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Title: 1H NMR Shows Slow Phospholipid Flip-Flop in Gel and Fluid Bilayers

Abstract

We measured the transbilayer diffusion of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in large unilamellar vesicles, in both the gel (L β') and fluid (L α) phases. The choline resonance of headgroup-protiated DPPC exchanged into the outer leaflet of headgroup-deuterated DPPC-d13 vesicles was monitored using 1H NMR spectroscopy, coupled with the addition of a paramagnetic shift reagent. This allowed us to distinguish between the inner and outer bilayer leaflet of DPPC, to determine the flip-flop rate as a function of temperature. Flip-flop of fluid-phase DPPC exhibited Arrhenius kinetics, from which we determined an activation energy of 122 kJ mol –1. In gel-phase DPPC vesicles, flip-flop was not observed over the course of 250 h. Here, our findings are in contrast to previous studies of solid-supported bilayers, where the reported DPPC translocation rates are at least several orders of magnitude faster than those in vesicles at corresponding temperatures. Finally, we reconcile these differences by proposing a defect-mediated acceleration of lipid translocation in supported bilayers, where long-lived, submicron-sized holes resulting from incomplete surface coverage are the sites of rapid transbilayer movement.

Authors:
ORCiD logo [1];  [2];  [3];  [1];  [4];  [5]; ORCiD logo [1]
  1. Univ. of Graz (Austria). Inst. of Molecular Biosciences, Biophysics Division; BioTechMed-Graz (Austria)
  2. Univ. of Tennessee, Knoxville, TN (United States). Bredesen Center, Dept. of Physics and Astronomy; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Joint Inst. for Biological Sciences, Biology and Soft Matter Division
  3. Univ. of South Florida, Tampa, FL (United States). Dept. of Physics
  4. Stony Brook Univ., NY (United States). Dept. of Biochemistry and Cell Biology
  5. Univ. of Tennessee, Knoxville, TN (United States). Bredesen Center, Dept. of Physics and Astronomy; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biology and Soft Matter Division; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Shull Wollan Center-A Joint Inst. for Neutron Sciences; Univ. of Tennessee, Knoxville, TN (United States). Dept. of Physics and Astronomy
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States)
Sponsoring Org.:
Austrian Science Fund (FWF); National Science Foundation (NSF); USDOE Office of Science (SC)
OSTI Identifier:
1342412
Alternate Identifier(s):
OSTI ID: 1352553; OSTI ID: 1362240
Grant/Contract Number:
AC05-00OR22725; 7394
Resource Type:
Journal Article: Published Article
Journal Name:
Langmuir
Additional Journal Information:
Journal Volume: 33; Journal Issue: 15; Journal ID: ISSN 0743-7463
Publisher:
American Chemical Society
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Marquardt, Drew, Heberle, Frederick A., Miti, Tatiana, Eicher, Barbara, London, Erwin, Katsaras, John, and Pabst, Georg. 1H NMR Shows Slow Phospholipid Flip-Flop in Gel and Fluid Bilayers. United States: N. p., 2017. Web. doi:10.1021/acs.langmuir.6b04485.
Marquardt, Drew, Heberle, Frederick A., Miti, Tatiana, Eicher, Barbara, London, Erwin, Katsaras, John, & Pabst, Georg. 1H NMR Shows Slow Phospholipid Flip-Flop in Gel and Fluid Bilayers. United States. doi:10.1021/acs.langmuir.6b04485.
Marquardt, Drew, Heberle, Frederick A., Miti, Tatiana, Eicher, Barbara, London, Erwin, Katsaras, John, and Pabst, Georg. Fri . "1H NMR Shows Slow Phospholipid Flip-Flop in Gel and Fluid Bilayers". United States. doi:10.1021/acs.langmuir.6b04485.
@article{osti_1342412,
title = {1H NMR Shows Slow Phospholipid Flip-Flop in Gel and Fluid Bilayers},
author = {Marquardt, Drew and Heberle, Frederick A. and Miti, Tatiana and Eicher, Barbara and London, Erwin and Katsaras, John and Pabst, Georg},
abstractNote = {We measured the transbilayer diffusion of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in large unilamellar vesicles, in both the gel (Lβ') and fluid (Lα) phases. The choline resonance of headgroup-protiated DPPC exchanged into the outer leaflet of headgroup-deuterated DPPC-d13 vesicles was monitored using 1H NMR spectroscopy, coupled with the addition of a paramagnetic shift reagent. This allowed us to distinguish between the inner and outer bilayer leaflet of DPPC, to determine the flip-flop rate as a function of temperature. Flip-flop of fluid-phase DPPC exhibited Arrhenius kinetics, from which we determined an activation energy of 122 kJ mol–1. In gel-phase DPPC vesicles, flip-flop was not observed over the course of 250 h. Here, our findings are in contrast to previous studies of solid-supported bilayers, where the reported DPPC translocation rates are at least several orders of magnitude faster than those in vesicles at corresponding temperatures. Finally, we reconcile these differences by proposing a defect-mediated acceleration of lipid translocation in supported bilayers, where long-lived, submicron-sized holes resulting from incomplete surface coverage are the sites of rapid transbilayer movement.},
doi = {10.1021/acs.langmuir.6b04485},
journal = {Langmuir},
number = 15,
volume = 33,
place = {United States},
year = {Fri Jan 20 00:00:00 EST 2017},
month = {Fri Jan 20 00:00:00 EST 2017}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1021/acs.langmuir.6b04485

Citation Metrics:
Cited by: 5works
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  • We measured the transbilayer diffusion of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in large unilamellar vesicles, in both the gel (L β') and fluid (L α) phases. The choline resonance of headgroup-protiated DPPC exchanged into the outer leaflet of headgroup-deuterated DPPC-d13 vesicles was monitored using 1H NMR spectroscopy, coupled with the addition of a paramagnetic shift reagent. This allowed us to distinguish between the inner and outer bilayer leaflet of DPPC, to determine the flip-flop rate as a function of temperature. Flip-flop of fluid-phase DPPC exhibited Arrhenius kinetics, from which we determined an activation energy of 122 kJ mol –1. In gel-phase DPPC vesicles,more » flip-flop was not observed over the course of 250 h. Here, our findings are in contrast to previous studies of solid-supported bilayers, where the reported DPPC translocation rates are at least several orders of magnitude faster than those in vesicles at corresponding temperatures. Finally, we reconcile these differences by proposing a defect-mediated acceleration of lipid translocation in supported bilayers, where long-lived, submicron-sized holes resulting from incomplete surface coverage are the sites of rapid transbilayer movement.« less
  • We measured the transbilayer diffusion of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in large unilamellar vesicles, in both the gel (L β') and fluid (L α) phases. The choline resonance of headgroup-protiated DPPC exchanged into the outer leaflet of headgroup-deuterated DPPC-d13 vesicles was monitored using 1H NMR spectroscopy, coupled with the addition of a paramagnetic shift reagent. This allowed us to distinguish between the inner and outer bilayer leaflet of DPPC, to determine the flip-flop rate as a function of temperature. Flip-flop of fluid-phase DPPC exhibited Arrhenius kinetics, from which we determined an activation energy of 122 kJ mol –1. In gel-phase DPPC vesicles,more » flip-flop was not observed over the course of 250 h. Our findings are in contrast to previous studies of solid-supported bilayers, where the reported DPPC translocation rates are at least several orders of magnitude faster than those in vesicles at corresponding temperatures. We reconcile these differences by proposing a defect-mediated acceleration of lipid translocation in supported bilayers, where long-lived, submicron-sized holes resulting from incomplete surface coverage are the sites of rapid transbilayer movement.« less
  • Galactosyl- and glucosylceramide, globoside, and dihydrolactosylceramide, bearing (2,2-{sup 2}H{sub 2})stearic acid, have been studied at a concentration of 10 mol % in bilayers of dimyristoylphosphatidylcholine by {sup 2}H NMR. The quadrupolar splittings {Delta}v{sub Q} of the C2 deuterons were measured at several temperatures in the range of 30-60{degree}C. Spin-lattice relaxation times T{sub 1} of C2 deuterons were determined in the same temperature range for all lipids but globoside. T{sub 1} values for the GlcCer and GalCer systems increased with temperature, indicating that the motions responsible for relaxation were in the short correlation time regime. T{sub 1} for deuterons at themore » acyl chain C2-position of LaCer was observed to decrease with increasing temperature, indicating that the motion(s) dominating relaxation are in the long correlation time regime. Thus the mobility of the acyl chain at the 2-position is reduced in the LacCer with respect to GlcCer and GalCer.« less
  • Polyunsaturated fatty acids are widely distributed components of biological membranes and are believed to be involved in many biological functions. However, the mechanisms by which they act on a molecular level are not understood. To further investigate the unique properties of {omega}3 polyunsaturated phospholipid bilayers, deuterium nuclear ming tic resonance ({sup 2}H NMR) studies have been made of the liquid-crystalline (L{sub {alpha}}) and gel phases of a homologous series of mixed-chain phosphatidylcholines containing docosahexaenoic acid: (per-{sup 2}H-n:0) (22:6) PC, where n = 12, 14, 16, and 18. The moments of the {sup 2}H NMR lineshapes have been evaluated, and formmore » these the warming and cooling main phase transition temperatures were determined. The transition temperatures of the mixed-chain series were found to be significantly lower ing those of the corresponding lipids in the disaturated series, di(per-{sup 2}H-n:0)PC, with hystereses ranging form 2 to 14 C. Distinct effects of the docosahexaenoyl chain on bilayer order were found, though these effects varied across the mixed-chain series. In evaluating the moment data, an empirical method for normalizing the moments with respect to differences in temperature was applied, in addition to using the reduced temperature method. For the systems studied here, the method of normalization haing significant effect on the interpretation of the moment data.« less
  • The binding location for the hydrophobic ions tetraphenylphosphonium (TPP/sup +/) and tetraphenylboron (TPB/sup -/) was studied in sonicated phosphatidylcholine (PC) vesicles by measuring time-dependent and steady-state intermolecular /sup 1/H nuclear Overhauser effects (NOE's). Intermolecular cross-relaxation was also investigated by two-dimensional NOE spectroscopy. Information on the distance and order parameter dependence of the NOE's was obtained from a simple simulation of the NOE's in the alkyl chain region. Taken together, the NOE data and the simulation provide strong evidence that TPB/sup -/ and TPP/sup +/, at low concentrations (less than or equal to 10 mol %), are localized in the alkylmore » chain region of the bilayer. At these lower concentrations of TPP/sup +/ or TPB/sup -/, no significant effect on lipid /sup 13/C T/sub 1/ or T/sub 2/ relaxation rates is detected. The proposed location is consistent with the expected free energy profiles for hydrophobic ions and with the carbonyl oxygens or interfacial water as the source of the membrane dipole potential. At higher ion/lipid ratios (greater than or equal to 20 mol %), TPB/sup -//lipid NOE's increase. This results from a specific association of TPB/sup -/ with the choline head group.« less