We have characterised two biologically important protein complexes. Firstly, we have captured and analysed a complex of the biotin protein ligase (BPL) and the biotinylation domain (BCCPΔ67) of acetyl-CoA carboxylase (ACC) from the hyperthermophile <i>Aquifex aeolicus. </i>The genes encoding both BPL and BCCPΔ67 were overexpressed in <i>E. coli</i> and purified to homogeneity. Isolation of milligram amounts of both recombinant proteins allowed us to perform kinetic analysis of the BPL enzyme using steady-state techniques. Furthermore, a chemically crosslinked complex of BPL and BCCPΔ67 was isolated and a comprehensive mutational study has identified a salt bridge between the two proteins which is important for heterodimerisation. The role of a conserved ‘thumb-like’ motif in BCCPΔ67 was also investigated by mutagenesis. Our results suggest an interaction between the biotin moiety and the ‘thumb’ reduces the propensity of BPL and holo-BCCPΔ67 to heterodimerise. In a separate project we have investigate the relationship between the tertiary structure and biological activity of a novel β-defensin related peptide (Defrl). Defensins are cationic antimicrobial peptides which have a characteristic six cysteine motif and are important components of the innate immune system Defr1 is a polymorphism of mouse β-defensin 8 and contains only 5 cysteines. Against a panel of pathogens, we found that oxidized synthetic Defr1 had significantly higher activity than its reduced form, and the oxidized and reduced forms of its six-cysteine containing analogue (Defrl Y5C). Using non-denaturing gel electrophoresis and high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) we observed Defrl and Defrl Y5C dimers.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:643238 |
| Date | January 2005 |
| Creators | Clarke, David J. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/11989 |
Page generated in 0.0019 seconds