Research

Euan Pyle

An integrated structural biology approach for elucidating transient protein interactions

Euan Pyle - 3rd year PhD

Abstract

Living cells integrate an immense diversity of functional modules composed of large macromolecular assemblies of proteins, nucleic acids and lipids. However, structural analysis of many functional assemblies by traditional structural biology approaches remains a challenge, limiting our understanding of how they evolved, how they function and how they can be modulated. This is a particularly challenging problem with membrane protein complexes. Here we propose to integrate biochemical methods, native mass spectrometry (MS) experiments, and protein imaging techniques with a state-of-the-art modelling strategy to structurally characterize heterogeneous and transient membrane protein complexes. Bringing together these experiments into a hybrid methodology will allow us to determine their three-dimensional architectures and to capture the transient interactions involved in membrane protein complex formation.


In this project we will develop a hybrid structural biology methodology capable of integrating diverse datasets for studying heterogeneous and dynamic protein complexes. This technology will build upon a ground-breaking strategy, capable of combining various MS-based experiments, for structural modelling of soluble protein complexes (Politis et al., Nat Methods, 2014). The innovation is the development and adaptation of these technologies to the study of membrane protein complexes. In particular a novel combination of MS techniques namely hydrogen-deuterium exchange (HDX)-MS and native MS coupled with ion mobility will be integrated together with super-resolution imaging into a hybrid workflow for probing the transient interactions observed in membrane-embedded proteins.


We will apply the above technologies to characterize the structure and dynamics of membrane protein assemblies, starting from more basic targets (UapA, a membrane transport protein known to form a homodimer) and moving on to challenging transmembrane assemblies and their signalling partners such as G-protein-coupled receptors (GPCRs). These receptors are targeted by half of all modern drugs, yet the dynamic structural changes following ligand binding are largely unknown.  Despite intense efforts, the number of solved structures represent only ~4% of the pharmacologically relevant GPCRs. Here, you will develop the experimental and computational tools to probe the structure and dynamics of GPCRs upon binding of ligand and other GPCR-interacting biomolecules.