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

Abstract

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-mum-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 degrees 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 (17)O NMR study of reversible binding effects of mono- and divalent ions on the chemical shift properties of the Leu(10) carbonyl oxygen of transmembrane pore-forming peptide gramicidin A (gA). We further compare the (17)O 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.

Polarization-dependent fluorescence of proteins bound to nanopore-confined lipid bilayers

“…Previously, it has been shown that lipid bilayers and membrane proteins can be assembled in functional conformations inside macroscopically aligned nanochannels of anodic aluminum oxide ͑AAO͒ membranes. [5][6][7][8][9][10] Recently, we have reported on subwavelength hybrid metallo-dielectric structures for the enhancement of Raman spectroscopic signals 11,12 and one might wonder whether the same applies to fluorescence signals. Others also pointed to the fluorescence enhancing properties of such platforms albeit for optical beams at normal incidence.…”

“…For the experiments we employed zwitterionic lipid bilayers that could be incorporated into nanoporous aluminum oxide membranes with minimal perturbation to the bilayer properties. [5][6][7][8][9][10] The bilayers were doped with 0.5-5.0 mol % of biotinylated lipids and exposed to 5-͑4,6-dichlorotriazinyl͒aminofluorescein ͑DTAF͒-a conjugated streptavidin-to mimic protein docking to the membrane surface. Fluorescence signals were measured as a function of the angle between the optical polarization state of the incident beams and the orientations of macroscopically ordered AAO nanochannels.…”

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

“…A variety of pertinent information about integral membrane proteins can be obtained in aligned lipid bilayers [1,2]. The incorporation of a protein into an aligned lipid bilayer reveals unique structural information such as peptide tilt angle and lipid chain dynamics [3]. 2 H NMR spectroscopy of deuterated acyl chains of lipids is a powerful approach to monitor the dynamic properties inside lipid bilayers.…”

“…Aligned bicelles are prepared by mixing long and short chain phospholipids such as DMPC and DHPC (1,2-dihexanoyl-sn-glycero-3-phosphocholine) under optimized experimental conditions [7,8]. Drawbacks to the bicelle alignment procedure include the inability to perform experiments requiring the removal or replacement of solvent after the bicelle sample is created, low sample stability of non ether-linked lipids after being heated, the inability of some lipids (such as POPC) to align in the magnetic field of the NMR spectrometer, and the limited range of alignment temperatures for a given bicelle composition [3].…”

“…Since the discovery that bilayers could be aligned in AAO substrates, membrane proteins have been successfully incorporated into the AAO substrate lipid system for investigation with NMR and EPR spectroscopy [3,10,11,13]. The surface area of exposed lipids in the nanopores is comparable to a glass plate preparation, but there are many advantages of using the AAO substrate system for protein alignment in a lipid bilayer [13].…”