Distance measurements and conformational analysis of sn-2-arachidonoylglycerol-membrane sample by ²H-³¹P REDOR NMR.

The purpose of these studies is to determine the intermolecular distances that define the location, orientation, and conformation of 2-AG in palmitoyl-oleoyl-phosphatidylcholine (POPC) lipid bilayers using rotational-echo double-resonance (REDOR) NMR. All five protons on the glycerol backbone of 2-AG were replaced with ²H and the distance between the deuterons and naturally occurring ³¹P on the POPC lipid headgroup determined with REDOR. To determine the distance from each deuteron to the phosphorus, the POPC headgroup was arranged in a hexagonal array. The 2-AG intercalates between the lipid molecules and the ²H labels, resulting in an average distance of z directly above or below the center of the parallelogram of the four phosphorus atoms P₁, P₂, P₃, and P₄. For different z values, the ²H-³¹P inter-nuclear distances were 7.6-9.1 Å (²H-³¹P₁ and ²H-³¹P₃₁) and 4.4-6.7 Å (²H-³¹P₂ and ²H-³¹P₄). Each result involved the calculations and summation of 893,101 terms. Based on the curve-fitting parameters, the calculations with z = 0 fits the data the best, which means these methylene ²H atoms are at the same level as the phosphate group of the POPC lipid bilayer. Molecular dynamic simulation data suggested that the ²H atoms at the glycerol backbone of 2-AG are involved in an extended H-bonding network with the phosphorus atoms after 10-ns simulation.