| Summary: | The circadian cycle plays an important role in homeostasis by regulating the gene expression of rate-limiting enzymes and controlling the release of hormones. This means that disruption to this cycle can lead to metabolic and psychological disorders, such as diabetes or depression. In this thesis, major components of the circadian cycles have been studied using a combination of in vitro and in vivo techniques. CLOCK, NPAS2 and BMAL1 are transcription factors which utilise PAS domains to regulate their dimerisation and the binding of DNA via their helix-loop-helix domains. Constructs of the human forms of these proteins were expressed in E. coli and purified under native conditions. This permitted the study of the heme and carbon monoxide (CO) binding properties of the isolated domains from CLOCK and NPAS2 proteins. Similar heme binding characteristics were observed for both proteins, with a K[subscript D] values for heme binding in the micromolar range, biphasic dissociation curves and comparable UV-visible absorption maxima. All PAS domains tested were shown to bind CO but none reacted with oxygen to form a ferrous-oxy species. The heme chemistry in CLOCK and NPAS2 was examined further by determining the reduction potential of the heme iron. This was done using a novel method developed through the adaption of a previously described equilibrium reaction procedure, which was then altered by the addition of enzymes to remove oxygen, reduce the heme iron and reduce a redox dye. From the known reduction potential of the redox dye, it was possible to derive the reduction potential of the heme group. The results showed that heme bound to CLOCK and NPAS2 has a relatively low reduction potential, a property which likely contributes to the lack of a ferrous-oxy species. DNA-binding and functional assays were also used to show that the binding of heme observed in vitro has a physiological role. The DNA-binding behaviour of CLOCK and NPAS2 constructs were shown to be regulated by the addition of heme in vitro. Furthermore, the use of a heme synthesis inhibitor in a report assay demonstrated for the first time a direct link between intracellular heme concentrations and the transcriptional activity of the CLOCK:BMAL1 complex. It was seen that higher heme concentrations promoted the transcriptional activity of CLOCK and BMAL1. This is consistent with the observation made in vitro that heme disrupts the interaction between the inactive CLOCK:CLOCK homodimer and DNA.
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