Flat-coil probe for NMR spectroscopy of oriented membrane samples

A probe optimized for NMR studies of oriented lipid bilayer samples has been developed and demonstrated to be effective in singleand double-resonance experiments with a variety of nuclei. This probe is characterized by having a flat radiofrequency coil wrapped directly around a pair of square glass plates between which the actual sample is sandwiched. The very high efficiency of the probe results from the favorable filling factor, which in turn allows additional gains in efficiency through miniaturization of all capacitors, leads, and connectors. The square sample has a high ratio of area to circumference, reducing edge effects and improving the macroscopic orientation of the lipids. Neither lipid nor protein components of membranes are amenable to structural analysis by multidimensional solution NMR studies because of their slow overall reorientation rates. However, the structures of immobile peptides and proteins can be determined at atomic resolution by solid-state NMR methods when it is possible to orient the samples with respect to the magnetic field of the NMR spectrometer (I4). In favorable cases, spontaneous uniaxial orientation occurs due to interactions of the sample with the large external magnetic field. Magnetic alignment enabled the structure of the coat protein of the filamentous bacteriophage fd to be determined by solid-state NMR spectroscopy ( 1, 2). Some specific lipid mixtures and protein-containing membrane fragments have been found to orient in magnetic fields (5, 6). However, spontaneous sample alignment is exceptional and cannot be expected to work for most bilayer samples of interest. A commonly used and effective technique for aligning membranes involves the deposition of a mixture of lipids and proteins onto glass plates followed by application of pressure, shear, or centrifugal forces ( 7-1 I ) . A very high degree of orientation can be achieved when small amounts of lipids are placed between glass plates with relatively large overall surface area and the level of hydration subsequently reduced. Typically, a large number of rectangular glass plates are stacked in parallel so that a reasonable amount of material can be placed in the cylindrical coil of the probe (12-14). The preparation of large stacks of glass plates containing lipids has a number of serious limitations. It is laborious with difficulties arising from the lipid-containing material being extruded between the plates, the application of uniform pressure over the plates, and misalignment of the glass plates themselves. In addition, the overall filling factor of the coil is worse with a rectangular sample placed in a round coil.