Lougher, Matthew J. (2015) Functional and structural insights into MmyJ, An ArsR-like transcriptional repressor. PhD thesis, University of Warwick.

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Abstract

MmyJ is a protein encoded in the methylenomycin antibiotic gene cluster in Streptomyces coelicolor A3(2). It was identified as a novel member of the ArsR family of transcription repressors. In depth bioinformatic analyses were carried out to compare this to other members of the ArsR family, leading to high confidence of its classification. An expression system was engineered to investigate MmyJ in vitro, leading to the discovery of the presence of a covalently bonded dimer, which is unusual for ArsR family proteins and as such was thought to be an artefact of purification. A C49S mutant was then also engineered without the capacity to form covalent dimers, such that the two variants could be investigated side by side. Work was carried out to investigate the stability of MmyJ, and it was found that its secondary structure denatured above temperatures of 40oC and that the protein is only robust to a single freeze/thaw cycle. Electrophoretic mobility shift assays were then used to prove that MmyJ binds specifically to a 13-1-13 semi conserved inverted repeat overlapping the -35 region of both mmyJ and mmr promoters. This indicates that MmyJ not only regulates its own expression, but also that of Mmr, an efflux pump that removes methylenomycin A from the cell upon biosynthesis. It was also demonstrated that methylenomycin A caused complete dissociation of the MmyJ:DNA complex when present in a 20 times molar excess, hence suggesting that the methylenomycin A resistance mechanism is triggered in the presence of methylenomycin A. This is the first reported instance of an ArsR family protein sensing a non-metallic ligand. Structural insights into MmyJ were also sought, with a homology model produced by Phyre2 analysis complimented by circular dichroism analysis of recombinant MmyJ. X-ray diffraction data were obtained to a resolution of 2.1 A over 300o, but the phase of these data has yet to be determined, so the crystal structure has not yet been solved.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QR Microbiology
Library of Congress Subject Headings (LCSH):	Antifreeze proteins, Proteins -- Structure, Protein engineering
Official Date:	25 September 2015
Dates:	Date Event 25 September 2015 Submitted
Institution:	University of Warwick
Theses Department:	Molecular Organisation and Assembly in Cells
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Corre, Christophe ; Lewandowski, Józef R. ; Mitchell, Daniel E.
Sponsors:	Engineering and Physical Sciences Research Council
Extent:	xxv, 157 leaves : illustrations (colour), charts.
Language:	eng

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