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45340-AC4
Solid-State NMR Studies of Aligned Phospholipid Bilayer Nanotube Arrays

Gary A. Lorigan, Miami University

ACS PRF

Aligning lipid bilayers in nanoporous anodized aluminum oxide (AAO) is a new method to help study membrane proteins by electron paramagnetic resonance (EPR) and solid-state nuclear magnetic resonance (NMR) spectroscopic methods. The ability to maintain hydration, sample stability, and compartmentalization over long periods of time, and to easily change solvent composition are major advantages of this new method. To date, 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) has been the only phospholipid used for membrane protein studies with AAO substrates. The different properties of lipids with varying chain lengths require modified sample preparation procedures to achieve well formed bilayers within the lining of the AAO substrates. For this project a simple methodology to incorporate large quantities of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), DMPC, and 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) phospholipids inside AAO substrate nanopores of varying sizes. 2H and 31P solid-state NMR were used to confirm the alignment of each lipid and compare the efficiency of alignment. This ACS PRF funded project is the first step in standardizing the use of AAO substrates as a tool in NMR and EPR and will be useful for future structural studies of membrane proteins. Additionally, solid-state NMR and EPR data suggest possible applications of nanoporous aluminum oxide in future vesicle fusion studies.

Additionally, funds from this proposal have been used to develop new membrane protein alignment procedures for EPR spectroscopic studies. For the first time the determination of the helical tilt of an integral membrane peptide inserted into aligned phospholipids bilayer nanotube arrays using spin label EPR spectroscopy. Also, we demonstrate how the helical tilt of the peptide can be easily calculated using the hyperfine splitting values gleaned from a perpendicularly aligned bilayer in phospholipid bilayer nanotube arrays. EPR spectral simulations were used to verify the method. This method holds a great deal of promise for antimicrobial peptides.

This project has resulted in two publications:

Electron paramagnetic resonance studies of an integral membrane peptide inserted into aligned phospholipid bilayer nanotube arrays

Ethan S. Karp, Johnson, J. Inbaraj, Mikhail Laryukin, and Gary A. Lorigan

J. Am. Chem. Soc. 128, 12070-12071 (2006).

Characterization of lipid bilayer formation in aligned nanoporous aluminum oxide nanotube arrays

Ethan S. Karp, Justin P. Newstadt, Shidong Chu, and Gary A. Lorigan

J. Mag. Reson. 187, 112-119 (2007).

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