Susceptibility corrections in solid-state NMR experiments with oriented membrane samples. Part I: applications

Chemical shift referencing of solid-state NMR experiments on oriented membranes has to compensate for bulk magnetic susceptibility effects that are associated with the non-spherical sample shape, as described in the accompanying paper [J. Magn. Reson. 164 (2003) 115-127]. The resulting frequency deviations can be on the order of 10 ppm, which is serious for nuclei with a narrow chemical shift anisotropy such as H-1 or C-13, and in some cases even F-19. Two referencing schemes are proposed here to compensate for these effects: A flat (0.4 mm) glass container with an isotropic reference molecule dissolved in a thin film of liquid is stacked on top of the oriented membrane sample. Alternatively, the intrinsic proton signal of the hydrated lipid can be used for chemical shift referencing. Further aspects related to magnetic susceptibility are discussed, such as air gaps in susceptibility-matched probeheads, the benefits of shimming, and limitations in the accuracy of orientational constraints. A biological application is illustrated by a series of experiments on the antimicrobial peptide PGLa, aimed at understanding its concentration-dependent membranolytic effect. To address a wide range of molar peptide/lipid ratios between 1:3000 and 1:8, multilayers of hydrated DMPC containing a F-19-labeled peptide were oriented between stacked glass plates. Maintaining an approximately constant amount of peptide gives rise to thick samples (18 plates) at low, and thin samples (3 plates) at high peptide/lipid ratio. Accurate referencing was critical to reveal a small but significant change over 5 ppm in the anisotropic chemical shift of the F-19 label on the peptide, indicative of a change in the orientation and/or dynamics of PGLa in the membrane. (C) 2003 Elsevier Inc. All rights reserved.