Developing Carbene Footprinting Mass Spectrometry to Interrogate Protein-Ligand Interactions

Lloyd, James (2023) Developing Carbene Footprinting Mass Spectrometry to Interrogate Protein-Ligand Interactions. PhD thesis, University of Nottingham.

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The interaction of proteins with small molecules or other proteins is essential to almost every cellular process. Because of this importance and the potential implication in disease pathogenesis, proteins are attractive drug targets. Improved knowledge of these relationships greatly benefits our understanding of key biological processes and aids in drug discovery efforts. Techniques that provide structural information on a short time scale and employ small amounts of sample are highly desired. Carbene footprinting is a recently developed mass spectrometry-based chemical labelling technique that probes protein interactions and conformation. The work reported in this thesis aims to further develop the application of carbene footprinting to the study of protein structure and interactions by mapping the contact interfaces of several clinically relevant protein systems.

Using the methodology, the contact surface between the human eukaryotic initiation factor 4A (eIF4A) and small molecular inhibitor hippuristanol was accurately mapped to the protein’s C-terminal domain (CTD). The technique was successfully employed to study interactions between members of the inflammasome human Gasdermin D (hGSDMD) and hCaspase 1 (C285A) which revealed direct occupancy of the hCaspase-1 active-site by hGSDMD for the first time. Carbene footprinting was also applied to the hGSDMD pore-forming N terminal domain (NTD) assembled in liposomes and then compared to the soluble monomer which showed masking effects consistent with oligomeric assembly and insertion into the lipid bilayer. Interactions between Caspase-1 (C285A) and the specific inhibitor O-desethyl-belnacasan (VRT-043198) were studied by carbene footprinting which revealed that the small molecule non-covalently occupied the active-site of a C285A mutant. Carbene footprinting was also applied to two alpha helical membrane proteins. The archaeal multidrug and toxic compound extrusion (MATE) transporter PfMATE was employed to optimise the labelling workflow to insoluble protein systems which notably revealed covalent modification of detergent molecules. Carbene labelling was also applied to the G protein-coupled receptor (GPCR) beta-1 adrenergic receptor (β1AR). Activated and inactivated ternary β1AR nanobody (Nb) complexes highlighted binding of small molecules to the extracellular binding cavity as well as differential labelling changes on intracellular transmembrane helices (TMs) that indicated varying activation states of the receptor.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Oldham, Neil
Keywords: proteins, protein interactions, drug targeting, carbene footprinting, mass spectrometry
Subjects: Q Science > QD Chemistry > QD 71 Analytical chemistry
Q Science > QP Physiology > QP501 Animal biochemistry
R Medicine > RM Therapeutics. Pharmacology
Faculties/Schools: UK Campuses > Faculty of Science > School of Chemistry
Item ID: 76803
Depositing User: Lloyd, James
Date Deposited: 25 Jan 2024 14:03
Last Modified: 25 Jan 2024 14:03

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