Abstract
Quantitative analysis of protein complexes in their native cellular environment provides unbiased information for mechanistic understanding of various cellular processes. Particularly for protein complexes formed by transient protein-protein interactions, single cell analysis emerges as a powerful method for revealing the heterogeneity that plays important roles in spatiotemporal regulation of biological functions. In this chapter, single cell pull-down (SiCPull) is introduced for characterizing protein complexes formed in live cells at a single molecule level. This chapter starts with a brief introduction on fluorescence-based methods used for detecting protein complexes. Multidisciplinary techniques required for establishing SiCPull are elaborated with respect to surface biofunctionalization for protein capturing, micropatterning, and single molecule fluorescence analysis. Applications of SiCPull for determining the stability and stoichiometry of protein complexes are demonstrated for proteins involved in type I interferon signaling. Further applications of SiCPull are highlighted for label-free protein characterization in visible to infrared (IR) and terahertz (THz) spectral regions. At the end of the chapter, future developments toward high throughput SiCPull are discussed.
This is a preview of subscription content, log in via an institution to check access.
References
Aggarwal V, Ha T (2014) Single-molecule pull-down (SiMPull) for new-age biochemistry. BioEssays 36(11):1109–1119. https://doi.org/10.1002/bies.201400090
Agrawal A, Cho SH, Zandi O, Ghosh S, Johns RW, Milliron DJ (2018) Localized surface plasmon resonance in semiconductor nanocrystals. Chem Rev 118(6):3121–3207. https://doi.org/10.1021/acs.chemrev.7b00613
Albizu L, Cottet M, Kralikova M, Stoev S, Seyer R, Brabet I, Roux T, Bazin H, Bourrier E, Lamarque L, Breton C, Rives ML, Newman A, Javitch J, Trinquet E, Manning M, Pin JP, Mouillac B, Durroux T (2010) Time-resolved FRET between GPCR ligands reveals oligomers in native tissues. Nat Chem Biol 6(8):587–594. https://doi.org/10.1038/nchembio.396
Baumdick M, Gelléri M, Uttamapinant C, Beránek V, Chin JW, Bastiaens PIH (2018) A conformational sensor based on genetic code expansion reveals an autocatalytic component in EGFR activation. Nat Commun 9(1):3847. https://doi.org/10.1038/s41467-018-06299-7
Bettenhausen M, Römer F, Witzigmann B, Flesch J, Kurre R, Korneev S, Piehler J, You C, Kazmierczak M, Guha S, Capellini G, Schröder T (2018) Germanium plasmon enhanced resonators for label-free terahertz protein sensing. Frequenz 72(3–4):113. https://doi.org/10.1515/freq-2018-0009
Bhagawati M, You C, Piehler J (2013) Quantitative real-time imaging of protein–protein interactions by LSPR detection with micropatterned gold nanoparticles. Anal Chem 85(20):9564–9571. https://doi.org/10.1021/ac401673e
Bhagawati M, Terhorst TME, Füsser F, Hoffmann S, Pasch T, Pietrokovski S, Mootz HD (2019) A mesophilic cysteine-less split intein for protein trans-splicing applications under oxidizing conditions. Proc Natl Acad Sci U S A 116(44):22164–22172. https://doi.org/10.1073/pnas.1909825116
Bhagawati M, Hoffmann S, Höffgen KS, Piehler J, Busch KB, Mootz HD (2020) In cellulo protein semi-synthesis from endogenous and exogenous fragments using the ultra-fast split Gp41-1 intein. Angew Chem Int Ed Eng. https://doi.org/10.1002/anie.202006822
Cabukusta B, Kohlen JA, Richter CP, You C, Holthuis JC (2016) Monitoring changes in the oligomeric state of a candidate endoplasmic reticulum (ER) ceramide sensor by single-molecule photobleaching. J Biol Chem 291(47):24735–24746. https://doi.org/10.1074/jbc.M116.749812
Camp T, Mehta K, Sligar SG, Zhang K (2020) Molecular orientation determination in nanodiscs at the single-molecule level. Anal Chem 92(2):2229–2236. https://doi.org/10.1021/acs.analchem.9b04950
Cassonnet P, Rolloy C, Neveu G, Vidalain P-O, Chantier T, Pellet J, Jones L, Muller M, Demeret C, Gaud G, Vuillier F, Lotteau V, Tangy F, Favre M, Jacob Y (2011) Benchmarking a luciferase complementation assay for detecting protein complexes. Nat Methods 8(12):990–992. https://doi.org/10.1038/nmeth.1773
Chen Y, Müller JD, So PT, Gratton E (1999) The photon counting histogram in fluorescence fluctuation spectroscopy. Biophys J 77(1):553–567. https://doi.org/10.1016/s0006-3495(99)76912-2
Chen L, Holman H-YN, Hao Z, Bechtel HA, Martin MC, Wu C, Chu S (2012) Synchrotron infrared measurements of protein phosphorylation in living single PC12 cells during neuronal differentiation. Anal Chem 84(9):4118–4125. https://doi.org/10.1021/ac300308x
Choudhary D, Mossa A, Jadhav M, Cecconi C (2019) Bio-molecular applications of recent developments in optical tweezers. Biomolecules 9(1). https://doi.org/10.3390/biom9010023
Chun W, Waldo GS, Johnson GV (2011) Split GFP complementation assay for quantitative measurement of tau aggregation in situ. Methods Mol Biol 670:109–123. https://doi.org/10.1007/978-1-60761-744-0_9
Cisse I, Okumus B, Joo C, Ha T (2007) Fueling protein–DNA interactions inside porous nanocontainers. Proc Natl Acad Sci 104(31):12646–12650. https://doi.org/10.1073/pnas.0610673104
Clarke S, Pinaud F, Beutel O, You C, Piehler J, Dahan M (2010) Covalent monofunctionalization of peptide-coated quantum dots for single-molecule assays. Nano Lett 10(6):2147–2154. https://doi.org/10.1021/nl100825n
Conti Nibali V, Havenith M (2014) New insights into the role of water in biological function: studying solvated biomolecules using terahertz absorption spectroscopy in conjunction with molecular dynamics simulations. J Am Chem Soc. https://doi.org/10.1021/ja504441h
Drees C, Raj AN, Kurre R, Busch KB, Haase M, Piehler J (2016) Engineered upconversion nanoparticles for resolving protein interactions inside living cells. Angew Chem Int Ed 55(38):11668–11672. https://doi.org/10.1002/anie.201603028
Fernandez-Leiro R, Scheres SHW (2016) Unravelling biological macromolecules with cryo-electron microscopy. Nature 537(7620):339–346. https://doi.org/10.1038/nature19948
Flesch J, Kappen M, Drees C, You C, Piehler J (2020) Self-assembly of robust gold nanoparticle monolayer architectures for quantitative protein interaction analysis by LSPR spectroscopy. Anal Bioanal Chem 412(14):3413–3422. https://doi.org/10.1007/s00216-020-02551-6
Fricke F, Dietz MS, Heilemann M (2015) Single-molecule methods to study membrane receptor oligomerization. ChemPhysChem 16(4):713–721. https://doi.org/10.1002/cphc.201402765
Gaiduk A, Kühnemuth R, Antonik M, Seidel CA (2005) Optical characteristics of atomic force microscopy tips for single-molecule fluorescence applications. ChemPhysChem 6(5):976–983. https://doi.org/10.1002/cphc.200400485
Gandhi H, Worch R, Kurgonaite K, Hintersteiner M, Schwille P, Bökel C, Weidemann T (2014) Dynamics and interaction of interleukin-4 receptor subunits in living cells. Biophys J 107(11):2515–2527. https://doi.org/10.1016/j.bpj.2014.07.077
Garoli D, Yamazaki H, Maccaferri N, Wanunu M (2019) Plasmonic nanopores for single-molecule detection and manipulation: toward sequencing applications. Nano Lett 19(11):7553–7562. https://doi.org/10.1021/acs.nanolett.9b02759
Gavutis M, Lata S, Lamken P, Müller P, Piehler J (2005) Lateral ligand-receptor interactions on membranes probed by simultaneous fluorescence-interference detection. Biophys J 88(6):4289–4302
Gavutis M, Lata S, Piehler J (2006) Probing 2-dimensional protein–protein interactions on model membranes. Nat Protoc 1:2091. https://doi.org/10.1038/nprot.2006.270
Hansen S, Stüber JC, Ernst P, Koch A, Bojar D, Batyuk A, Plückthun A (2017) Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity. Sci Rep 7(1):16292. https://doi.org/10.1038/s41598-017-15711-z
Helma J, Cardoso MC, Muyldermans S, Leonhardt H (2015) Nanobodies and recombinant binders in cell biology. J Cell Biol 209(5):633–644. https://doi.org/10.1083/jcb.201409074
Howorka S, Hesse J (2014) Microarrays and single molecules: an exciting combination. Soft Matter 10(7):931–941. https://doi.org/10.1039/C3SM52561A
Hughes AJ, Spelke DP, Xu Z, Kang C-C, Schaffer DV, Herr AE (2014) Single-cell western blotting. Nat Methods advance online publication. https://doi.org/10.1038/nmeth.2992
Jain A, Liu R, Ramani B, Arauz E, Ishitsuka Y, Ragunathan K, Park J, Chen J, Xiang YK, Ha T (2011) Probing cellular protein complexes using single-molecule pull-down. Nature 473(7348):484–488. http://www.nature.com/nature/journal/v473/n7348/abs/10.1038-nature10016-unlocked.html#supplementary-information
Jain A, Arauz E, Aggarwal V, Ikon N, Chen J, Ha T (2014) Stoichiometry and assembly of mTOR complexes revealed by single-molecule pulldown. Proc Natl Acad Sci 111(50):17833–17838. https://doi.org/10.1073/pnas.1419425111
Kam LC, Shen K, Dustin ML (2013) Micro- and nanoscale engineering of cell signaling. Annu Rev Biomed Eng 15(1):305–326. https://doi.org/10.1146/annurev-bioeng-071811-150050
Kang C-C, Lin J-MG, Xu Z, Kumar S, Herr AE (2014) Single-cell western blotting after whole-cell imaging to assess cancer chemotherapeutic response. Anal Chem 86(20):10429–10436. https://doi.org/10.1021/ac502932t
Kazmierczak M, Flesch J, Mitzloff J, Capellini G, Klesse WM, Skibitzki O, You C, Bettenhausen M, Witzigmann B, Piehler J, Schroeder T, Guha S (2018) Stable and selective self-assembly of α-lipoic acid on Ge(001) for biomolecule immobilization. J Appl Phys 123(17):175305. https://doi.org/10.1063/1.5022532
Keppler A, Pick H, Arrivoli C, Vogel H, Johnsson K (2004) Labeling of fusion proteins with synthetic fluorophores in live cells. Proc Natl Acad Sci U S A 101(27):9955–9959. https://doi.org/10.1073/pnas.0401923101
Kirchhofer A, Helma J, Schmidthals K, Frauer C, Cui S, Karcher A, Pellis M, Muyldermans S, Casas-Delucchi CS, Cardoso MC, Leonhardt H, Hopfner KP, Rothbauer U (2010) Modulation of protein properties in living cells using nanobodies. Nat Struct Mol Biol 17(1):133–138. https://doi.org/10.1038/nsmb.1727
Kosol S, Contreras-Martos S, Cedeno C, Tompa P (2013) Structural characterization of intrinsically disordered proteins by NMR spectroscopy. Molecules 18(9):10802–10828. https://doi.org/10.3390/molecules180910802
Lee H-W, Kyung T, Yoo J, Kim T, Chung C, Ryu JY, Lee H, Park K, Lee S, Jones WD, Lim D-S, Hyeon C, Do Heo W, Yoon T-Y (2013) Real-time single-molecule co-immunoprecipitation analyses reveal cancer-specific Ras signalling dynamics. Nat Commun 4:1505. http://www.nature.com/ncomms/journal/v4/n2/suppinfo/ncomms2507_S1.html
Leonetti MD, Sekine S, Kamiyama D, Weissman JS, Huang B (2016) A scalable strategy for high-throughput GFP tagging of endogenous human proteins. Proc Natl Acad Sci 113(25):E3501–E3508. https://doi.org/10.1073/pnas.1606731113
Löchte S, Waichman S, Beutel O, You C, Piehler J (2014) Live cell micropatterning reveals the dynamics of signaling complexes at the plasma membrane. J Cell Biol 207(3):407–418. https://doi.org/10.1083/jcb.201406032
Los GV, Encell LP, McDougall MG, Hartzell DD, Karassina N, Zimprich C, Wood MG, Learish R, Ohana RF, Urh M, Simpson D, Mendez J, Zimmerman K, Otto P, Vidugiris G, Zhu J, Darzins A, Klaubert DH, Bulleit RF, Wood KV (2008) HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol 3(6):373–382. https://doi.org/10.1021/cb800025k
MacBeath G, Schreiber SL (2000) Printing proteins as microarrays for high-throughput function determination. Science 289(5485):1760–1763. https://doi.org/10.1126/science.289.5485.1760
Marx V (2017) Probes: FRET sensor design and optimization. Nat Methods 14(10):949–953. https://doi.org/10.1038/nmeth.4434
Medina MÁ, Schwille P (2002) Fluorescence correlation spectroscopy for the detection and study of single molecules in biology. BioEssays 24(8):758–764. https://doi.org/10.1002/bies.10118
Medintz IL, Clapp AR, Mattoussi H, Goldman ER, Fisher B, Mauro JM (2003) Self-assembled nanoscale biosensors based on quantum dot FRET donors. Nat Mater 2(9):630–638. https://doi.org/10.1038/nmat961
Mihoubi W, Sahli E, Gargouri A, Amiel C (2017) FTIR spectroscopy of whole cells for the monitoring of yeast apoptosis mediated by p53 over-expression and its suppression by Nigella sativa extracts. PLoS One 12(7):e0180680. https://doi.org/10.1371/journal.pone.0180680
Neubrech F, Huck C, Weber K, Pucci A, Giessen H (2017) Surface-enhanced infrared spectroscopy using resonant nanoantennas. Chem Rev 117(7):5110–5145. https://doi.org/10.1021/acs.chemrev.6b00743
Overington JP, Al-Lazikani B, Hopkins AL (2006) How many drug targets are there? Nat Rev Drug Discov 5(12):993–996. https://doi.org/10.1038/nrd2199
Philippi M, You C, Richter CP, Schmidt M, Thien J, Liße D, Wollschläger J, Piehler J, Steinhart M (2019) Close-packed silane nanodot arrays by capillary nanostamping coupled with heterocyclic silane ring opening. RSC Adv 9(43):24742–24750. https://doi.org/10.1039/C9RA03440D
Phizicky E, Bastiaens PIH, Zhu H, Snyder M, Fields S (2003) Protein analysis on a proteomic scale. Nature 422(6928):208–215
Piehler J, Brecht A, Valiokas R, Liedberg B, Gauglitz G (2000) A high-density poly(ethylene glycol) polymer brush for immobilization on glass-type surfaces. Biosens Bioelectron 15(9–10):473–481
Pleiner T, Bates M, Görlich D (2017) A toolbox of anti–mouse and anti–rabbit IgG secondary nanobodies. J Cell Biol 217(3):1143–1154. https://doi.org/10.1083/jcb.201709115
Salazar-Cavazos E, Nitta CF, Mitra ED, Wilson BS, Lidke KA, Hlavacek WS, Lidke DS (2020) Multisite EGFR phosphorylation is regulated by adaptor protein abundances and dimer lifetimes. Mol Biol Cell 31(7):695–708. https://doi.org/10.1091/mbc.E19-09-0548
Schwab EH, Pohl TL, Haraszti T, Schwaerzer GK, Hiepen C, Spatz JP, Knaus P, Cavalcanti-Adam EA (2015) Nanoscale control of surface immobilized BMP-2: toward a quantitative assessment of BMP-mediated signaling events. Nano Lett 15(3):1526–1534. https://doi.org/10.1021/acs.nanolett.5b00315
Shaner NC, Lambert GG, Chammas A, Ni Y, Cranfill PJ, Baird MA, Sell BR, Allen JR, Day RN, Israelsson M, Davidson MW, Wang J (2013) A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat Methods 10(5):407–409. https://doi.org/10.1038/nmeth.2413
Tiede C, Bedford R, Heseltine SJ, Smith G, Wijetunga I, Ross R, AlQallaf D, Roberts APE, Balls A, Curd A, Hughes RE, Martin H, Needham SR, Zanetti-Domingues LC, Sadigh Y, Peacock TP, Tang AA, Gibson N, Kyle H, Platt GW, Ingram N, Taylor T, Coletta LP, Manfield I, Knowles M, Bell S, Esteves F, Maqbool A, Prasad RK, Drinkhill M, Bon RS, Patel V, Goodchild SA, Martin-Fernandez M, Owens RJ, Nettleship JE, Webb ME, Harrison M, Lippiat JD, Ponnambalam S, Peckham M, Smith A, Ferrigno PK, Johnson M, McPherson MJ, Tomlinson DC (2017) Affimer proteins are versatile and renewable affinity reagents. elife 6:e24903. https://doi.org/10.7554/eLife.24903
Ulbrich MH, Isacoff EY (2007) Subunit counting in membrane-bound proteins. Nat Methods 4(4):319–321. http://www.nature.com/nmeth/journal/v4/n4/suppinfo/nmeth1024_S1.html
Urizar E, Yano H, Kolster R, Galés C, Lambert N, Javitch JA (2011) CODA-RET reveals functional selectivity as a result of GPCR heteromerization. Nat Chem Biol 7(9):624–630. https://doi.org/10.1038/nchembio.623
Wang X, Li S, Yan C, Liu P, Ding J (2015) Fabrication of RGD micro/nanopattern and corresponding study of stem cell differentiation. Nano Lett 15(3):1457–1467. https://doi.org/10.1021/nl5049862
Waichman S, You C, Beutel O, Bhagawati M, Piehler J (2011) Maleimide photolithography for single-molecule protein-protein interaction analysis in micropatterns. Anal Chem 83(2):501–508. https://doi.org/10.1021/ac1021453
Wedeking T, Löchte S, Richter CP, Bhagawati M, Piehler J, You C (2015) Single cell GFP-trap reveals stoichiometry and dynamics of cytosolic protein complexes. Nano Lett 15(5):3610–3615. https://doi.org/10.1021/acs.nanolett.5b01153
Weidemann T, Mücksch J, Schwille P (2014) Fluorescence fluctuation microscopy: a diversified arsenal of methods to investigate molecular dynamics inside cells. Curr Opin Struct Biol 28:69–76. https://doi.org/10.1016/j.sbi.2014.07.008
Wenta N, Strauss H, Meyer S, Vinkemeier U (2008) Tyrosine phosphorylation regulates the partitioning of STAT1 between different dimer conformations. Proc Natl Acad Sci 105(27):9238–9243. https://doi.org/10.1073/pnas.0802130105
Wilmes S, Hafer M, Vuorio J, Tucker JA, Winkelmann H, Löchte S, Stanly TA, Pulgar Prieto KD, Poojari C, Sharma V, Richter CP, Kurre R, Hubbard SR, Garcia KC, Moraga I, Vattulainen I, Hitchcock IS, Piehler J (2020) Mechanism of homodimeric cytokine receptor activation and dysregulation by oncogenic mutations. Science 367(6478):643–652. https://doi.org/10.1126/science.aaw3242
Yang J, Stark GR (2008) Roles of unphosphorylated STATs in signaling. Cell Res 18(4):443–451. https://doi.org/10.1038/cr.2008.41
Yeom KH, Heo I, Lee J, Hohng S, Kim VN, Joo C (2011) Single-molecule approach to immunoprecipitated protein complexes: insights into miRNA uridylation. EMBO Rep 12(7):690–696. https://doi.org/10.1038/embor.2011.100
You C, Piehler J (2016) Functional protein micropatterning for drug design and discovery. Expert Opin Drug Discovery 11(1):105–119. https://doi.org/10.1517/17460441.2016.1109625
You C, Wilmes S, Beutel O, Lochte S, Podoplelowa Y, Roder F, Richter C, Seine T, Schaible D, Uze G, Clarke S, Pinaud F, Dahan M, Piehler J (2010) Self-controlled monofunctionalization of quantum dots for multiplexed protein tracking in live cells. Angew Chem Int Ed Eng 49(24):4108–4112. https://doi.org/10.1002/anie.200907032
You C, Wilmes S, Richter CP, Beutel O, Lisse D, Piehler J (2013) Electrostatically controlled quantum dot monofunctionalization for interrogating the dynamics of protein complexes in living cells. ACS Chem Biol 8(2):320–326. https://doi.org/10.1021/cb300543t
You C, Richter CP, Löchte S, Wilmes S, Piehler J (2014) Dynamic submicroscopic signaling zones revealed by pair correlation tracking and localization microscopy. Anal Chem 86(17):8593–8602. https://doi.org/10.1021/ac501127r
Zeug A, Woehler A, Neher E, Ponimaskin EG (2012) Quantitative intensity-based FRET approaches–a comparative snapshot. Biophys J 103(9):1821–1827. https://doi.org/10.1016/j.bpj.2012.09.031
Zheng Z, Daniel WL, Giam LR, Huo F, Senesi AJ, Zheng G, Mirkin CA (2009) Multiplexed protein arrays enabled by polymer pen lithography: addressing the inking challenge. Angew Chem Int Ed 48(41):7626–7629. https://doi.org/10.1002/anie.200902649
Zhu H, Bilgin M, Bangham R, Hall D, Casamayor A, Bertone P, Lan N, Jansen R, Bidlingmaier S, Houfek T, Mitchell T, Miller P, Dean RA, Gerstein M, Snyder M (2001) Global analysis of protein activities using proteome chips. Science 293(5537):2101–2105. https://doi.org/10.1126/science.1062191
Acknowledgment
The authors thank the intramural funding from Osnabrück University within the profile line “Integrated Science” and funding from NSFC (31870976) for support.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Nature Singapore Pte Ltd
About this entry
Cite this entry
Philippi, M., Li, Z., Bhagawati, M., You, C. (2021). Single Cell Pull-Down for Characterization of Protein Complexes. In: Santra, T.S., Tseng, FG. (eds) Handbook of Single Cell Technologies. Springer, Singapore. https://doi.org/10.1007/978-981-10-4857-9_43-1
Download citation
DOI: https://doi.org/10.1007/978-981-10-4857-9_43-1
Received:
Accepted:
Published:
Publisher Name: Springer, Singapore
Print ISBN: 978-981-10-4857-9
Online ISBN: 978-981-10-4857-9
eBook Packages: Springer Reference EngineeringReference Module Computer Science and Engineering