DogDots: a new approach to labelling of membrane proteins for tomographic analysis

Membrane proteins are crucial to cellular function, such as recognition of - and interaction with - the extracellular environment and neighbouring cells, and controlling membrane dynamics, cellular signalling and transport of molecules and ions across membranes. Membrane protein malfunction underlies many diseases and disorders (in humans, animals and plants), and ~60% of current drug targets are membrane proteins. Determination of membrane protein structures, mainly by X-ray crystallography and single-particle cryo-electron microscopy (cryoEM), has provided detailed insights into their function and molecular interactions. However, membrane protein structures are typically determined after purification and stabilisation in non-native environments, hindering determination of physiologically relevant states necessary for understanding mechanism-of-action. The next revolution in membrane protein structural biology will be their structure determination in a native cellular environment by tomography, and many laboratories (including ours) are rapidly investing in tomography facilities for in-situ structural biology research. A major challenge in this field is how to detect and localise the membrane protein(s)-of-interest in a crowded membrane environment, and new technologies for this are urgently needed. Here, we propose to develop DogCatcher-quantum dots (DogDots), which will allow us to rapidly and specifically deliver quantum dots to membrane proteins that incorporate a short peptide (DogTag) within an extracellular loop. As a model system, we will use TRPC5 ion channels, which have important roles in health and disease. We have a strong track record of studying TRPC5 structure (by cryoEM) and have previously shown the feasibility of specifically labelling TRPC5 with DogCatcher. Using a workflow that builds complexity we will demonstrate how DogDots can be used to pinpoint TRPC5 in proteoliposomes, membrane-derived liposomes, and intact cell membrane, by both fluorescent and electron microscopy. Using Leeds' state-of-the-art Astbury Structure Laboratory, we will use DogDots to determine the first structure of TRPC5 in its cellular environment, at sub-nanometre resolution. We will also determine in-situ structures of TRPC5 in the presence of potent and selective activators, which may lead to the first structural insights into TRPC5's open channel state. This technology can be translated to any membrane protein with an extracellular loop, and working with our academic and industrial networks, we will develop DogDots as a versatile technology for structural studies of membrane proteins, thereby transforming our understanding of these important biomolecules.