CcdB : Stability, folding and application to design novel antibacterials

Abstract

The primary amino acid sequence typically dictates the ultimate conformation of the protein. Mutations in the sequence demonstrate neutral, positive or negative effects on the structure-function relationship of the protein. Destabilising mutations often reduce the soluble levels of the protein in vivo, leading to complete or partial loss of its function. Global suppressors are diverse compensatory mutations that can alleviate the detrimental effects of multiple destabilised, inactive mutants, despite being physically distant from the site of the original inactive mutations. The molecular mechanisms responsible for suppression are still unknown, despite the characterisation of suppressors in several proteins since the late 1980s. Another modulator of a protein’s structure is the pH of the solution. It has long been known that proteins form molten globules at acidic pH conditions, which are compact denatured states with fluctuating tertiary structures. CcdB (Controller of Cell Division or Death B), a 101-residue homodimeric toxin which is a part of the CcdA:CcdB toxin:antitoxin module and poisons intracellular Gyrase, has been utilised as the model protein of choice in the course of the studies. Chapter 1 presents the importance of global suppressors, gives an overview of the role of pH on the protein’s structure and discusses various Gyrase inhibitors and outlines how the CcdB:Gyrase interaction can be utilised to design antibacterial peptides based on the toxin’s sequence. Chapter 2 provides a detailed thermodynamic and kinetic investigation of a global suppressor in CcdB. The studies show that the suppressor restores the stability and function of inactive mutants by marginally enhancing their apparent thermodynamic stabilities and lowering their unfolding rates. Chapter 3 shows the broad-spectrum antibacterial action of a novel CcdB-derived peptide on E.coli and pathogenic strains of S.aureus, S.Typhimurium, as well as a multi-drug resistant clinical isolate of A.baumannii. Chapter 4 reports a preliminary investigation of the low pH-induced molten globule-like state of CcdB using NMR spectroscopy.