ROTATIONAL RESONANCE NMR DETERMINATION OF INTRAMOLECULAR AND INTERMOLECULAR DISTANCE CONSTRAINTS IN DIPALMITOYLPHOSPHATIDYLCHOLINE BILAYERS

Rotational resonance (RR) NMR methods are explored for determining intramolecular and intermolecular distances between C-13-labeled sites in membrane bilayers. Specific C-13 labels have been incorporated into dipalmitoylphosphatidylcholine (DPPC) and a hydrophobic peptide corresponding to the transmembrane domain of human glycophorin A. The exchange of magnetization between these labels in the RR NMR experiment can be related to their internuclear separation. Intramolecular magnetization exchange rates have been measured between C-13 labels incorporated at the 1-position of the sn-1 acyl chain (1-[1-C-13]) and the 2-position of the sn-2 acyl chain (2-[2-C-13]) of DPPC. These positions are 5.3-5.5 Angstrom apart in the crystal structure of dimyristoylphosphatidylcholine (DMPC), but are estimated to be 4.5-5.0 Angstrom apart in DPPC below the subphase transition temperature. These results are consistent with a smaller axial displacement between the sn-1 and sn-2 acyl chains than the similar to 3.6-Angstrom displacement observed in the DMPC crystal structure. Intermolecular magnetization transfer rates have been measured between 1,-2[2-C-13]DPPC and 2-[1-C-13]DPPC and between 1,2-[1-C-13]DPPC and 2-[2-C-13]DPPC. In addition, intermolecular magnetization exchange rates have been measured between 1,2-[2-C-13]DPPC and the C-13-OH position of Y-93 in the glycophorin transmembrane domain. These intermolecular distance measurements demonstrate that the relative orientation and location of membrane lipids and peptides can be established using RR NMR methods.