University of Dundee

The use of fully deuterated systems for the investigation of membrane associated proteins by EPR

In most genomes 20–30% of all genes encode membrane proteins and yet are represented by less than 3% of determined protein structures. Membrane proteins are seen as a crucial target for therapeutic intervention. The small number of structures of membrane proteins available is indicative of inherent problems in crystallisation. Nanometre distance measurements by EPR (PELDOR or DEER)1 are becoming increasingly popular for studying protein tertiary and quaternary structures and have been postulated to be a promising method for the investigation of membrane proteins. The sensitivity and distance range of EPR crucially depends on spin relaxation times (i.e. phase memory times) and EPR on membrane proteins has suffered from very fast relaxation, diminishing its usefulness. This fast relaxation is in part caused by aggregation of the proteins within the lipid environment, leading to the relaxation of electron spins by other spins within the aggregate. This can be minimised either by diluting the labelled proteins with unlabelled protein or by the use of Nano-disk technology producing singly occupied membrane-like disks. In spite of these approaches, relaxation is still a major problem.

We have developed the use of protein deuteration to increase the phase memory time in EPR studies. This approach has been a great success, allowing PELDOR data to be gathered at up to 60 μs (six- to eightfold the times achievable previously). This approach has almost doubled the commonly cited distance limit of ~80 Å. A second important advantage from the increased phase memory time is greatly enhanced sensitivity.

This project intends to extend the use of total system deuteration to the study of membrane proteins. Some work has been done in this direction but to date no studies of relaxation in fully deuterated membrane systems have been reported. The project would involve the use of fully deuterated lipids, isolated from fully deuterated E.coli paired with Nano-disk technology (with the scope of fully deuterating the Nano-disk forming girdle proteins) to study relaxation in spin-labelled, fully deuterated membrane proteins. The project would also take advantage of state of the art pulse forming technologies to maximize the relaxation time and sensitivity.

Although the distances that are generally measured in membrane-associated proteins are relatively small, the much larger distance measurement capability of a fully deuterated system will lead to an increased resolution of the distance distribution and thus a much larger reliability of the shape of the distance distribution. In addition, the much enhanced sensitivity of such systems will allow much more dilute samples to be used and provide enhanced signal. The project would also investigate the application of fully deuterated membrane systems for the investigation of membrane immersion depth, lipid interaction and solvent accessibility studies.

The student will be integrated into the St Andrews-Dundee EPR group which has an outstanding track record in EPR applications especially for distance measurements. Expertise ranges from production of optimised samples (mutagenesis, protein deuteration, reconstitution of membrane proteins) to data analysis and structural modelling and interpretation. We have the UK’s largest density of state-of-the-art pulse EPR facilities dedicated to structural biology applications.

The student will receive cutting-edge training in a wide range of skills: molecular biology and mutagenesis, protein purification, Nano-disk membrane assembly and spin labelling. The student will receive world class training in EPR spectroscopy and data handling and hands on EPR data generation and analysis.

References:

1) Pannier M,  Veit S,  Godt A,  Jeschke G,  Spiess H.W (2000) Dead-Time Free Measurement of Dipole–Dipole Interactions between Electron Spins, J. Magn. Reson. 2000, 142, 331-340

2) El Mkami, H., and Norman, D. G. (2015) EPR Distance Measurements in Deuterated Proteins, Methods in Enzymology.

3) Dastvan, B. E. Bode, M. P. R. Karuppiah, A. Marko, S. Lyubenova, H. Schwalbe, T. F. Prisner, Optimization of Transversal Relaxation of Nitroxides for PELDOR Spectroscopy in Phospholipid Membranes J. Phys. Chem. B 2010, 114, 13507-13516.