A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2013. / The forkhead box (FOX) proteins are a family of transcription factors that interact with DNA via a winged helix motif that forms part of the forkhead domain. The FOXP (FOXP1-4) subfamily is unique in the family in that the forkhead domains of these proteins exhibit domain swapping where structural elements are exchanged via extension of the hinge-loop region. The FOXP subfamily members have high sequence homology, yet wild-type FOXP3 is a stable domain-swapped dimer in solution whereas FOXP1 and FOXP2 exist in a monomer/domain-swapped dimer equilibrium. A single amino acid difference is observed in the hinge region of the FOXP subfamily. This corresponds to Tyr540 in FOXP2 and Phe373 in FOXP3. We propose that it is the phenylalanine residue in FOXP3 that shifts the equilibrium towards dimer. Here we use FOXP2 to investigate the effect of a mutation, Y540F, on the structure and dimerisation propensity of the FOXP subfamily. Crystals for the Y540F variant in the presence of DNA have been obtained to demonstrate conclusively that domain swapping occurs. Size-exclusion chromatography indicates that the wild type FOXP2 forkhead domain is almost entirely monomeric at concentrations less than 100 μM. The Y540F variant is shown to stabilise the dimer and the ratio between monomer and dimer is concentration-dependent. DNA binding assays suggest that the Y540F variant binds less favourably to the cognate binding sequence than does the WT FOXP2 forkhead domain. Taken together, these findings suggest that domain swapping may modulate DNA binding.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/14851 |
| Date | 02 July 2014 |
| Creators | Perumal, Kershia |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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