Ancestor reconstruction within the CcdB family of gyrase inhibitors

Abstract

To understand the evolution of a protein the crucial element to concentrate on is mutation. Mutation influences protein structure, stability and function; hence mutations are very important in the gradual development of protein. The CcdB toxin inhibits DNA gyrase by preventing the strand passage during replication and transcription in prokaryotic organisms. Amino acid residues responsible for the activity of the CcdB toxin vary between CcdB families while the binding amino acid residue on their gyrase target remains the same. The aim of the work was to study how gyrase-CcdB interaction is achieved in different CcdB families. As it was observed, the C-terminal residues of the CcdB toxin are exclusively necessary in the CcdB-gyrase interaction. The amino acid in C-terminal region of CcdB were mutated and constructs composed of gyrA14 and ccdB mutants were generated and transformed into E. coli strains to study the CcdB toxicity. Protein expression and purification were performed to study the protein interaction between the CcdB toxin and gyrase.
The expression of the complexes resulted in higher production of gyrase than of CcdB. This unexpected result has prevented us to study the interaction of these proteins. One possible explanation could be the dissociation of complexes. By changing the purification strategy, by purifying each protein separately and subsequently incubating them together complex formation could be formed in Vitro.

Date of Award	1 Feb 2017
Original language	English
Awarding Institution	Vrije Universiteit Brussel
Supervisor	Remy Loris (Promotor)