NMR and Structural Studies of Membrane Proteins

Our laboratory is studying the structure-function relationships of macro-molecular assemblies and biological systems at the molecular level using new 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 are 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 international laboratories, we are studying biological ion channels, 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 interdisciplinary 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 2005 Publications :

• Dimerisation of N-acetyl-L-tyrosine ethyl ester and Ab peptides via formation of dityrosine. Ali, F.E. Leung, A., Cherny, R., Mavros, C., Barnham, K.J., Separovic F. and Barrow, C.J. (2005) Free Radical Research (in press).
• Direct visualization of membrane leakage induced by the antibiotic peptides: Maculatin, Citropin and Aurein. Ambroggio, E.E., Separovic, F., Bowie, J.H., Fidelio, G.D. and Bagatolli, L.A. (2005) Biophys. J 89, 1874-1881.
• Solid-state NMR of membrane-active proteins and peptides. Gehman, J.D. and Separovic, F. (2005) in Handbook of Modern Magnetic Resonance I (T. Asakura, H. Saito and I. Ando, eds.) Kluwer Academic Publishers, London, U.K. (in press).
• Surface behavior and lipid interaction of Alzheimer b-amyloid peptide 1-42: a membrane-disrupting peptide. Ambroggio, E.E., Kim, D.H., Separovic, F., Barrow, C.J., Barnham, K.J., Bagatolli, L.A. and Fidelio, G.D. (2005) Biophys. J. 88, 2706-2713.
• 29Si MAS NMR study of structural ordering in aluminosilicate geopolymer gels. Duxson, P., Provis, J.L., Lukey, G.C., Separovic, F. and van Deventer, J.S.J. (2005) 21, 3028-3036.
• Developments in hyphenated spectroscopic methods in natural product profiling. Urban, S. and Separovic, F. (2005) in Frontiers in Drug Design and Discovery 1 (Atta-ur-Rahman, G. Caldwell, eds.) Bentham Science Publishers. pp 113-166.
• The role of Ab peptides in Alzheimers disease. Tickler, A.K., Wade, J.D. and Separovic, F. (2005) Protein & Peptide Lett. 12, 513-519.
• The conformational flexibility of the antibiotic virginiamycin M1. Dang, J., Metzger, R.P., Brownlee, R.T.C., Ng, C.A., Bergdahl, M. and Separovic, F. (2005) Eur. Biophys. J. 34, 383-388.
• Interaction of the eukaryotic pore-forming cytolysin equinatoxin II with model membranes: 19F NMR studies. Anderluh, G., Razpotnik, A., Podlesek, Z., Macek, P., Separovic, F. and Norton, R.S. (2005) J. Mol. Biol. 347, 27-39.
• Matrix method for analysis of selective NMR pulses. Tesiram, Y.A. and Separovic, F. (2005) Concepts in Magnetic Resonance 25A, 1-17.
• Methionine regulates copper/hydrogen peroxide oxidation products of Ab. Ali, F.E. Separovic, F., Barrow, C.J., Cherny, R.A., Fraser, F., Bush, A.I., Masters, C.L. and Barnham, K.J. (2005) J. Peptide Science 11, 353-360.