Developing novel methods to watch membrane proteins move

MACMILLAN_U23DTP

We study the architecture and functional dynamics of membrane proteins, especially many medically-relevant membrane transport systems.

There is increasing evidence that membrane proteins do not act alone, but that they are organised as nano-machineries which function through the concerted action of individual components with high precision and specificity observed in both time and space.

We are seeking to unravel the principles underlying the architecture and dynamics of these protein nano-machineries as well as their function.

Our experimental approach focuses on the use of magnetic resonance techniques specifically Electron Paramagnetic Resonance (EPR) and Nuclear Magnetic Resonance (NMR) spectroscopy in combination with molecular biological, and biochemical approaches.

This project will study a specific sodium symporter transporter LeuT, a small amino-acid transporter from Aquifex aeolicus, which is a structural homologue of the human neurotransmitter transporters for dopamine, serotonin, norepinephrine and amino butyric acid.

These human transporters are implicated in several diseased states including depression, anxiety and attention-deficit hyperactivity disorder. Several prescribed medications target these transporters and illicit street drugs like cocaine or amphetamine interact with them.

However, the LeuT transporter is highly dynamic and the development of medically relevant SLC6 inhibitors relies on understanding the distribution of conformational states.

Recent structural studies have proposed large scale conformational changes and we aim to probe the functional dynamics of this protein family using a combination of state-of-the-art magnetic resonance techniques as well as techniques to purify and stabilise the protein using novel SMA lipid particles.

The successful candidate will receive world class training and become competent in a wide range of biochemical and biophysical techniques working within a team environment using state-of-the-art equipment to study an important family of membrane transporters with societal impact.

References

Colas C, Ung PM & Schlessinger A, SLC Transporters: Structure, Function, and Drug Discovery. Med. Chem. Commun. 7, 1069–1081 (2016).

Hall J, Sohail A, Cabrita EJ, Macdonald C, Stockner T, Sitte HH, Angulo J, MacMillan F, Saturation transfer difference NMR on the integral trimeric membrane transport protein GltPh determines cooperative substrate binding Scientific Reports 10, 16483 (2020).

Barber-Zucker S, Hall J, Froes A, Kolusheva A, MacMillan F* & Zarivach R*, The cation diffusion facilitator protein MamM’s cytoplasmic domain exhibits metal-type dependent binding modes and discriminates against Mn2+ J. Biol. Chem. 295, 16614-16629 (2020).

Ross MO, MacMillan F, Wang J, Nisthal A, Lawton TJ, Olafson BD, Mayo SL, Rosenzweig AC, Hoffman BM, Particulate methane monooxygenase contains only mononuclear copper centers. Science 364, 566-570 (2019).