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Flow-Through Lipid Nanotube Arrays for Structure-Function Studies of Membrane Proteins by Solid-State NMR Spectroscopy

Eduard Y. Chekmenev ^* , Peter L. Gor'kov ^*, Timothy A. Cross ^*  , Ali M. Alaouie  and Alex I. Smirnov 

^* The Center for Interdisciplinary Magnetic Resonance, National High Magnetic Field Laboratory, Tallahassee, Florida 32310; Department of Chemistry & Biochemistry, and Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32310; and Department of Chemistry, North Carolina State University, Raleigh, NC 27695-8204

Correspondence: Address reprint requests to Alex I. Smirnov, Dept. of Chemistry, North Carolina State University, 2620 Yarbrough Dr., Campus Box 8204, Raleigh, NC 27695-8204. Tel.: 919-513-437; Fax: 919-513-7353; E-mail: alex_smirnov{at}ncsu.edu.

A novel method for studying membrane proteins in a native lipid bilayer environment by solid-state NMR spectroscopy is 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 period of up to a few hours 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.