Bacterial back talk - unfolding and molecular interaction studies in Staphylococcal proteins and engineering structure-informed antibiotics

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Staphylococci are extremely common human commensals, which have been implicated in systemic infections in hospitals, biofilm formation in surgical devices and implants, industrial fouling and food spoilage. The accessory gene regulation (Agr) in staphylococci controls important bacterial functions, such as virulence and adhesion to surfaces (biofilm formation). AgrB is a membrane endopeptidase responsible for pre-processing of the signalling molecule, AIP, which mediates bacterial communications and quorum sensing. The AIP response cascade is a two component system including a membrane receptor AgrC, which phosphorylates the nucleotide-binding partner AgrA. Pharmacological regulation of the Agr protein is an attractive target for bacterial control, attenuating virulence and biofilm inhibition and a new opportunity for antimicrobial design.We have developed a new molecular recognition assay, which reports on substrate or ligand binding through increased protein stability to degradation. Thermal unfolding of proteins is a gradual process, in which thermal fluctuations increase in frequency and magnitude with temperature and can be monitored by circular dichroism, small angle neutron or X-ray scattering. NMR spectroscopy and high performance computing complement the studies with detailed atomic level structural information on the interaction between the Agr proteins and their substrates and regulatory ligands. In this project we will develop a large scale screen for protein/ligand interactions based on the thermal denaturation assay and will validate it against existing libraries of Agr modulators. Using recombinant protein from current research project on Agr, we will:a) use a combination of scattering techniques and CD to monitor changes in protein structure and overall conformation and will develop the tool with reference to critical behavior;b) develop a sample environment at partner institution ISIS for the large scale screen in conjunction with beamlines SANS2D and ZOOM; develop analytical framework for rapid data interpretation;c) characterize ligand/receptor interactions using solid state NMR, molecular docking and simulations.The programme relies on advanced biophysical, structural and computational tools for molecular analysis, which are combined in complementary, yet partially overlapping groups to ensure successful completion of the work.