Area per Lipid and Cholesterol Interactions in Membranes from Separated Local-Field 13 C NMR Spectroscopy

Abstract

Investigations of lipid membranes using NMR spectroscopy generally require isotopic labeling, often precluding structural studies of complex lipid systems. Solid-state (13)C magic-angle spinning NMR spectroscopy at natural isotopic abundance gives site-specific structural information that can aid in the characterization of complex biomembranes. Using the separated local-field experiment DROSS, we resolved (13)C-(1)H residual dipolar couplings that were interpreted with a statistical mean-torque model. Liquid-disordered and liquid-ordered phases were characterized according to membrane thickness and average cross-sectional area per lipid. Knowledge of such structural parameters is vital for molecular dynamics simulations, and provides information about the balance of forces in membrane lipid bilayers. Experiments were conducted with both phosphatidylcholine (dimyristoylphosphatidylcholine (DMPC) and palmitoyloleoylphosphatidylcholine (POPC)) and egg-yolk sphingomyelin (EYSM) lipids, and allowed us to extract segmental order parameters from the (13)C-(1)H residual dipolar couplings. Order parameters were used to calculate membrane structural quantities, including the area per lipid and bilayer thickness. Relative to POPC, EYSM is more ordered in the ld phase and experiences less structural perturbation upon adding 50% cholesterol to form the lo phase. The loss of configurational entropy is smaller for EYSM than for POPC, thus favoring its interaction with cholesterol in raftlike lipid systems. Our studies show that solid-state (13)C NMR spectroscopy is applicable to investigations of complex lipids and makes it possible to obtain structural parameters for biomembrane systems where isotope labeling may be prohibitive.

Molecular simulations of the effects of phospholipid and cholesterol peroxidation on lipid membrane properties

“…In simulations, an area per lipid of 0.613  0.009 nm 2 was obtained for pure POPC at 310 K. It compares well with the value of 0.66  0.04 nm 2 , which was found by interpolation of the experimental data of Leftin et al [45]. It is also close to the value of 0.638 nm 2 reported in the original parametrization [28].…”

“…For the POPC/cholesterol (2:1) mixture at 310 K, an average area per lipid of 0.42  0.01 nm 2 was found in simulations. A value of 0.451  0.009 nm 2 has been measured for a 1:1 mixture at 321 K [45].…”

Deuterium NMR of Raft Model Membranes Reveals Domain-Specific Order Profiles and Compositional Distribution

“…In contrast, more atomistic information of lipid bilayers is obtainable from solid-state 2 H NMR spectroscopy, which provides quadrupole couplings of deuterated phospholipids and Chol, consequently yielding direct information regarding their membrane dynamics and the effect of Chol on the membranes (21)(22)(23)(24)(25)(26)(27)(28). However, most 2 H NMR studies of lipid bilayers have utilized per-or multideuterated lipid molecules, which would hamper the application of 2 H NMR to complex membrane systems.…”

The effect of gramicidin inclusions on the local order of membrane components

“…Resonance assignments followed that of previously published data [24,27,22,30,31], using the C ω−n convention, where n is the total number of segments, decreasing from the terminal methyl segment, C ω , to the upper carbonyl segment C 1 . This representation permits a segment-by-segment comparison of the chain regions.…”

“…The absolute value of the segmental order parameter is an additional scaling factor χ of the static dipolar coupling into the measured dipolar coupling. Since the static dipolar coupling, on the order of 20 kHz, is not known with high precision for each carbon, we have adjusted it empirically in the case of DMPC, by comparing it to previously determined values [27,22,30]. …”