Determination of Interbilayer and Transbilayer Lipid Transfers by Time-Resolved Small-Angle Neutron Scattering

Nakano, Minoru; Fukuda, Masahiro; Kudo, Takayuki; Endo, Hitoshi; Handa, Tetsurou

doi:10.1103/physrevlett.98.238101

Cited by 131 publications

(205 citation statements)

References 35 publications

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“…5), from which the thermodynamic activation parameters at 37 C were calculated, using the method described by Homan and Pownall (25) ( Table 2). Intrinsic flip-flop of C 6 NBD-phospholipids in peptide-free LUVs was unfavorable both enthalpically and entropically, which agreed with previous results obtained for 1,2-dimyristoyl-snglycero-3-phosphocholine (26). EDEM1 reduced the activation free energy (DDG z ¼ -3.88, -8.36, and -7.82 kJ/mol for C 6 NBD-PC, C 6 NBD-PE, and C 6 NBD-PS, respectively).…”

Section: Kinetics Of Edem1-mediated Flipsupporting

confidence: 91%

Membrane-Spanning Sequences in Endoplasmic Reticulum Proteins Promote Phospholipid Flip-Flop

Nakao

Ikeda

Ishihama

et al. 2016

Biophysical Journal

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The mechanism whereby phospholipids rapidly flip-flop in the endoplasmic reticulum (ER) membrane remains unknown. We previously demonstrated that the presence of a hydrophilic residue in the center of the model transmembrane peptide sequence effectively promoted phospholipid flip-flop and that hydrophilic residues composed 4.5% of the central regions of the membrane-spanning sequences of human ER membrane proteins predicted by SOSUI software. We hypothesized that ER proteins with hydrophilic residues might play a critical role in promoting flip-flop. Here, we evaluated the flip rate of fluorescently labeled lipids in vesicles containing each of the 11 synthetic peptides of membrane-spanning sequences, using a dithionite-quenching assay. Although the flippase activities of nine peptides were unexpectedly low, the peptides based on the EDEM1 and SPAST proteins showed enhanced flippase activity with three different fluorescently labeled lipids. The substitution of hydrophobic Ala with His or Arg in the central region of the EDEM1 or SPAST peptides, respectively, attenuated their ability to flip phospholipids. Interestingly, substituting Ala with Arg or His at a location outside of the central region of EDEM1 or SPAST, respectively, also affected the enhancement of flip-flop. These results indicated that both Arg and His are important for the ability of these two peptides to increase the flip rates. The EDEM1 peptide exhibited high activity at significantly low peptide concentrations, suggesting that the same side positioning of Arg and His in α-helix structure is critical for the flip-flop promotion and that the EDEM1 protein is a candidate flippase in the ER.

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Section: Kinetics Of Edem1-mediated Flipsupporting

confidence: 91%

Membrane-Spanning Sequences in Endoplasmic Reticulum Proteins Promote Phospholipid Flip-Flop

Nakao

Ikeda

Ishihama

et al. 2016

Biophysical Journal

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“…39 Previously, we have succeeded in characterizing interbilayer and transbilayer lipid transfers of DMPC with time-resolved small-angle neutron scattering (TR-SANS). 40 The transfer rate of DMPC in LUV was 0.276 h −1 , which is slightly smaller than that of Chol obtained in the present study (1.11 h −1 ). The energy barrier for the lipid transfer is the exposure of hydrophobic region of the lipid into aqueous media (hydrophobic hydration) on the dissociation process.…”

Section: ■ Discussioncontrasting

confidence: 77%

Kinetic and Thermodynamic Analysis of Cholesterol Transfer between Phospholipid Vesicles and Nanodiscs

2015

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We investigated interparticle transfer of cholesterol (Chol) between large unilamellar vesicles (LUVs) and phospholipid bilayer nanodiscs. The Chol transfer rate from LUVs to nanodiscs was decreased by an increase in the Chol content or incorporation of sphingomyelin in donor phosphatidylcholine/Chol LUVs but was not influenced by the lipid composition of acceptor particles. These results suggest that Chol dissociation from the lipid bilayer into aqueous phase is the rate-limiting step of the transfer and that the process depends on the fluidity of the donor membranes. The Chol dissociation rate from nanodiscs was faster than that from LUVs with similar lipid composition. Chol preferably partitioned to LUVs rather than nanodiscs, which is consistent with the faster dissociation rate from nanodiscs. The activation energy of Chol dissociation from nanodiscs was 1.7 kJ/mol lower than that from LUV, which was brought by increased (less negative) activation entropy and enthalpy. In addition, fluorescence lifetime and anisotropy data revealed that the lipid bilayer of nanodiscs is more tightly packed than that of LUVs. These results suggest that the tighter lipid packing in nanodiscs destabilizes the Chol-containing bilayer by reducing the entropy, which facilitates Chol dissociation.

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“…Instead, TR-SANS relies on monitoring changes in the neutron scattering length density (SLD) accompanying the exchange of molecules between hydrogenated and deuterated particles. This principle has been used to follow content exchange among copolymer micelles2930 and, specifically, lipid transfer among MSP nanodiscs31 or LUVs32. We prepared SMALPs that harboured either hydrogenated or deuterated DMPC (h- and d-SMALPs, respectively) in Tris buffer containing 42.8% ( v / v ) D 2 O, which matches the SLD of nanodiscs composed of equal amounts of h- and d-DMPC (h/d-SMALPs).…”

Section: Resultsmentioning

confidence: 99%

“…3a). This allowed a comparison with MSP nanodiscs31 and LUVs32, which exhibit lipid transfer solely by monomer diffusion. For both of the latter, the pronounced enthalpic penalty incurred upon formation of the transition state can be ascribed to the disruption of dispersive acyl-chain and polar headgroup interactions2627.…”

Section: Resultsmentioning

confidence: 99%

“…3b). At high c L , k obs plateaus for monomer diffusion among MSP nanodiscs31 and LUVs32, whereas collisional exchange among SMALPs, small unilamellar vesicles (SUVs)25, phosphocholine/taurocholate (PC/TC) mixed micelles24, and Triton X-100 (TX-100) micelles34 leads to a steady rise in k obs with c L . For PC/TC micelles, where transfer takes place through a combination of monomer diffusion and second-order (“bimolecular”) as well as third-order (“termolecular”) collisions, k obs displays a particularly pronounced increase at high c L .…”

Section: Resultsmentioning

confidence: 99%

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Fast Collisional Lipid Transfer Among Polymer-Bounded Nanodiscs

Arenas

Danielczak

Martel

et al. 2017

Sci Rep

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Some styrene/maleic acid (SMA) copolymers solubilise membrane lipids and proteins to form polymer-bounded nanodiscs termed SMA/lipid particles (SMALPs). Although SMALPs preserve a lipid-bilayer core, they appear to be more dynamic than other membrane mimics. We used time-resolved Förster resonance energy transfer and small-angle neutron scattering to determine the kinetics and the mechanisms of phospholipid transfer among SMALPs. In contrast with vesicles or protein-bounded nanodiscs, SMALPs exchange lipids not only by monomer diffusion but also by fast collisional transfer. Under typical experimental conditions, lipid exchange occurs within seconds in the case of SMALPs but takes minutes to days in the other bilayer particles. The diffusional and second-order collisional exchange rate constants for SMALPs at 30 °C are kdif = 0.287 s−1 and kcol = 222 M−1s−1, respectively. Together with the fast kinetics, the observed invariability of the rate constants with probe hydrophobicity and the moderate activation enthalpy of ~70 kJ mol−1 imply that lipids exchange through a “hydrocarbon continuum” enabled by the flexible nature of the SMA belt surrounding the lipid-bilayer core. Owing to their fast lipid-exchange kinetics, SMALPs represent highly dynamic equilibrium rather than kinetically trapped membrane mimics, which has important implications for studying protein/lipid interactions in polymer-bounded nanodiscs.

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Determination of Interbilayer and Transbilayer Lipid Transfers by Time-Resolved Small-Angle Neutron Scattering

Cited by 131 publications

References 35 publications

Membrane-Spanning Sequences in Endoplasmic Reticulum Proteins Promote Phospholipid Flip-Flop

Membrane-Spanning Sequences in Endoplasmic Reticulum Proteins Promote Phospholipid Flip-Flop

Kinetic and Thermodynamic Analysis of Cholesterol Transfer between Phospholipid Vesicles and Nanodiscs

Fast Collisional Lipid Transfer Among Polymer-Bounded Nanodiscs

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