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Title: Structural and functional studies of protein complexes
Author: Clarke, David J.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2005
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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 Aquifex aeolicus. The genes encoding both BPL and BCCPΔ67 were overexpressed in E. coli 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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available