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

Drew Marquardt; Frederick A. Heberle; Tatiana Miti; Barbara Eicher; Erwin London; John Katsaras; Georg Pabst

doi:10.1021/acs.langmuir.6b04485

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

Drew Marquardt, Frederick A. Heberle, Tatiana Miti, Barbara Eicher, Erwin London, John Katsaras, Georg Pabst

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103 Scopus citations

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. 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.

Original language	English
Pages (from-to)	3731-3741
Number of pages	11
Journal	Langmuir
Volume	33
Issue number	15
DOIs	https://doi.org/10.1021/acs.langmuir.6b04485
State	Published - Apr 18 2017

Funding

The authors thank Santa Jansone-Popova for NMR technical assistance. This work acknowledges support from the Austrian Science Fund (FWF) project P27083 (to G.P.); the University of Tennessee\u2212Oak Ridge National Laboratory (ORNL) Joint Institute for Biological Sciences (to F.A.H.); the National Science Foundation (NSF) DMR 1404985 (to E.L.); and the Laboratory Directed Research and Development Program award 7394 (to J.K. and F.A.H.) from ORNL, managed by UT-Battelle, LLC, under US Department of Energy (DOE) contract no. DE-AC05-00OR22725. SAXS, DLS, and DSC measurements were supported by DOE scientific user facilities.

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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 (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. 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.",

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AU - Eicher, Barbara

AU - London, Erwin

AU - Katsaras, John

AU - Pabst, Georg

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N2 - 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. 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.

AB - 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. 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.

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

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