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Circadian Regulation Through The Binding of Heme to Drosophila Melanogaster PER-PAS

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posted on 2019-09-04, 10:14 authored by Raoof J. A. Maaroof
In Drosophila, the clock gene period (per), is an integral component of the circadian clock and acts via a negative auto regulatory feedback loop. Comparative analyses of per genes in insects and mammals have revealed that they may function in similar ways. PERIOD proteins are central components of the Drosophila and mammalian circadian clocks. Their function is controlled by daily changes in synthesis, cellular localization, phosphorylation, degradation, as well as specific interaction with other clock components. A comparison between the Drosophila and the mammalian circadian clock system reveals many similarities and differences amongst them. In Drosophila there are two period genes (per1, per2). PERIOD is a transcriptional regulatory factor involved in metazoan circadian rhythms. In Drosophila melanogaster, PERIOD2 (dPER2) dimerises with TIMELESS (TIM) and the complex enters the nucleus and disrupts the DNA binding of the transcriptional activator heterodimer CLOCK/CYCLE (or dBMAL1). The dCLOCK/dBMAL1 complex is a transcriptional activator for both per and tim genes and hence PER and TIM inhibit their own expression through a negative feedback mechanism. In more recent years, the role of heme in biology appears to also include a regulatory function in the cell; some proteins involved in transcription, like the CLOCK proteins, are known to interact with heme via their PAS domains. This work endeavours to investigate the effect of heme on the dPER protein. In this work, we have cloned and expressed constructs of Drosophila per2 in E. coli; including the PAS-A, PAS-B, PAS-AB and PAS- ABα domains of the protein. These structures were designed in three trials. In each trial, the different regions of the per gene was expressed to identify the heme binding domain and characterise the binding. Constructs were designed with a range of purification tags (His, GST, MBP and trial without tag) to enable soluble protein expression. We have used difference absorption spectroscopy to examine whether the dPER-PAS fragments (dPER-PAS-A, dPER-PAS-B, dPER-PAS-AB and dPER-PAS-ABα) are able to bind heme. The difference absorption spectra obtained after addition of increasing amounts of heme to dPER-PAS-A, dPER- PAS-B, dPER-PAS-AB and dPER-PAS-ABα show that heme can form a complex with all four domains above with a Soret band at ~ 423nm. To support the finding of heme binding to these dPER-PAS fragments, Electron Paramagnetic Resonance (EPR) was performed. EPR showed binding between heme and a cysteine within the dPER domain in a 1:1 ratio. There are eight cysteines in the dPER domain. To find out whether this interaction is specific, two single mutants were made for each of dPER-PAS-A (C312A, C369A), dPER-PAS-B (C455A, C467A), dPER-PAS-AB (C312A, C455A) and dPER-PAS-ABα (C312A, C455A). There was no significant change to the heme binding properties of mutant domains as measured by UV-visible spectroscopy. This suggests that the heme binding occurs in one of the cysteines that we have yet to look at in this study. Additionally, we attempted to make dPER-PAS-A, dPER-PAS-B, dPER-PAS-AB, and dPER-PAS-AB crystals complexed with heme to identify the site and the amino acids responsible for heme binding. Conditions resulting in crystalline dPER-PAS-A were discovered. Overall, in this project we have shown that the dPER protein is capable of binding heme. Each of the domains studied here (PAS-A, PAS-B, PAS-AB) are capable, on their own, of heme binding. We have demonstrated that the binding of heme to dPER is via a cysteine residue, however loss of cysteine through mutagenesis at positions C312, C369, C455, and C467, is not sufficient to ablate the heme binding activity of dPER.

History

Supervisor(s)

Raven, Emma; Hudson, Andrew

Date of award

2019-08-07

Author affiliation

Department of Chemistry

Awarding institution

University of Leicester

Qualification level

  • Doctoral

Qualification name

  • PhD

Language

en

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