Cellular homeostasis depends on the rapid, ATP-independent translocation of newly synthesized lipids across the endoplasmic reticulum (ER) membrane. Lipid translocation is facilitated by membrane proteins known as scramblases, a few of which have recently been identified in the ER. Our previous structure of the translocon-associated protein (TRAP) bound to the Sec61 translocation channel revealed local membrane thinning, suggesting that the Sec61/TRAP complex might be involved in lipid scrambling. Using complementary fluorescence spectroscopy assays, we detected nonselective scrambling by reconstituted translocon complexes. This activity was unaffected by Sec61 inhibitors that block its lateral gate, suggesting a second lipid scrambling pathway within the complex. Molecular dynamics simulations indicate that the trimeric TRAP subunit forms this alternative route, facilitating lipid translocation via a "credit card" mechanism, using a crevice lined with polar residues to shield lipid head groups from the hydrophobic membrane interior. Kinetic and thermodynamic analyses confirmed that local membrane thinning enhances scrambling efficiency and that both Sec61 and TRAP scramble phosphatidylcholine faster than phosphatidylethanolamine and phosphatidylserine, reflecting the intrinsic lipid flip-flop tendencies of these lipid species. As the Sec61 scrambling site lies in the lateral gate region, it is likely inaccessible during protein translocation, in line with our experiments on Sec61-inhibited samples. Hence, our findings suggest that the metazoan-specific trimeric TRAP bundle is a viable candidate for lipid scrambling activity that is insensitive to the functional state of the translocon.

Central European Institute of Technology Masaryk University Kamenice 5 CZ 62500 Brno Czech Republic

Department of Physical and Macromolecular Chemistry Charles University Hlavova 8 CZ 12800 Prague 2 Czech Republic

Institute of Biotechnology HiLIFE University of Helsinki FI 00790 Helsinki Finland

J Heyrovský Institute of Physical Chemistry CZ 18223 Prague 8 Czech Republic

National Centre for Biomolecular Research Faculty of Science Masaryk University Kamenice 5 CZ 62500 Brno Czech Republic

Onego Bio Hämeentie 157 FI 00560 Helsinki Finland

Unit of Physics University of Tampere FI 33720 Tampere Finland

Zobrazit více v PubMed

Van Meer G.; Voelker D. R.; Feigenson G. W. Membrane Lipids: Where They Are and How They Behave. Nat. Rev. Mol. Cell Biol. 2008, 9, 112–124. 10.1038/nrm2330. PubMed DOI PMC

Jacquemyn J.; Cascalho A.; Goodchild R. E. The Ins and Outs of Endoplasmic Reticulum-Controlled Lipid Biosynthesis. EMBO Rep. 2017, 18, 1905–1921. 10.15252/embr.201643426. PubMed DOI PMC

Sunshine H.; Iruela-Arispe M. L. Membrane Lipids and Cell Signaling. Curr. Opin. Lipidol. 2017, 28, 408.10.1097/MOL.0000000000000443. PubMed DOI PMC

Walther T. C.; Farese R. V. Jr Lipid Droplets and Cellular Lipid Metabolism. Annu. Rev. Biochem. 2012, 81, 687–714. 10.1146/annurev-biochem-061009-102430. PubMed DOI PMC

Sezgin E.; Levental I.; Mayor S.; Eggeling C. The Mystery of Membrane Organization: Composition, Regulation and Roles of Lipid Rafts. Nat. Rev. Mol. Cell Biol. 2017, 18, 361–374. 10.1038/nrm.2017.16. PubMed DOI PMC

Hossein A.; Deserno M. Spontaneous Curvature, Differential Stress, and Bending Modulus of Asymmetric Lipid Membranes. Biophys. J. 2020, 118, 624–642. 10.1016/j.bpj.2019.11.3398. PubMed DOI PMC

Fagone P.; Jackowski S. Membrane Phospholipid Synthesis and Endoplasmic Reticulum Function. J. Lipid Res. 2009, 50, S311–S316. 10.1194/jlr.R800049-JLR200. PubMed DOI PMC

Kobayashi T.; Menon A. K. Transbilayer Lipid Asymmetry. Curr. Biol. 2018, 28, R386–R391. 10.1016/j.cub.2018.01.007. PubMed DOI

Marquardt D.; Heberle F. A.; Miti T.; Eicher B.; London E.; Katsaras J.; Pabst G. 1H NMR Shows Slow Phospholipid Flip-Flop in Gel and Fluid Bilayers. Langmuir 2017, 33, 3731–3741. 10.1021/acs.langmuir.6b04485. PubMed DOI PMC

Lorent J.; Levental K.; Ganesan L.; Rivera-Longsworth G.; Sezgin E.; Doktorova M.; Lyman E.; Levental I. Plasma Membranes Are Asymmetric in Lipid Unsaturation, Packing and Protein Shape. Nat. Chem. Biol. 2020, 16, 644–652. 10.1038/s41589-020-0529-6. PubMed DOI PMC

Allhusen J. S.; Conboy J. C. he Ins and Outs of Lipid Flip-Flop. Acc. Chem. Res. 2017, 50, 58–65. 10.1021/acs.accounts.6b00435. PubMed DOI

Contreras F.-X.; Sánchez-Magraner L.; Alonso A.; Goñi F. M. Transbilayer (Flip-Flop) Lipid Motion and Lipid Scrambling in Membranes. FEBS Lett. 2010, 584, 1779–1786. 10.1016/j.febslet.2009.12.049. PubMed DOI

Sanyal S.; Menon A. K. Flipping Lipids: Why an Whats the Reason For?. ACS Chem. Biol. 2009, 4, 895–909. 10.1021/cb900163d. PubMed DOI PMC

Backer J. M.; Dawidowicz E. A. Reconstitution of a Phospholipid Flippase From Rat Liver Microsomes. Nature 1987, 327, 341–343. 10.1038/327341a0. PubMed DOI

Bishop W. R.; Bell R. M. Assembly of the Endoplasmic Reticulum Phospholipid Bilayer: The Phosphatidylcholine Transporter. Cell 1985, 42, 51–60. 10.1016/S0092-8674(85)80100-8. PubMed DOI

Menon A. K.; Watkins W. E.; Hrafnsdóttir S. Specific Proteins Are Required to Translocate Phosphatidylcholine Bidirectionally Across the Endoplasmic Reticulum. Curr. Biol. 2000, 10, 241–252. 10.1016/S0960-9822(00)00356-0. PubMed DOI

Chang Q.-l.; Gummadi S. N.; Menon A. K. Chemical Modification Identifies Two Populations of Glycerophospholipid Flippase in Rat Liver ER. Biochemistry 2004, 43, 10710–10718. 10.1021/bi049063a. PubMed DOI

Menon I.; Huber T.; Sanyal S.; Banerjee S.; Barré P.; Canis S.; Warren J. D.; Hwa J.; Sakmar T. P.; Menon A. K. Opsin Is a Phospholipid Flippase. Curr. Biol. 2011, 21, 149–153. 10.1016/j.cub.2010.12.031. PubMed DOI PMC

Suzuki J.; Umeda M.; Sims P. J.; Nagata S. Calcium-Dependent Phospholipid Scrambling by TMEM16F. Nature 2010, 468, 834–838. 10.1038/nature09583. PubMed DOI

Brunner J. D.; Lim N. K.; Schenck S.; Duerst A.; Dutzler R. X-Ray Structure of a Calcium-Activated TMEM16 Lipid Scramblase. Nature 2014, 516, 207–212. 10.1038/nature13984. PubMed DOI

Kalienkova V.; Mosina V. C.; Paulino C. The Groovy TMEM16 Family: Molecular Mechanisms of Lipid Scrambling and Ion Conduction. J. Mol. Biol. 2021, 433, 16694110.1016/j.jmb.2021.166941. PubMed DOI

Gyobu S.; Ishihara K.; Suzuki J.; Segawa K.; Nagata S. Characterization of the Scrambling Domain of the TMEM16 Family. Proc. Natl. Acad. Sci. U.S.A. 2017, 114, 6274–6279. 10.1073/pnas.1703391114. PubMed DOI PMC

Bethel N. P.; Grabe M. Atomistic Insight Into Lipid Translocation by a TMEM16 Scramblase. Proc. Natl. Acad. Sci. U.S.A. 2016, 113, 14049–14054. 10.1073/pnas.1607574113. PubMed DOI PMC

Feng Z.; Di Zanni E.; Alvarenga O.; Chakraborty S.; Rychlik N.; Accardi A. In or Out of the Groove? Mechanisms of Lipid Scrambling by TMEM16 Proteins. Cell Calcium 2024, 121, 10289610.1016/j.ceca.2024.102896. PubMed DOI PMC

Huang D.; et al. TMEM41B Acts as an ER Scramblase Required for Lipoprotein Biogenesis and Lipid Homeostasis. Cell Metab. 2021, 33, 1655–1670. 10.1016/j.cmet.2021.05.006. PubMed DOI

Bushell S. R.; et al. The Structural Basis of Lipid Scrambling and Inactivation in the Endoplasmic Reticulum Scramblase TMEM16K. Nat. Commun. 2019, 10, 3956.10.1038/s41467-019-11753-1. PubMed DOI PMC

Tsuji T.; Cheng J.; Tatematsu T.; Ebata A.; Kamikawa H.; Fujita A.; Gyobu S.; Segawa K.; Arai H.; Taguchi T.; Nagata S.; Fujimoto T. Predominant Localization of Phosphatidylserine at the Cytoplasmic Leaflet of the ER, and Its TMEM16K-Dependent Redistribution. Proc. Natl. Acad. Sci. U.S.A. 2019, 116, 13368–13373. 10.1073/pnas.1822025116. PubMed DOI PMC

Li Y. E.; et al. TMEM41B and VMP1 Are Scramblases and Regulate the Distribution of Cholesterol and Phosphatidylserine. J. Cell. Biol. 2021, 220, e20210310510.1083/jcb.202103105. PubMed DOI PMC

Ghanbarpour A.; Valverde D. P.; Melia T. J.; Reinisch K. M. A Model for a Partnership of Lipid Transfer Proteins and Scramblases in Membrane Expansion and Organelle Biogenesis. Proc. Natl. Acad. Sci. U.S.A. 2021, 118, e210156211810.1073/pnas.2101562118. PubMed DOI PMC

Reinisch K. M.; Chen X.-W.; Melia T. J. “VTT”--Domain Proteins VMP1 and TMEM41B Function in Lipid Homeostasis Globally and Locally as ER Scramblases. Contact 2021, 4, 2515256421102449410.1177/25152564211024494. PubMed DOI PMC

Li D.; Rocha-Roa C.; Schilling M. A.; Reinisch K. M.; Vanni S. Lipid Scrambling Is a General Feature of Protein Insertases. Proc. Natl. Acad. Sci. U.S.A. 2024, 121, e231947612110.1073/pnas.2319476121. PubMed DOI PMC

Bartoš L.; Menon A. K.; Vácha R. Insertases Scramble Lipids: Molecular Simulations of MTCH2. Structure 2024, 32, 505–510. 10.1016/j.str.2024.01.012. PubMed DOI PMC

Karki S.; Javanainen M.; Rehan S.; Tranter D.; Kellosalo J.; Huiskonen J. T.; Happonen L.; Paavilainen V. Molecular View of ER Membrane Remodeling by the SEC61/Trap Translocon. EMBO Rep. 2023, 24, e5791010.15252/embr.202357910. PubMed DOI PMC

Pauwels E.; Shewakramani N. R.; De Wijngaert B.; Camps A.; Provinciael B.; Stroobants J.; Kalies K.-U.; Hartmann E.; Maes P.; Vermeire K.; Das K. Structural Insights Into Trap Association With Ribosome-Sec61 Complex and Translocon Inhibition by a CADA Derivative. Sci. Adv. 2023, 9, eadf079710.1126/sciadv.adf0797. PubMed DOI PMC

Jaskolowski M.; Jomaa A.; Gamerdinger M.; Shrestha S.; Leibundgut M.; Deuerling E.; Ban N. Molecular Basis of the Trap Complex Function in ER Protein Biogenesis. Nat. Struct. Mol. Biol. 2023, 30, 770.10.1038/s41594-023-00990-0. PubMed DOI PMC

Gemmer M.; Chaillet M. L.; van Loenhout J.; Cuevas Arenas R.; Vismpas D.; Grollers-Mulderij M.; Koh F. A.; Albanese P.; Scheltema R. A.; Howes S. C.; Kotecha A.; Fedry J.; Forster F. Visualization of Translation and Protein Biogenesis at the ER Membrane. Nature 2023, 614, 160–167. 10.1038/s41586-022-05638-5. PubMed DOI PMC

Li X.; Itani O. A.; Haataja L.; Dumas K. J.; Yang J.; Cha J.; Flibotte S.; Shih H.-J.; Delaney C. E.; Xu J.; Qi L.; Arvan P.; Liu M.; Hu P. J. Requirement for Translocon-Associated Protein (TRAP)α In Insulin Biogenesis. Sci. Adv. 2019, 5, eaax029210.1126/sciadv.aax0292. PubMed DOI PMC

Nguyen D.; Stutz R.; Schorr S.; Lang S.; Pfeffer S.; Freeze H. H.; Förster F.; Helms V.; Dudek J.; Zimmermann R. Proteomics Reveals Signal Peptide Features Determining the Client Specificity in Human TRAP-Dependent ER Protein Import. Nat. Commun. 2018, 9, 3765.10.1038/s41467-018-06188-z. PubMed DOI PMC

Pomorski T.; Menon A. Lipid Flippases and Their Biological Functions. Cell. Mol. Life Sci. 2006, 63, 2908–2921. 10.1007/s00018-006-6167-7. PubMed DOI PMC

Vehring S.; Pakkiri L.; Schröer A.; Alder-Baerens N.; Herrmann A.; Menon A. K.; Pomorski T. Flip-Flop of Fluorescently Labeled Phospholipids in Proteoliposomes Reconstituted With Saccharomyces Cerevisiae Microsomal Proteins. Eukaryot. Cell 2007, 6, 1625–1634. 10.1128/EC.00198-07. PubMed DOI PMC

Watkins W. E. III; Menon A. Reconstitution of Phospholipid Flippase Activity from E. coli Inner Membrane: A Test of the Protein Translocon as a Candidate Flippase. Biol. Chem. 2002, 383, 1435–1440. 10.1515/BC.2002.162. PubMed DOI

Rehan S.; et al. Signal Peptide Mimicry Primes Sec61 for Client-Selective Inhibition. Nat. Chem. Biol. 2023, 19, 1054–1062. 10.1038/s41589-023-01326-1. PubMed DOI PMC

Jahn H.; Bartoš L.; Dearden G. I.; Dittman J. S.; Holthuis J. C.; Vácha R.; Menon A. K. Phospholipids Are Imported Into Mitochondria by VDAC, a Dimeric Beta Barrel Scramblase. Nat. Commun. 2023, 14, 8115.10.1038/s41467-023-43570-y. PubMed DOI PMC

Malvezzi M.; Chalat M.; Janjusevic R.; Picollo A.; Terashima H.; Menon A. K.; Accardi A. Ca2+-Dependent Phospholipid Scrambling by a Reconstituted TMEM16 Ion Channel. Nat. Commun. 2013, 4, 2367.10.1038/ncomms3367. PubMed DOI PMC

Kubelt J.; Menon A. K.; Müller P.; Herrmann A. Transbilayer Movement of Fluorescent Phospholipid Analogues in the Cytoplasmic Membrane of Escherichia Coli. Biochemistry 2002, 41, 5605–5612. 10.1021/bi0118714. PubMed DOI

Itskanov S.; Wang L.; Junne T.; Sherriff R.; Xiao L.; Blanchard N.; Shi W. Q.; Forsyth C.; Hoepfner D.; Spiess M.; Park E. A Common Mechanism of Sec61 Translocon Inhibition by Small Molecules. Nat. Chem. Biol. 2023, 19, 1063.10.1038/s41589-023-01337-y. PubMed DOI PMC

Zong G.; et al. Ipomoeassin F Binds Sec61α To Inhibit Protein Translocation. J. Am. Chem. Soc. 2019, 141, 8450–8461. 10.1021/jacs.8b13506. PubMed DOI PMC

Paatero A. O.; Kellosalo J.; Dunyak B. M.; Almaliti J.; Gestwicki J. E.; Gerwick W. H.; Taunton J.; Paavilainen V. O. Apratoxin Kills Cells by Direct Blockade of the Sec61 Protein Translocation Channel. Cell. Chem. Biol. 2016, 23, 561–566. 10.1016/j.chembiol.2016.04.008. PubMed DOI

Russo A. Understanding the Mammalian Trap Complex Function(s). Open Biol. 2020, 10, 190244.10.1098/rsob.190244. PubMed DOI PMC

Webb B.; Sali A. Comparative Protein Structure Modeling Using MODELLER. Curr. Protoc. Bioinformatics 2016, 54, 5–6. 10.1002/cpbi.3. PubMed DOI PMC

Souza P. C.; et al. Martini 3: A General Purpose Force Field for Coarse-Grained Molecular Dynamics. Nat. Methods 2021, 18, 382–388. 10.1038/s41592-021-01098-3. PubMed DOI

De Jong D. H.; Baoukina S.; Ingólfsson H. I.; Marrink S. J. Performance Using a Shorter Cutoff and GPUs. Comput. Phys. Commun. 2016, 199, 1–7. 10.1016/j.cpc.2015.09.014. DOI

Khelashvili G.; Menon A. K. Phospholipid Scrambling by G Protein-Coupled Receptors. Annu. Rev. Biophys. 2022, 51, 39–61. 10.1146/annurev-biophys-090821-083030. PubMed DOI PMC

Morra G.; Razavi A. M.; Pandey K.; Weinstein H.; Menon A. K.; Khelashvili G. Mechanisms of Lipid Scrambling by the G Protein-Coupled Receptor Opsin. Structure 2018, 26, 356–367. 10.1016/j.str.2017.11.020. PubMed DOI PMC

Berg B. V. D.; Clemons W. M. Jr; Collinson I.; Modis Y.; Hartmann E.; Harrison S. C.; Rapoport T. A. X-Ray Structure of a Protein-Conducting Channel. Nature 2004, 427, 36–44. 10.1038/nature02218. PubMed DOI

Voorhees R. M.; Fernández I. S.; Scheres S. H.; Hegde R. S. Structure of the Mammalian Ribosome-Sec61 Complex to 3.4 Å Resolution. Cell 2014, 157, 1632–1643. 10.1016/j.cell.2014.05.024. PubMed DOI PMC

Voorhees R. M.; Hegde R. S. Structure of the Sec61 Channel Opened by a Signal Sequence. Science 2016, 351, 88–91. 10.1126/science.aad4992. PubMed DOI PMC

Gamayun I.; O’Keefe S.; Pick T.; Klein M.-C.; Nguyen D.; McKibbin C.; Piacenti M.; Williams H. M.; Flitsch S. L.; Whitehead R. C.; Swanton E.; Helms V.; High S.; Zimmermann R.; Cavalié A. Eeyarestatin Compounds Selectively Enhance Sec61-Mediated Ca2+ Leakage From the Endoplasmic Reticulum. Cell Chem. Biol. 2019, 26, 571–583. 10.1016/j.chembiol.2019.01.010. PubMed DOI PMC

Luesch H.; Paavilainen V. O. Natural Products as Modulators of Eukaryotic Protein Secretion. Nat. Prod. Rep. 2020, 37, 717–736. 10.1039/C9NP00066F. PubMed DOI PMC

Bennett W. D.; Tieleman D. P. The Importance of Membrane Defects–Lessons from Simulations. Acc. Chem. Res. 2014, 47, 2244–2251. 10.1021/ar4002729. PubMed DOI

Watanabe H.; Hanashima S.; Yano Y.; Yasuda T.; Murata M. Passive Translocation of Phospholipids in Asymmetric Model Membranes: Solid-State 1H NMR Characterization of Flip-Flop Kinetics Using Deuterated Sphingomyelin and Phosphatidylcholine. Langmuir 2023, 39, 15189–15199. 10.1021/acs.langmuir.3c01650. PubMed DOI

Nakano M.; Fukuda M.; Kudo T.; Endo H.; Handa T. Determination of Interbilayer and Transbilayer Lipid Transfers by Time-Resolved Small-Angle Neutron Scattering. Phys. Rev. Lett. 2007, 98, 238101.10.1103/PhysRevLett.98.238101. PubMed DOI

Porcar L.; Gerelli Y. On the Lipid Flip-Flop and Phase Transition Coupling. Soft Matter 2020, 16, 7696–7703. 10.1039/D0SM01161D. PubMed DOI

Marrink S. J.; Tieleman D. P. Perspective on the Martini Model. Chem. Soc. Rev. 2013, 42, 6801–6822. 10.1039/c3cs60093a. PubMed DOI

Javanainen M.; Martinez-Seara H.; Vattulainen I. Excessive Aggregation of Membrane Proteins in the Martini Model. PLoS One 2017, 12, e0187936.10.1371/journal.pone.0187936. PubMed DOI PMC

Klein W.; Rutz C.; Eckhard J.; Provinciael B.; Specker E.; Neuenschwander M.; Kleinau G.; Scheerer P.; von Kries J.-P.; Nazare M.; Nazaré M.; Vermeire K.; Schülein R. Use of a Sequential High Throughput Screening Assay to Identify Novel Inhibitors of the Eukaryotic SRP-Sec61 Targeting/Translocation Pathway. PLoS One 2018, 13, e0208641.10.1371/journal.pone.0208641. PubMed DOI PMC

Qi Y.; Ingólfsson H. I.; Cheng X.; Lee J.; Marrink S. J.; Im W. CHARMM-GUI martini maker for coarse-grained simulations with the Martini force field. J. Chem. Theory Comput. 2015, 11, 4486–4494. 10.1021/acs.jctc.5b00513. PubMed DOI

Páll S.; Zhmurov A.; Bauer P.; Abraham M.; Lundborg M.; Gray A.; Hess B.; Lindahl E. Heterogeneous Parallelization and Acceleration of Molecular Dynamics Simulations in GROMACS. J. Chem. Phys. 2020, 153, 134110.10.1063/5.0018516. PubMed DOI

Abraham M. J.; Murtola T.; Schulz R.; Páll S.; Smith J. C.; Hess B.; Lindahl E. GROMACS: High Performance Molecular Simulations Through Multi-Level Parallelism From Laptops to Supercomputers. SoftwareX 2015, 1, 19–25. 10.1016/j.softx.2015.06.001. DOI

Páll S.; Hess B. A Flexible Algorithm for Calculating Pair Interactions on SIMD Architectures. Comput. Phys. Commun. 2013, 184, 2641–2650. 10.1016/j.cpc.2013.06.003. DOI

Bussi G.; Donadio D.; Parrinello M. Canonical Sampling Through Velocity Rescaling. J. Chem. Phys. 2007, 126, 014101.10.1063/1.2408420. PubMed DOI

Parrinello M.; Rahman A. Polymorphic Transitions in Single Crystals: A New Molecular Dynamics Method. J. Appl. Phys. 1981, 52, 7182–7190. 10.1063/1.328693. DOI

Lindahl V.; Lidmar J.; Hess B. Accelerated Weight Histogram Method for Exploring Free Energy Landscapes. J. Chem. Phys. 2014, 141, 044110.10.1063/1.4890371. PubMed DOI

MacCallum J. L.; Tieleman D. P. Computer Simulation of the Distribution of Hexane in a Lipid Bilayer: Spatially Resolved Free Energy, Entropy, and Enthalpy Profiles. J. Am. Chem. Soc. 2006, 128, 125–130. 10.1021/ja0535099. PubMed DOI

Gapsys V.; de Groot B. L.; Briones R. Computational Analysis of Local Membrane Properties. J. Compt. Aid. Mol. Des. 2013, 27, 845–858. 10.1007/s10822-013-9684-0. PubMed DOI PMC

Klauda J. B.; Venable R. M.; Freites J. A.; O’Connor J. W.; Tobias D. J.; Mondragon-Ramirez C.; Vorobyov I.; MacKerell A. D. Jr; Pastor R. W. Update of the CHARMM All-Atom Additive Force Field for Lipids: Validation on Six Lipid Types. J. Phys. Chem. B 2010, 114, 7830–7843. 10.1021/jp101759q. PubMed DOI PMC

Darden T.; York D.; Pedersen L. Particle Mesh Ewald: An N·log(N) Method for Ewald Sums in Large Systems. J. Chem. Phys. 1993, 98, 10089–10092. 10.1063/1.464397. DOI

Essmann U.; Perera L.; Berkowitz M. L.; Darden T.; Lee H.; Pedersen L. G.; Smooth A. Particle Mesh Ewald Method. J. Chem. Phys. 1995, 103, 8577–8593. 10.1063/1.470117. DOI

Nosé S. A Unified Formulation of the Constant Temperature Molecular Dynamics Methods. J. Chem. Phys. 1984, 81, 511–519. 10.1063/1.447334. DOI

Hoover W. G. Canonical Dynamics: Equilibrium Phase-Space Distributions. Phys. Rev. A 1985, 31, 1695.10.1103/PhysRevA.31.1695. PubMed DOI

Hess B.; Bekker H.; Berendsen H. J.; Fraaije J. G. LINCS: A Linear Constraint Solver for Molecular Simulations. J. Comput. Chem. 1997, 18, 1463–1472. 10.1002/(SICI)1096-987X(199709)18:12<1463::AID-JCC4>3.0.CO;2-H. DOI

Hess B. P-LINCS: A Parallel Linear Constraint Solver for Molecular Simulation. J. Chem. Theory Comput. 2008, 4, 116–122. 10.1021/ct700200b. PubMed DOI

Miyamoto S.; Kollman P. A. SETTLE: An Analytical Version of the SHAKE and RATTLE Algorithm for Rigid Water Models. J. Comput. Chem. 1992, 13, 952–962. 10.1002/jcc.540130805. DOI

Walter P.; Blobel G. Preparation of Microsomal Membranes for Cotranslational Protein Translocation. Methods Enzymol. 1983, 96, 84–93. 10.1016/S0076-6879(83)96010-X. PubMed DOI

Vermeire K.; Allan S.; Provinciael B.; Hartmann E.; Kalies K.-U. Ribonuclease-Neutralized Pancreatic Microsomal Membranes From Livestock for in Vitro Co-Translational Protein Translocation. Anal. Biochem. 2015, 484, 102–104. 10.1016/j.ab.2015.05.019. PubMed DOI

Brunner J. D.; Schenck S. Preparation of Proteoliposomes With Purified TMEM16 Protein for Accurate Measures of Lipid Scramblase Activity. Intracellular Lipid Transport: Methods and Protocols 2019, 1949, 181–199. 10.1007/978-1-4939-9136-5_14. PubMed DOI

Rigaud J.-L.; Lévy D. Reconstitution of Membrane Proteins Into Liposomes. Methods Enzymol. 2003, 372, 65–86. 10.1016/S0076-6879(03)72004-7. PubMed DOI

Geertsma E. R.; Nik Mahmood N.; Schuurman-Wolters G. K.; Poolman B. Membrane Reconstitution of ABC Transporters and Assays of Translocator Function. Nat. Protoc. 2008, 3, 256–266. 10.1038/nprot.2007.519. PubMed DOI

Perez-Riverol Y.; Bai J.; Bandla C.; García-Seisdedos D.; Hewapathirana S.; Kamatchinathan S.; Kundu D. J.; Prakash A.; Frericks-Zipper A.; Eisenacher M.; Walzer M.; Wang S.; Brazma A.; Vizcaíno J. The PRIDE Database Resources in 2022: A Hub for Mass Spectrometry-Based Proteomics Evidences. Nucleic Acids Res. 2022, 50, D543–D552. 10.1093/nar/gkab1038. PubMed DOI PMC