle and it was not possible to separate specific binding from non-specific hemin-detergent interactions, but this lead needs to be explored further.