Concepts and Methods of Solid-State NMR Spectroscopy Applied to Biomembranes

Concepts of solid-state NMR spectroscopy and applications to fluid membranes are reviewed in this paper. Membrane lipids with ²H-labeled acyl chains or polar head groups are studied using ²H NMR to yield knowledge of their atomistic structures in relation to equilibrium properties. This review demonstrates the principles and applications of solid-state NMR by unifying dipolar and quadrupolar interactions and highlights the unique features offered by solid-state ²H NMR with experimental illustrations. For randomly oriented multilamellar lipids or aligned membranes, solid-state ²H NMR enables <i>direct</i> measurement of residual quadrupolar couplings (RQCs) due to individual C–²H-labeled segments. The distribution of RQC values gives nearly complete profiles of the segmental order parameters <i>S</i>_CD^(<i>i</i>) as a function of acyl segment position (<i>i</i>). Alternatively, one can measure residual dipolar couplings (RDCs) for natural abundance lipid samples to obtain segmental <i>S</i>_CH order parameters. A theoretical mean-torque model provides acyl-packing profiles representing the cumulative chain extension along the normal to the aqueous interface. Equilibrium structural properties of fluid bilayers and various thermodynamic quantities can then be calculated, which describe the interactions with cholesterol, detergents, peptides, and integral membrane proteins and formation of lipid rafts. One can also obtain direct information for membrane-bound peptides or proteins by measuring RDCs using magic-angle spinning (MAS) in combination with dipolar recoupling methods. Solid-state NMR methods have been extensively applied to characterize model membranes and membrane-bound peptides and proteins, giving unique information on their conformations, orientations, and interactions in the natural liquid-crystalline state

Variations in ¹⁵N relaxation rates <i>R</i>_1Z and <i>R</i>₂ and the ratios <i>R</i>₂/<i>R</i>_1Z support the predicted secondary structure of OEP16 and backbone assignments.

<p>(A) ¹⁵N spin-lattice <i>R</i>_1Z relaxation rates, (B) ¹⁵N transverse <i>R</i>₂ relaxation rates, and (C) <i>R</i>₂/<i>R</i>_1Z ratios. The suggested TM helical regions are indicated by the red bars above the plot with helical breaks in yellow and solvent-exposed helices in green. Relaxation rates for U-¹⁵N-labeled OEP16 in SDS micelles were obtained at 600 MHz at 310 K with sample conditions identical to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078116#pone-0078116-g001" target="_blank">Figure 1</a>. Higher <i>R</i>₂<i>/R</i>_1Z values are located in the central part of the TM helices possibly indicating relatively rigid regions of the protein.</p

High-Resolution NMR Reveals Secondary Structure and Folding of Amino Acid Transporter from Outer Chloroplast Membrane

<div><p>Solving high-resolution structures for membrane proteins continues to be a daunting challenge in the structural biology community. In this study we report our high-resolution NMR results for a transmembrane protein, outer envelope protein of molar mass 16 kDa (OEP16), an amino acid transporter from the outer membrane of chloroplasts. Three-dimensional, high-resolution NMR experiments on the ¹³C, ¹⁵N, ²H-triply-labeled protein were used to assign protein backbone resonances and to obtain secondary structure information. The results yield over 95% assignment of N, H_N, CO, C_α, and C_β chemical shifts, which is essential for obtaining a high resolution structure from NMR data. Chemical shift analysis from the assignment data reveals experimental evidence for the first time on the location of the secondary structure elements on a per residue basis. In addition <i>T</i>_1<i>Z</i> and <i>T₂</i> relaxation experiments were performed in order to better understand the protein dynamics. Arginine titration experiments yield an insight into the amino acid residues responsible for protein transporter function. The results provide the necessary basis for high-resolution structural determination of this important plant membrane protein.</p></div