KEYWORDS: solid state NMR, magic-angle spinning, 13C-1H NMR, 2D, structure, dynamics, fluid phase model membranes, dipolar couplings, J-couplings, magnitude and sign of order parameters, Dipolar Recoupling On-axis with Scaling and Shape preservation (DROSS), natural abundance dimyristoylphosphatidylcholine
John D. Gross, Dror E. Warschawski, Robert G. Griffin
ABSTRACT: Novel two-dimensional magic-angle spinning (2D MAS) NMR experiments designed to measure the magnitudes and signs of 13C-1H dipolar interactions in fluid phase lipid bilayers are presented: MAS is employed throughout the experiments while dipolar recoupling is achieved (by radio frequency irradiation) during the evolution period. The pulse sequence has been called DROSS for Dipolar Recoupling On-axis with Scaling and Shape preservation. Multiple 13C-1H dipolar couplings are measured for a natural abundance sample of La phase dimyristoylphosphatidylcholine (DMPC). The magnitudes of 13C-1H dipolar interactions are determined by fitting numerical simulations of recoupled powder lineshapes with experimental data while the signs of these interactions are obtained by monitoring the build-up of antiphase magnetization by 13C detection. A comparison of the order parameters obtained by 13C-1H dipolar recoupling with those previously obtained for DMPC by 2H NMR indicates that dipolar recoupling is a viable method for determining segmental order in fluid phase lipid bilayers without recourse to isotopic enrichment. Measurement of the signs of 13C-1H dipolar couplings provides additional structural information that is unavailable through 2H NMR. The results obtained for DMPC suggest that the accuracy of the dipolar recoupling experiments presented in this work is competitive with that of previous techniques which require switched-angle spinning for the measurement of the magnitudes and signs of 13C-1H dipolar interactions in lipid bilayers.
REFERENCE: J. D. Gross, D. E. Warschawski and R. G. Griffin, Journal of the American Chemical Society 119:796-802 (1997)
2D 13C-1H DROSS spectrum of natural abundance DMPC at 28C, spinning at 8kHz. (A) Acyl chain region; (B) Head group and glycerol region.
KEYWORDS: solid state NMR, 13C-NMR, 1H-decoupling, magic-angle spinning, structure, dynamics, fluid phase model membranes, isotopically labeled peptides, gramicidin A
Dror E. Warschawski, John D. Gross, Robert G. Griffin
ABSTRACT: In the past fifteen years, interferences between molecular dynamics and coherent manipulation of nuclear magnetization in nuclear magnetic resonance (NMR) experiments such as spin decoupling, cross-polarization or magic-angle spinning (MAS) have been identified and studied carefully. Recent experiments performed in our laboratory on model compounds have provided insight into the nature of a perturbation responsible for the broadening of 13C and 15N signals, namely the interference of some molecular motion with 1H-decoupling. The same effect is demonstrated here for the first time in the case of a membrane peptide, gramicidin A (gA), in an hydrated lipid bilayer. Early attempts to obtain high-resolution MAS NMR spectra of peptides and proteins in liquid-crystalline membranes were unsuccessful because of problems associated with the dynamics of the molecules. Subsequent studies have circumvented these issues by performing experiments at very low temperatures, where motion is quenched. Unfortunately, information regarding structure and dynamics under physiological conditions is lost. The present experiment provides the first successful attempt to observe high-resolution solid-state 13C NMR spectra of a Calpha moiety in membrane peptide, in an hydrated lipid bilayer in the Lalpha phase. The samples studied here are synthesized and purified specifically 2H,13C,15N-labeled gA reconstituted into lipid bilayers. The development of strategies to circumvent the broadening effect allows us to extract relevant data concerning the dynamics of gA under physiological conditions. Similar strategies could be useful for structural and dynamical studies of other membrane peptides in hydrated bilayers.
REFERENCE: D. E. Warschawski, J. D. Gross and R. G. Griffin, Journal de Chimie Physique 95:460-466 (1998)
79.9 MHz 13C-MAS-NMR spectra of: a) NCCD2-Gly gA/DMPC/D2O with 90 kHz 1H-decoupling. T = 25°C, wr = 4.7 kHz, r. d. = 15 s, 2048 scans; b) NCCH2-Gly gA/DMPC/D2O with 90 kHz 1H-decoupling. T = 25°C, wr = 6.5 kHz, r. d. = 10 s, 2048 scans.