NMR and Structural Studies of Membrane Proteins

Our laboratory is studying the structure-function relationships of macromolecular assemblies and biological systems at the molecular level through the use of powerful solid-state NMR (nuclear magnetic resonance) methods. We are employing these techniques to determine the structure and dynamics of membrane polypeptides, both as dehydrated powders and when reconstituted into lipid membranes for biomolecular engineering applications.

The structures of many biological systems cannot be obtained by traditional methods. Many systems are simply too large for solution-state NMR or have not been crystallized for X-ray diffraction. Solid-state NMR experiments have been designed for the structural determination of molecular systems that do not lend themselves to solution-state NMR and crystallographic methods. For example, structures of powder samples can be determined and compared to single crystal X-ray structures of the same compound. Similarly, colloidal and membrane dispersions which undergo anisotropic motion and protein complexes which precipitate out of solution are amenable to solid-state NMR studies.

Structural information for such systems has been obtained using novel solid-state NMR methods. These methods can be applied to study the structure and dynamics of crystalline powders and biological membranes. Our primary research interest is the determination of the structure and dynamics of membrane components in situ, using solid-state NMR as the main technique.

We have determined the molecular structure of the antibiotic gramicidin A and the bee toxin melittin within phospholipid membranes using solid-state NMR spectroscopy. Both gramicidin A and melittin form membrane ion channels and the techniques used to study these polypeptides are being extended to other integral membrane proteins. Together with researchers from CSIRO, industry and other international laboratories, we are studying biological ion channels, amyloid peptides, toxins and antibiotics in membranes, polymer coatings and resins, silk fibres and protein-sugar complexes with pharmaceutical applications.

Future advancements in bio/nano-technology require an inter-disciplinary approach and insight is needed from studies of biology at the molecular level. In collaboration with other multidisciplinary research groups we are providing insight into the biophysical chemistry and structure-function relationships of membrane-active peptides and proteins relevant to disease states and treatments.

Selected Publications :

1. Separovic F.; Smith R.; Yannoni C.S.; Cornell B.A., J. Amer. Chem. Soc. 1990, 112, 8324-8
2. Smith R.; Separovic F.; Milne T.J.; Whittaker A.; Bennett F.M.; Cornell B.A.; Makriyannis A., J. Mol. Biol. 1994, 241, 456-66
3. Separovic F.; Gehrmann J.; Milne T.; Cornell B.A.; Lin S.; Smith R., Biophys. J. 1994, 67, 1495-500
4. Separovic F.; Gawrisch K., Biophys. J. 1996, 71, 274-82
5. Separovic, F.; Ashida, J.; Woolf, T.; Smith, R.; Terao, T., Chem. Phys. Lett. 1999, 303, 493-8
6. Lam, Y.-H.; Wassall, S.R.; Morton, C.J.; Smith, R.; Separovic, F., Biophys. J. 2001, 81, 2752-2761
7. Bonev, B.B.; Lam, Y-H.; Anderluch, G.; Watts, A.; Norton, R.S.; Separovic F., Biophys. J. 2003, 84 2382-92
8. de Planque, M.R.R.; Rijkers, D.T.S.; Fletcher, J.; Liskamp, R.M.K.; Separovic F., Biochim., Biophys. Acta. 2004, 1665, 40-47
9. Lau, T.L.; Ambroggio, E.E.; Tew, D.J.; Cappai, R.; Masters, C.L.; Fidelio, G.D.; Barnham, K.J.; Separovic, F., J. Mol. Biol., 2005, 356, 759-770
10. Drechsler, A.; Potrich, C.; Sabo, J.K.; Frisanco, M.; Guella, G.; Serra, M.D.; Anderluh, G.; Separovic, F.; Norton, R.S., Biochemistry, 2006, 45, 1818-1828