SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
Flow-through Lipid Nanotube Arrays for Structure-function Studies of Membrane Proteins by Solid-state NMR Spectroscopy
Eduard Y Chekmenev 1, Peter L Gor'kov 2, Timothy A. Cross 3, Ali M Alaouie 4 and Alex I. Smirnov 4*
1 Florida State University
2 National High Magnetic Field Laboratory
3 Florida State Univ.
4 North Carolina State University
* To whom correspondence should be addressed. E-mail: alex_smirnov{at}ncsu.edu.
Submitted on March 16, 2006
Revised on April 17, 2006
Accepted on 11 July 2006
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Abstract |
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A novel method for studying membrane proteins in a native lipid bilayer environment by solid-state NMR spectroscopy is being described and tested. Anodic aluminum oxide (AAO) substrates with flow-through 175 nm wide and 60 µm long nanopores were employed to form macroscopically aligned peptide containing lipid bilayers that are fluid and highly hydrated. We demonstrate that the surfaces of both leaflets of such bilayers are fully accessible to aqueous solutes. Thus, high hydration levels as well as pH and desirable ion and/or drug concentrations could be easily maintained and modified as desired in a series of experiments with the same sample. The method allows for membrane protein NMR experiments in a broad pH range that could be extended to as low as 1 and as high as 12 units for a few hour periods and temperatures as high as 70 °C without losing the lipid alignment or bilayers from the nanopores. We demonstrate the utility of this method by a solid state 19.6 T 17O NMR study of reversible binding effects of mono- and divalent ions on the chemical shift properties of the Leu10 carbonyl oxygen of transmembrane pore-forming peptide gramicidin A (gA). We further compare the 17O shifts induced by binding metal ions to the binding of protons in the pH range from 1 to 12 and find a significant difference. This unexpected result points to a difference in mechanisms for ion and proton conduction by the gA pore. We believe that a large number of solid-state NMR based studies, including structure-function, drug screening, proton exchange, pH and other titration experiments, will benefit significantly from the method described here.
Key Words:
17O chemical shift, gramicidin, ion channel, lipid nanotube array, solid-state NMR, substrate-supported bilayers
