Real-time imaging of structure and dynamics of transmembrane biomolecules by FRET-induced single-molecule fluorescence attenuation

Dongfei Ma; Wenqing Hou; Chenguang Yang; Shuxin Hu; Weijing Han; Ying Lu

doi:10.52601/bpr.2021.210030

Volume 7 Issue 6

Dec. 2021

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Dongfei Ma, Wenqing Hou, Chenguang Yang, Shuxin Hu, Weijing Han, Ying Lu. Real-time imaging of structure and dynamics of transmembrane biomolecules by FRET-induced single-molecule fluorescence attenuation[J]. Biophysics Reports, 2021, 7(6): 490-503. doi: 10.52601/bpr.2021.210030

Citation:

Dongfei Ma, Wenqing Hou, Chenguang Yang, Shuxin Hu, Weijing Han, Ying Lu. Real-time imaging of structure and dynamics of transmembrane biomolecules by FRET-induced single-molecule fluorescence attenuation[J]. Biophysics Reports, 2021, 7(6): 490-503. doi: 10.52601/bpr.2021.210030

Citation:

Dongfei Ma, Wenqing Hou, Chenguang Yang, Shuxin Hu, Weijing Han, Ying Lu. Real-time imaging of structure and dynamics of transmembrane biomolecules by FRET-induced single-molecule fluorescence attenuation[J]. Biophysics Reports, 2021, 7(6): 490-503. doi: 10.52601/bpr.2021.210030

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Real-time imaging of structure and dynamics of transmembrane biomolecules by FRET-induced single-molecule fluorescence attenuation

doi: 10.52601/bpr.2021.210030

Dongfei Ma^{1, 2},
Wenqing Hou^{2, 3},
Chenguang Yang^{2, 3},
Shuxin Hu²,
Weijing Han¹,
Ying Lu^{1, 2, 3
,
,}

1.
Songshan Lake Materials Laboratory, Dongguan, Guangdong 523808, China
2.
Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China
3.
University of Chinese Academy of Sciences, Beijing 100049, China

Funds: This work was supported by the National Natural Science Foundation of China (11974411), the Outstanding Young Scholars of the National Natural Science Foundation of China (12022409), the CAS Key Research Program of Frontier Sciences (ZDBS-LY-SLH015), the CAS Youth Innovation Promotion Association (2017015), and China Postdoctoral Science Foundation (2020M680728). The authors also gratefully acknowledge the support of the K. C. Wong Education Foundation.

More Information

Corresponding author: yinglu@iphy.ac.cn (Y. Lu)
Received Date: 29 July 2021
Accepted Date: 29 December 2021

Available Online: 14 March 2022

Publish Date: 31 December 2021

Abstract

Abstract

Tracking the transmembrane topology and conformational dynamics of membrane proteins is key to understand their functions. It is however challenging to monitor position changes of individual proteins in cell membranes with high sensitivity and high resolution. We review on three single-molecule fluorescence imaging methods — SIFA, LipoFRET and QueenFRET — recently developed in our lab for studying the dynamics of membrane proteins. They can be applied, progressively, to investigate membrane proteins in solid-supported lipid bilayers, artificial liposome membranes and live-cell plasma membranes. The techniques take advantage of the energy transfer from a fluorophore to a cloud of quenchers and are able to extract in real time positions and position changes of a single fluorophore-labeled protein in the direction normal to the membrane surface. The methods have sub-nanometer precision and have proved powerful to investigate biomolecules interacting with bio-membranes.
- Single-molecule fluorescence imaging,
- Dynamics of membrane proteins

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References(91)

References

[1]	Almen MS, Nordstrom KJV, Fredriksson R, Schioth HB (2009) Mapping the human membrane proteome: a majority of the human membrane proteins can be classified according to function and evolutionary origin. BMC Biology 7: 50. https://doi.org/10.1186/1741-7007-7-50
[2]	Andersson R, Safari C, Bath P, Bosman R, Shilova A, Dahl P, Ghosh S, Dunge A, Kjeldsen-Jensen R, Nan J, Shoeman RL, Kloos M, Doak RB, Mueller U, Neutze R, Branden G (2019) Well-based crystallization of lipidic cubic phase microcrystals for serial X-ray crystallography experiments. Acta Crystallogr D Struct Biol 75(Pt 10): 937-946
[3]	Asher WB, Geggier P, Holsey MD, Gilmore GT, Pati AK, Meszaros J, Terry DS, Mathiasen S, Kaliszewski MJ, McCauley MD, Govindaraju A, Zhou Z, Harikumar KG, Jaqaman K, Miller LJ, Smith AW, Blanchard SC, Javitch JA (2021) Single-molecule FRET imaging of GPCR dimers in living cells. Nat Methods 18(4): 397−405 doi: 10.1038/s41592-021-01081-y
[4]	Avelar-Freitas BA, Almeida VG, Pinto MC, Mourao FA, Massensini AR, Martins-Filho OA, Rocha-Vieira E, Brito-Melo GE (2014) Trypan blue exclusion assay by flow cytometry. Braz J Med Biol Res 47(4): 307−315 doi: 10.1590/1414-431X20143437
[5]	Basu A, Karmakar P, Karmakar S (2020) Supported planar single and multiple bilayer formation by DOPC vesicle rupture on mica substrate: a mechanism as revealed by atomic force microscopy study. J Membr Biol 253(3): 205−219 doi: 10.1007/s00232-020-00117-2
[6]	Bebarova M (2012) Advances in patch clamp technique: towards higher quality and quantity. Gen Physiol Biophys 31(2): 131−140 doi: 10.4149/gpb_2012_016
[7]	Bennett DL, Clark AJ, Huang J, Waxman SG, Dib-Hajj SD (2019) The role of voltage-gated sodium channels in pain signaling. Physiol Rev 99(2): 1079−1151 doi: 10.1152/physrev.00052.2017
[8]	Betz WJ, Angleson JK (1998) The synaptic vesicle cycle. Annu Rev Physiol 60: 347−363 doi: 10.1146/annurev.physiol.60.1.347
[9]	Bibow S, Hiller S (2019) A guide to quantifying membrane protein dynamics in lipids and other native-like environments by solution-state NMR spectroscopy. FEBS J 286(9): 1610−1623 doi: 10.1111/febs.14639
[10]	Boychuk VM, Kotsyubynsky VO, Bandura KV, Yaremiy IP, Fedorchenko SV (2019) Reduced graphene oxide obtained by Hummers and Marcano-Tour Methods: comparison of electrical properties. J Nanosci Nanotechnol 19(11): 7320−7329 doi: 10.1166/jnn.2019.16712
[11]	Bretscher MS, Raff MC (1975) Mammalian plasma membranes. Nature 258(5530): 43−49 doi: 10.1038/258043a0
[12]	Bronson JE, Fei J, Hofman JM, Gonzalez Jr RL, Wiggins CH (2009) Learning rates and states from biophysical time series: a Bayesian approach to model selection and single-molecule FRET data. Biophys J 97(12): 3196−3205 doi: 10.1016/j.bpj.2009.09.031
[13]	Calebiro D, Sungkaworn T (2018) Single-molecule imaging of GPCR interactions. Trends Pharmacol Sci 39(2): 109−122 doi: 10.1016/j.tips.2017.10.010
[14]	Caparotta M, Bustos DM, Masone D (2020) Order-disorder skewness in alpha-synuclein: a key mechanism to recognize membrane curvature. Phys Chem Chem Phys 22(9): 5255−5263 doi: 10.1039/C9CP04951G
[15]	Cheng Y (2018) Membrane protein structural biology in the era of single particle cryo-EM. Curr Opin Struct Biol 52: 58−63
[16]	Contreras FX, Sanchez-Magraner L, Alonso A, Goni FM (2010) Transbilayer (flip-flop) lipid motion and lipid scrambling in membranes. FEBS Lett 584(9): 1779−1786 doi: 10.1016/j.febslet.2009.12.049
[17]	de Lera Ruiz M, Kraus RL (2015) Voltage-gated sodium channels: structure, function, pharmacology, and clinical indications. J Med Chem 58(18): 7093−7118 doi: 10.1021/jm501981g
[18]	Diao J, Ishitsuka Y, Bae WR (2011) Single-molecule FRET study of SNARE-mediated membrane fusion. Biosci Rep 31(6): 457−463 doi: 10.1042/BSR20110011
[19]	Diao J, Su Z, Ishitsuka Y, Lu B, Lee KS, Lai Y, Shin YK, Ha T (2010) A single-vesicle content mixing assay for SNARE-mediated membrane fusion. Nat Commun 1: 54. https://doi.org/10.1038/ncomms1054
[20]	Feicht P, Biskupek J, Gorelik TE, Renner J, Halbig CE, Maranska M, Puchtler F, Kaiser U, Eigler S (2019) Brodie's or Hummers' method: oxidation conditions determine the structure of graphene oxide. Chemistry 25(38): 8955−8959 doi: 10.1002/chem.201901499
[21]	Ferhan AR, Yoon BK, Park S, Sut TN, Chin H, Park JH, Jackman JA, Cho NJ (2019) Solvent-assisted preparation of supported lipid bilayers. Nat Protoc 14(7): 2091−2118 doi: 10.1038/s41596-019-0174-2
[22]	Fessl T, Watkins D, Oatley P, Allen WJ, Corey RA, Horne J, Baldwin SA, Radford SE, Collinson I, Tuma R (2018) Dynamic action of the Sec machinery during initiation, protein translocation and termination. Elife 7: e35112. https://doi.org/10.7554/eLife.35112
[23]	Fish KN (2009) Total internal reflection fluorescence (TIRF) microscopy. Curr Protoc Cytom Chapter 12: Unit12−18
[24]	Fitzgerald GA, Terry DS, Warren AL, Quick M, Javitch JA, Blanchard SC (2019) Quantifying secondary transport at single-molecule resolution. Nature 575(7783): 528−534 doi: 10.1038/s41586-019-1747-5
[25]	Frederix PLTM, de Beer EL, Hamelink W, Gerritsen HC (2002) Dynamic Monte Carlo simulations to model FRET and photobleaching in systems with multiple donor-acceptor interactions. J Phys Chem B 106(26): 6793−6801 doi: 10.1021/jp014549b
[26]	Garcia-Nafria J, Tate CG (2020) Cryo-electron microscopy: moving beyond X-ray crystal structures for drug receptors and drug development. Annu Rev Pharmacol Toxicol 60: 51−71 doi: 10.1146/annurev-pharmtox-010919-023545
[27]	Gennis RB, Cantor CR (1972) Use of nonspecific dye labeling for singlet energy-transfer measurements in complex systems. A simple model. Biochemistry 11(13): 2509−2517 doi: 10.1021/bi00763a020
[28]	Ghosh A, Chizhik AI, Karedla N, Enderlein J (2021) Graphene- and metal-induced energy transfer for single-molecule imaging and live-cell nanoscopy with (sub)-nanometer axial resolution. Nat Protoc 16(7): 3695−3715 doi: 10.1038/s41596-021-00558-6
[29]	Ghosh A, Sharma A, Chizhik AI, Isbaner S, Ruhlandt D, Tsukanov R, Gregor I, Karedla N, Enderlein JJNP (2019) Graphene-based metal-induced energy transfer for sub-nanometre optical localization. Nat Photonics 13(12): 860−865 doi: 10.1038/s41566-019-0510-7
[30]	Goksu EI, Vanegas JM, Blanchette CD, Lin WC, Longo ML (2009) AFM for structure and dynamics of biomembranes. Biochim Biophys Acta 1788(1): 254−266 doi: 10.1016/j.bbamem.2008.08.021
[31]	Gu L, Li Y, Zhang S, Xue Y, Li W, Li D, Xu T, Ji W (2019) Molecular resolution imaging by repetitive optical selective exposure. Nat Methods 16(11): 1114−1118 doi: 10.1038/s41592-019-0544-2
[32]	Ha T, Enderle T, Ogletree DF, Chemla DS, Selvin PR, Weiss S (1996) Probing the interaction between two single molecules: Fluorescence resonance energy transfer between a single donor and a single acceptor. Proc Natl Acad Sci USA 93(13): 6264−6268 doi: 10.1073/pnas.93.13.6264
[33]	Haberl F, Lanig H, Clark T (2009) Induction of the tetracycline repressor: characterization by molecular-dynamics simulations. Proteins 77(4): 857−866 doi: 10.1002/prot.22505
[34]	Harms GS, Orr G, Montal M, Thrall BD, Colson SD, Lu HP (2003) Probing conformational changes of gramicidin ion channels by single-molecule patch-clamp fluorescence microscopy. Biophys J 85(3): 1826−1838 doi: 10.1016/S0006-3495(03)74611-6
[35]	Hellstrand E, Grey M, Ainalem ML, Ankner J, Forsyth VT, Fragneto G, Haertlein M, Dauvergne MT, Nilsson H, Brundin P, Linse S, Nylander T, Sparr E (2013) Adsorption of alpha-synuclein to supported lipid bilayers: positioning and role of electrostatics. ACS Chem Neurosci 4(10): 1339−1351 doi: 10.1021/cn400066t
[36]	Hou W, Ma D, He X, Han W, Ma J, Wang H, Xu C, Xie R, Fan Q, Ye F, Hu S, Li M, Lu Y (2021) Subnanometer-precision measurements of transmembrane motions of biomolecules in plasma membranes using quenchers in extracellular environment. Nano Lett 21(1): 485−491 doi: 10.1021/acs.nanolett.0c03941
[37]	Isbaner S, Karedla N, Kaminska I, Ruhlandt D, Raab M, Bohlen J, Chizhik A, Gregor I, Tinnefeld P, Enderlein J, Tsukanov R (2018) Axial colocalization of single molecules with nanometer accuracy using metal-induced energy transfer. Nano Lett 18(4): 2616−2622 doi: 10.1021/acs.nanolett.8b00425
[38]	Jackman JA, Cho NJ (2020) Supported lipid bilayer formation: beyond vesicle fusion. Langmuir 36(6): 1387−1400 doi: 10.1021/acs.langmuir.9b03706
[39]	Jager S, Brand L, Eggeling C (2003) New fluorescence techniques for high-throughput drug discovery. Curr Pharm Biotechnol 4(6): 463−476 doi: 10.2174/1389201033377382
[40]	Jefferson RE, Min D, Corin K, Wang JY, Bowie JU (2018) Applications of single-molecule methods to membrane protein folding studies. J Mol Biol 430(4): 424−437 doi: 10.1016/j.jmb.2017.05.021
[41]	Jensen MB, Bhatia VK, Jao CC, Rasmussen JE, Pedersen SL, Jensen KJ, Langen R, Stamou D (2011) Membrane curvature sensing by amphipathic helices: a single liposome study using alpha-synuclein and annexin B12. J Biol Chem 286(49): 42603−42614 doi: 10.1074/jbc.M111.271130
[42]	Kapanidis AN, Weiss SJTJocp (2002) Fluorescent probes and bioconjugation chemistries for single-molecule fluorescence analysis of biomolecules. J Chem Phys 117(24): 10953−10964 doi: 10.1063/1.1521158
[43]	Kim J, Cote LJ, Kim F, Huang J (2010) Visualizing graphene based sheets by fluorescence quenching microscopy. J Am Chem Soc 132(1): 260−267 doi: 10.1021/ja906730d
[44]	Krainer G, Hartmann A, Anandamurugan A, Gracia P, Keller S, Schlierf M (2018) Ultrafast protein folding in membrane-mimetic environments. J Mol Biol 430(4): 554−564 doi: 10.1016/j.jmb.2017.10.031
[45]	Krainer G, Keller S, Schlierf M (2019) Structural dynamics of membrane-protein folding from single-molecule FRET. Curr Opin Struct Biol 58: 124−137 doi: 10.1016/j.sbi.2019.05.025
[46]	Kweon DH, Kong B, Shin YK (2017) Hemifusion in synaptic vesicle cycle. Front Mol Neurosci 10: 65. https://doi.org/10.3389/fnmol.2017.00065
[47]	Latorraca NR, Venkatakrishnan AJ, Dror RO (2017) GPCR Dynamics: structures in motion. Chem Rev 117(1): 139−155 doi: 10.1021/acs.chemrev.6b00177
[48]	Lee J, Lee S, Ragunathan K, Joo C, Ha T, Hohng S (2010) Single-molecule four-color FRET. Angew Chem Int Ed Engl 122(51): 10118−10121 doi: 10.1002/ange.201005402
[49]	Leth-Larsen R, Lund RR, Ditzel HJ (2010) Plasma membrane proteomics and its application in clinical cancer biomarker discovery. Mol Cell Proteomics 9(7): 1369−1382 doi: 10.1074/mcp.R900006-MCP200
[50]	Li H, Cao X, Li B, Zhou X, Lu G, Liusman C, He Q, Boey F, Venkatraman SS, Zhang H (2011) Single-layer graphene oxide sheet: a novel substrate for dip-pen nanolithography. Chem Commun (Camb) 47(36): 10070−10072 doi: 10.1039/c1cc12648b
[51]	Li Y, Qian Z, Ma L, Hu S, Nong D, Xu C, Ye F, Lu Y, Wei G, Li M (2016) Single-molecule visualization of dynamic transitions of pore-forming peptides among multiple transmembrane positions. Nat Commun 7: 12906. https://doi.org/10.1038/ncomms12906
[52]	Lieberman RL, Peek ME, Watkins JD (2013) Determination of soluble and membrane protein structures by X-ray crystallography. Methods Mol Biol 955: 475−493
[53]	Lingwood D, Simons K (2010) Lipid rafts as a membrane-organizing principle. Science 327(5961): 46−50 doi: 10.1126/science.1174621
[54]	Ma D, Xu C, Hou W, Zhao C, Ma J, Huang X, Jia Q, Ma L, Diao J, Liu C, Li M, Lu Y (2019a) Detecting single-molecule dynamics on lipid membranes with quenchers-in-a-liposome FRET. Angew Chem Int Ed Engl 58(17): 5577−5581 doi: 10.1002/anie.201813888
[55]	Ma D, Hou W, Xu C, Zhao C, Ma J, Huang X, Jia Q, Ma L, Liu C, Li M (2020) Investigation of structure and dynamics of alpha-synuclein on membrane by quenchers-in-a-liposome fluorescence resonance energy transfer method. Acta Phys Sin 69(3): 038701. https://doi.org/10.7498/aps.69.20191607
[56]	Ma L, Hu S, He X, Yang N, Chen L, Yang C, Ye F, Wei T, Li M (2019b) Detection of tBid oligomerization and membrane permeabilization by graphene-based single-molecule surface-induced fluorescence attenuation. Nano Lett 19(10): 6937−6944 doi: 10.1021/acs.nanolett.9b02223
[57]	Ma L, Li Y, Ma J, Hu S, Li M (2018) Watching three-dimensional movements of single membrane proteins in lipid bilayers. Biochemistry 57(31): 4735−4740 doi: 10.1021/acs.biochem.8b00253
[58]	Machleidt T, Robers M, Hanson GT (2007) Protein labeling with FlAsH and ReAsH. Methods Mol Biol 356: 209−220
[59]	Mangadlao JD, Santos CM, Felipe MJ, de Leon AC, Rodrigues DF, Advincula RC (2015) On the antibacterial mechanism of graphene oxide (GO) Langmuir-Blodgett films. Chem Commun (Camb) 51(14): 2886−2889 doi: 10.1039/C4CC07836E
[60]	McKinney SA, Joo C, Ha TJ (2006) Analysis of single-molecule FRET trajectories using hidden Markov modeling. Biophys J 91(5): 1941−1951 doi: 10.1529/biophysj.106.082487
[61]	Meng X, Mezei M, Cui M (2014) Computational approaches for modeling GPCR dimerization. Curr Pharm Biotechnol 15(10): 996−1006 doi: 10.2174/1389201015666141013102515
[62]	Middleton ER, Rhoades E (2010) Effects of curvature and composition on alpha-synuclein binding to lipid vesicles. Biophys J 99(7): 2279−2288 doi: 10.1016/j.bpj.2010.07.056
[63]	Miller SE, Mathiasen S, Bright NA, Pierre F, Kelly BT, Kladt N, Schauss A, Merrifield CJ, Stamou D, Honing S, Owen DJ (2015) CALM regulates clathrin-coated vesicle size and maturation by directly sensing and driving membrane curvature. Dev Cell 33(2): 163−175 doi: 10.1016/j.devcel.2015.03.002
[64]	Milligan G, Ward RJ, Marsango S (2019) GPCR homo-oligomerization. Curr Opin Cell Biol 57: 40−47 doi: 10.1016/j.ceb.2018.10.007
[65]	Moghaddam-Taaheri P, Karlsson AJ (2018) Protein labeling in live cells for immunological applications. Bioconjug Chem 29(3): 680−685 doi: 10.1021/acs.bioconjchem.7b00722
[66]	Moon Y, Jun Y (2020) The Effects of regulatory lipids on intracellular membrane fusion mediated by dynamin-like GTPases. Front Cell Dev Biol 8: 518. https://doi.org/10.3389/fcell.2020.00518
[67]	Morandat S, Azouzi S, Beauvais E, Mastouri A, El Kirat K (2013) Atomic force microscopy of model lipid membranes. Anal Bioanal Chem 405(5): 1445−1461 doi: 10.1007/s00216-012-6383-y
[68]	Muller DJ, Sapra KT, Scheuring S, Kedrov A, Frederix PL, Fotiadis D, Engel A (2006) Single-molecule studies of membrane proteins. Curr Opin Struct Biol 16(4): 489−495 doi: 10.1016/j.sbi.2006.06.001
[69]	Nishida N, Osawa M, Takeuchi K, Imai S, Stampoulis P, Kofuku Y, Ueda T, Shimada I (2014) Functional dynamics of cell surface membrane proteins. J Magn Reson 241: 86−96 doi: 10.1016/j.jmr.2013.11.007
[70]	Prakash P, Gorfe AA (2019) Probing the conformational and energy landscapes of KRAS membrane orientation. J Phys Chem B 123(41): 8644−8652 doi: 10.1021/acs.jpcb.9b05796
[71]	Radic M, Pattanaik D (2018) Cellular and molecular mechanisms of anti-phospholipid syndrome. Front Immunol 9: 969. https://doi.org/10.3389/fimmu.2018.00969
[72]	Saheki Y, De Camilli P (2012) Synaptic vesicle endocytosis. Cold Spring Harb Perspect Biol 4(9): a005645. https://doi.org/10.1101/cshperspect.a005645
[73]	Sasmal DK, Lu HP (2014) Single-molecule patch-clamp FRET microscopy studies of NMDA receptor ion channel dynamics in living cells: revealing the multiple conformational states associated with a channel at its electrical off state. J Am Chem Soc 136(37): 12998−13005 doi: 10.1021/ja506231j
[74]	Schoch RL, Barel I, Brown FLH, Haran G (2018) Lipid diffusion in the distal and proximal leaflets of supported lipid bilayer membranes studied by single particle tracking. J Chem Phys 148(12): 123333. https://doi.org/10.1063/1.5010341
[75]	Sonnino S, Prinetti A (2013) Membrane domains and the "lipid raft" concept. Curr Med Chem 20(1): 4−21
[76]	Stahlberg H, Muller DJ, Suda K, Fotiadis D, Engel A, Meier T, Matthey U, Dimroth P (2001) Bacterial Na(+)-ATP synthase has an undecameric rotor. EMBO Rep 2(3): 229−233 doi: 10.1093/embo-reports/kve047
[77]	Strober W (2001) Trypan blue exclusion test of cell viability. Curr Protoc Immunol Appendix 3: Appendix 3B. doi: 10.1002/0471142735.ima03bs21
[78]	Tanaka Y, Schroit AJ (1983) Insertion of fluorescent phosphatidylserine into the plasma membrane of red blood cells. Recognition by autologous macrophages. J Biol Chem 258(18): 11335−11343
[79]	Thoma J, Sapra KT, Muller DJ (2018) Single-molecule force spectroscopy of transmembrane beta-barrel proteins. Annu Rev Anal Chem (Palo Alto Calif) 11(1): 375−395 doi: 10.1146/annurev-anchem-061417-010055
[80]	Thompson ST, Cass KH, Stellwagen E (1975) Blue dextran-sepharose: an affinity column for the dinucleotide fold in proteins. Proc Natl Acad Sci USA 72(2): 669−672 doi: 10.1073/pnas.72.2.669
[81]	van de Meent J-W, Bronson JE, Wiggins CH, Gonzalez Jr RL (2014) Empirical Bayes methods enable advanced population-level analyses of single-molecule FRET experiments. Biophys J 106(6): 1327−1337 doi: 10.1016/j.bpj.2013.12.055
[82]	van der Bliek AM, Shen Q, Kawajiri S (2013) Mechanisms of mitochondrial fission and fusion. Cold Spring Harb Perspect Biol 5(6): a011072. https://doi.org/10.1101/cshperspect.a011072
[83]	Volkman BF, Lipson D, Wemmer DE, Kern D (2001) Two-state allosteric behavior in a single-domain signaling protein. Science 291(5512): 2429−2433 doi: 10.1126/science.291.5512.2429
[84]	Vuojola J, Hyppanen I, Nummela M, Kankare J, Soukka T (2011) Distance and temperature dependency in nonoverlapping and conventional Forster resonance energy-transfer. J Phys Chem B 115(46): 13685−13694 doi: 10.1021/jp205564n
[85]	Wallrabe H, Periasamy A (2005) Imaging protein molecules using FRET and FLIM microscopy. Curr Opin Biotechnol 16(1): 19−27 doi: 10.1016/j.copbio.2004.12.002
[86]	Wang D, Agrawal A, Piestun R, Schwartz DK (2017) Enhanced information content for three-dimensional localization and tracking using the double-helix point spread function with variable-angle illumination epifluorescence microscopy. Appl Phys Lett 110(21): 211107. https://doi.org/10.1063/1.4984133
[87]	Wang M, Guo X, Yang X, Zhang B, Ren J, Liu A, Ran Y, Yan B, Chen F, Guddat LW, Hu J, Li J, Rao Z (2019) Mycobacterial dynamin-like protein IniA mediates membrane fission. Nat Commun 10(1): 3906. https://doi.org/10.1038/s41467-019-11860-z
[88]	Xu C, Ma W, Wang K, He K, Chen Z, Liu J, Yang K, Yuan B (2020) Correlation between single-molecule dynamics and biological functions of antimicrobial peptide melittin. J Phys Chem Lett 11(12): 4834−4841 doi: 10.1021/acs.jpclett.0c01169
[89]	Yao X, Fan X, Yan N (2020) Cryo-EM analysis of a membrane protein embedded in the liposome. Proc Natl Acad Sci USA 117(31): 18497−18503 doi: 10.1073/pnas.2009385117
[90]	Zeno WF, Thatte AS, Wang L, Snead WT, Lafer EM, Stachowiak JC (2019) Molecular mechanisms of membrane curvature sensing by a disordered protein. J Am Chem Soc 141(26): 10361−10371 doi: 10.1021/jacs.9b03927
[91]	Zhang H (2017) Thin-film hydration followed by extrusion method for liposome preparation. Methods Mol Biol 1522: 17−22