Deuterium nuclear magnetic resonance studies of the interaction between dimyristoylphosphatidylcholine and gramicidin A'.

Deuterium nuclear magnetic resonance spectra of dimyristoylphosphatidylcholines (DMPCs) specifically labeled with deuterium at one of positions 2’, 3’, 4’, 6’, 8’, lo’, 12’, or 14’ of the 2-chain have been recorded at 34.1 MHz in the presence of varying concentrations of the linear pentadecapeptide antibiotic gramicidin A’. Deuterium quadrupole splittings (AvQ) have been used to partially characterize the motion and hydrocarbon chain order of phospholipid in contact with the polypeptide surface. At lipid concentrations below 4 lipids/gramicidin molecule the quadrupole splitting of a terminal methyl-labeled DMPC collapses to a single line. The quadrupole splittings of the methylene labels are decreased, and the line shapes are dominated by large intrinsic line widths. The time constants characterizing the decays of the echo intensity ( T2,) are correspondingly reduced in the lipid-polypeptide complexes. At lipid-polypeptide molar ratios of greater than 15:1, the quadrupole splittings of the labels increase linearly with gramicidin concentration to a value I n recent years there has been much interest in developing and applying physical methods to study the structure of model and intact biological membranes. Information on the static structures of both types of membrane has been obtained by using X-ray diffraction (Engelman, 1971; Tardieu et al., 1973) and neutron diffraction techniques (Worcester & Franks, 1976; Buldt et al., 1978; D. L. Worcester, M. Meadows, D. Rice, and E. Oldfield, unpublished experiments), while information on the dynamic structures of these systems has been gained predominantly from a variety of magnetic resonance techniques [for example, Chapman & Salsbury (1966), Oldfield et al. (1971, 1972, 1976, 1978a,b) Gaffney & McConnell (1974), Davis et al. (1976), Lawaczeck et al. (1976), Gent et al. (1978), Mantsch et ai. (1977), Seelig (1977), Feigenson et al. (1977), Marsh et al. (1978), and Griffin et al. (1978)]. Although proteins normally constitute about one-half of a membrane’s dry weight, there have been remarkably few From the Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801. Received December 6, 1978. This work was supported by the U S . National Institutes of Health (Grant HL-19481) and in part by the U S . National Science Foundation (Grant PCM 76-01491), by the American Heart Association with funds provided in part by the Illinois Heart Association (Grant 77-1004), by the Illinois Heart Association (Grant N-6), and by the Alfred P. Sloan Foundation. *Alfred P. Sloan Research Fellow, 1978-1980. ‘Supported by a U S . National Institutes of Health, Cell and Molecular Biology Training Grant (Grant GM-07283). 0006-2960/79/0418-3272$01 .OO/O 30% greater than that of pure lipid. Between ratios of 15: 1 and 4:1, the splittings decrease slowly. At a ratio of about 4:l a rather abrupt transition occurs to the high gramicidin phase, and on the time scale of the deuterium N M R experiment ( 5.0 ps) lipid adjacent to polypeptide appears disordered (smaller Avq) compared to pure lipid. At all lipidprotein ratios investigated above the gel-to-liquid crystalline phase transition temperature of the pure lipid (Tc ) , the experimental spectrum is shown to be accurately simulated by using one quadrupole splitting together with a Lorentzian line broadening corresponding to the quadrupolar echo decay rate ( T T ~ ~ ) ’ . In some gramicidin-lecithin complexes, T2, values as short as 49 ps are observed. This implies in general for studies of protein-lipid organization in both model and biological membranes that T2e values should be determined routinely in order to eliminate spectral distortions due to relaxation. studies of protein-lipid interaction using physical techniques. Electron spin resonance (ESR)’ studies using nitroxide free-radical spin-labels have suggested that, at least on the time scale of s, there is a reduction in the rate of motion of a fraction of the hydrocarbon chains in a protein-containing lipid bilayer, the so-called “boundary lipid” or “annulus” (Jost et al., 1973; Hesketh et al., 1976).2 Calorimetric studies indicate a decrease in the enthalpy of the gel-to-liquid crystalline phase transition of zwitterionic phospholipids with intrinsic proteinor polypeptide-containing bilayers (Chapman et al., 1974; Papahadjopoulos et al., 1975; Curatolo et al., 1977), and Raman spectroscopy has shown a reduction of the number of gauche isomers at low concentrations of the polypeptide gramicidin A‘ (Chapman et al., 1977), which is in agreement with spin-label results which indicate an increase in chain segmental order parameters (Chapman et al., 1977; Cornel1 et al., 1978). More recently, deuterium quadrupole-echo Fourier transform nuclear magnetic resonance (NMR)’ studies of protein-lipid and polypeptide-lipid interactions in model and intact biological membranes have been reported (Old field et I Abbreviations used: DPPC, 1,2-dipalmitoyl-3-sn-phosphatidylcholine; DMPC, 1,2-dimyristoyl-3-sn-phosphatidylcholine; PPPC, l-palmitoyl2-palmitoleyl-3-sn-phosphatidylcholine; NMR, nuclear magnetic resonance. More recent studies by Jost & Griffith (1978) have interpreted the ESR results as indicating that “boundary lipid” is far more ordered than free bilayer lipid.