Principal Supervisor: Professor Mark Wheatley, School of Biosciences
Co-supervisor: Dr Sarah L. Horswell, School of Chemistry
PhD project title: Investigating G-protein-coupled receptor (GPCR) activation.
University of Registration: University of Birmingham
G-protein-coupled receptors (GPCRs) form the largest class of membrane proteins in the human genome with >800 unique GPCRs. They are central to cell signalling and are of great commercial value to the pharmaceutical industry worldwide, with ~50% of clinically-marketed drugs and ~25% of top-selling drugs targeting this receptor family. GPCRs are activated by a wide variety of agonists which differ with respect to chemical class, physical properties and size – from photons and small biogenic amines to peptides and large glycoproteins. Despite this diversity, these receptors exhibit a conserved protein architecture comprising a bundle of seven transmembrane (TM) helices linked by extracellular loops (ECLs) and intracellular loops (ICLs). Historically, it was envisaged that GPCRs were effectively on/off switches. Binding an agonist induced the ‘on’ conformation which stimulated activation of a specific G-protein to initiate an intracellular signal. In contrast, it was thought that antagonists merely occupied the binding site thereby preventing activation by agonist. It is now very clear that for GPCRs in general, agonist binding and receptor activation are multi-step processes involving a series of distinct receptor conformations and that different agonists can stabilise different active receptor structures. Static structures from crystallography therefore cannot inform us of the dynamic changes underlying GPCR activation. Obtaining detailed biophysical information on drug:GPCR interaction using purified GPCR protein would aid drug discovery and provide mechanistic insight into differences between blocking drugs (antagonists) and activating drugs (agonists). The major hurdle to biophysical analysis of GPCRs is their inherent instability in the surfactants/detergents required for their purification. We reported the first purification of pharmacologically-active GPCRs in the total absence of detergent in 2015, using styrene maleic acid (SMA) polymer to extract a nano-scale disc of native membrane bilayer encapsulating the GPCR. This puts us ahead of the field and opens new opportunities for applying biophysical techniques to study conformational changes associated with GPCR structure, function and activation.
- Characterise the ability of recently reported ‘second generation’ polymers (including DiBMALP and SMILP) to stabilise purified functional GPCR-nanoparticles.
- Develop enabling strategies, including surface tethering methodologies, for quantitative interrogation of ligand interaction with GPCR-nano-particles using a range of biophysical approaches including surface plasmon resonance and isothermal titration calorimetry.
- Exploiting the strategies developed in Objective ii, apply spectroscopic and imaging tools for following conformational changes in the GPCRs on binding, to develop a mechanistic understanding that may lead to rational drug design in the future.
A wide range of methods will be utilised in this project. Initially we will focus on the vasopressin/oxytocin family of receptors as we have extensive experience of studying this family using molecular pharmacological approaches including mutagenesis, peptide chemistry and pharmacological characterisation of mutant receptors in cultured cells by radioligand binding assays and second messenger assays. Receptors will have ‘tag’ sequences added to aid identification and purification and reactive (cysteine) residues introduced at specific sites. They will be SMALP-solubilised and purified. The resultant GPCR-SMALP will be investigated by a range of biophysical approaches for characterising proteins including vibrational spectroscopies, circular dichroism, analytical ultracentrifugation, surface plasmon resonance, isothermal titration calorimetry and also by a raft of fluorescence-based techniques designed to quantify specific conformational changes, including FRET. For tethered SMALPs, investigations will be made with surface infrared and Raman spectroscopies and atomic force microscopy.
- Wheatley et al., (2012) Brit. J. Pharmacol. 165, 1688-1703.
- Wheatley et al., (2016) Biochem. Soc. Trans. 44, 619-623.
- Madrid and Horswell (2013) Langmuir 29, 1695-1708.
- Madrid and Horswell (2014) Electrochim. Acta 146, 850-860.
BBSRC Strategic Research Priority: Molecules, Cells and Systems
Techniques that will be undertaken during the project:
- Molecular pharmacology
- Cell culture
- Radio-ligand binding assays
- Second messenger assays
- Molecular biology
- Site-directed mutagenesis,
- Western blot analysis
- Protein purification
- Surface plasmon resonance
- Isothermal titration calorimetry
- Fluorescence-based techniques including FRET