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High-Speed Force Spectroscopy for Single Protein Unfolding

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Nanoscale Imaging

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1814))

Abstract

Single-molecule force spectroscopy (SMFS) measurements allow for quantification of the molecular forces required to unfold individual protein domains. Atomic force microscopy (AFM) is one of the long-established techniques for force spectroscopy (FS). Although FS at conventional AFM pulling rates provides valuable information on protein unfolding, in order to get a more complete picture of the mechanism, explore new regimes, and combine and compare experiments with simulations, we need higher pulling rates and μs-time resolution, now accessible via high-speed force spectroscopy (HS-FS). In this chapter, we provide a step-by-step protocol of HS-FS including sample preparation, measurements and analysis of the acquired data using HS-AFM with an illustrative example on unfolding of a well-studied concatamer made of eight repeats of the titin I91 domain.

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References

  1. Kubelka J, Hofrichter J, Eaton WA (2004) The protein folding ‘speed limit’. Curr Opin Struct Biol 14(1):76–88

    Article  CAS  PubMed  Google Scholar 

  2. Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD (1994) Molecular biology of the cell, 3rd edn. Garland Publishing, New York

    Google Scholar 

  3. Bryngelson JD, Onuchic JN, Socci ND, Wolynes PG (1995) Funnels, pathways, and the energy landscape of protein folding: a synthesis. Proteins 21(3):167–195. https://doi.org/10.1002/prot.340210302

    Article  PubMed  CAS  Google Scholar 

  4. Kellermayer MS, Smith SB, Granzier HL, Bustamante C (1997) Folding-unfolding transitions in single titin molecules characterized with laser tweezers. Science 276(5315):1112–1116. https://doi.org/10.1126/science.276.5315.1112

    Article  PubMed  CAS  Google Scholar 

  5. Rief M, Gautel M, Oesterhelt F, Fernandez JM, Gaub HE (1997) Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276:1109–1112. https://doi.org/10.1126/science.276.5315.1109

    Article  CAS  PubMed  Google Scholar 

  6. del Rio A, Perez-Jimenez R, Liu R, Roca-Cusachs P, Fernandez JM, Sheetz MP (2009) Stretching single talin rod molecules activates vinculin binding. Science 323(5914):638–641. https://doi.org/10.1126/science.1162912

    Article  PubMed  CAS  Google Scholar 

  7. Johnson CP, Tang H-Y, Carag C, Speicher DW, Discher DE (2007) Forced unfolding of proteins within cells. Science 317(5838):663–666. https://doi.org/10.1126/science.1139857

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Dobson CM (2003) Protein folding and misfolding. Nature 426(6968):884–890

    Article  CAS  PubMed  Google Scholar 

  9. Schwaiger I, Kardinal A, Schleicher M, Noegel AA, Rief M (2004) A mechanical unfolding intermediate in an actin-crosslinking protein. Nat Struct Mol Biol 11(1):81–85

    Article  CAS  PubMed  Google Scholar 

  10. Marszalek PE, Lu H, Li H, Carrion-Vazquez M, Oberhauser aF, Schulten K, Fernandez JM (1999) Mechanical unfolding intermediates in titin modules. Nature 402(November):100–103. https://doi.org/10.1038/47083

    Article  CAS  PubMed  Google Scholar 

  11. Oberhauser AF, Hansma PK, Carrion-Vazquez M, Fernandez JM (2001) Stepwise unfolding of titin under force-clamp atomic force microscopy. Proc Natl Acad Sci U S A 98(2):468–472

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Oesterhelt F, Oesterhelt D, Pfeiffer M, Engel A, Gaub HE, Müller DJ (2000) Unfolding pathways of individual bacteriorhodopsins. Science 288(5463):143–146. https://doi.org/10.1126/science.288.5463.143

    Article  CAS  PubMed  Google Scholar 

  13. Woodside MT, Block SM (2014) Reconstructing folding energy landscapes by single-molecule force spectroscopy. Annu Rev Biophys 43:19

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Neupane K, Foster DA, Dee DR, Yu H, Wang F, Woodside MT (2016) Direct observation of transition paths during the folding of proteins and nucleic acids. Science 352(6282):239–242

    Article  CAS  PubMed  Google Scholar 

  15. Hughes ML, Dougan L (2016) The physics of pulling polyproteins: a review of single molecule force spectroscopy using the AFM to study protein unfolding. Rep Prog Phys 79(7):076601. https://doi.org/10.1088/0034-4885/79/7/076601

    Article  PubMed  CAS  Google Scholar 

  16. Ando T, Kodera N, Naito Y, Kinoshita T, Ky F, Toyoshima YY (2003) A high-speed atomic force microscope for studying biological macromolecules in action. ChemPhysChem 4(11):1196–1202. https://doi.org/10.1002/cphc.200300795

    Article  PubMed  CAS  Google Scholar 

  17. Viani MB, Schaffer TE, Chand A, Rief M, Gaub HE, Hansma PK (1999) Small cantilevers for force spectroscopy of single molecules. J Appl Phys 86:2258–2262

    Article  CAS  Google Scholar 

  18. Rico F, Gonzalez L, Casuso I, Puig-vidal M, Scheuring S (2013) High-speed force spectroscopy molecular dynamics simulations. Science 342:741–743. https://doi.org/10.1126/science.1239764

    Article  PubMed  CAS  Google Scholar 

  19. Yu H, Siewny MG, Edwards DT, Sanders AW, Perkins TT (2017) Hidden dynamics in the unfolding of individual bacteriorhodopsin proteins. Science 355(6328):945–950. https://doi.org/10.1126/science.aah7124

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Rigato A, Miyagi A, Scheuring S, Rico F (2017) High-frequency microrheology reveals cytoskeleton dynamics in living cells. Nat Phys 13:771–775

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Edwards DT, Faulk JK, Sanders AW, Bull MS, Walder R, LeBlanc MA, Sousa MC, Perkins TT (2015) Optimizing 1-mus-resolution single-molecule force spectroscopy on a commercial atomic force microscope. Nano Lett 15(10):7091–7098. https://doi.org/10.1021/acs.nanolett.5b03166

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Kassies R, van der Werf KO, Bennink ML, Otto C (2004) Removing interference and optical feedback artifacts in atomic force microscopy measurements by application of high frequency laser current modulation. Rev Sci Instrum 75(3):689–693. https://doi.org/10.1063/1.1646767

    Article  CAS  Google Scholar 

  23. Carrion-Vazquez M, Oberhauser AF, Fowler SB, Marszalek PE, Broedel SE, Clarke J, Fernandez JM (1999) Mechanical and chemical unfolding of a single protein: a comparison. Proc Natl Acad Sci 96(7):3694–3699. https://doi.org/10.1073/pnas.96.7.3694

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Ando T, Kodera N, Takai E, Maruyama D, Saito K, Toda A (2001) A high-speed atomic force microscope for studying biological macromolecules. Proc Natl Acad Sci 98(22):12468–12472. https://doi.org/10.1073/pnas.211400898

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Zakeri B, Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M (2012) Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A 109(12):E690–E697. https://doi.org/10.1073/pnas.1115485109

    Article  PubMed  PubMed Central  Google Scholar 

  26. Otten M, Ott W, Jobst MA, Milles LF, Verdorfer T, Pippig DA, Nash MA, Gaub HE (2014) From genes to protein mechanics on a chip. Nat Methods 11(11):1127–1130. https://doi.org/10.1038/nmeth.3099

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Schoeler C, Malinowska KH, Bernardi RC, Milles LF, Jobst MA, Durner E, Ott W, Fried DB, Bayer EA, Schulten K (2014) Ultrastable cellulosome-adhesion complex tightens under load. Nat Commun 5:5635

    Article  CAS  PubMed  Google Scholar 

  28. Pippig DA, Baumann F, Strackharn M, Aschenbrenner D, Gaub HE (2014) Protein-DNA chimeras for nano assembly. ACS Nano 8(7):6551–6555. https://doi.org/10.1021/nn501644w

    Article  PubMed  CAS  Google Scholar 

  29. Popa I, Berkovich R, Alegre-Cebollada J, Badilla CL, Rivas-Pardo JA, Taniguchi Y, Kawakami M, Fernandez JM (2013) Nanomechanics of HaloTag tethers. J Am Chem Soc 135(34):12762–12771. https://doi.org/10.1021/ja4056382

    Article  PubMed  CAS  Google Scholar 

  30. Zimmermann JL, Nicolaus T, Neuert G, Blank K (2010) Thiol-based, site-specific and covalent immobilization of biomolecules for single-molecule experiments. Nat Protoc 5(6):975–985. https://doi.org/10.1038/nprot.2010.49

    Article  CAS  PubMed  Google Scholar 

  31. Yin J, Lin AJ, Golan DE, Walsh CT (2006) Site-specific protein labeling by Sfp phosphopantetheinyl transferase. Nat Protoc 1(1):280–285. https://doi.org/10.1038/nprot.2006.43

    Article  PubMed  CAS  Google Scholar 

  32. Durner E, Ott W, Nash MA, Gaub HE (2017) Post-translational sortase-mediated attachment of high-strength force spectroscopy handles. ACS Omega 2(6):3064–3069. https://doi.org/10.1021/acsomega.7b00478

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Sumbul F, Marchesi A, Rico F (2018) History, rare, and multiple events of mechanical unfolding of repeat proteins, The Journal of chemical physics, 148:12 https://doi.org/10.1063/1.5013259

    Article  CAS  PubMed  Google Scholar 

  34. Zhang X, Rico F, Xu AJ, Moy VT (2009) Atomic force microscopy of protein–protein interactions. Springer, New York, NY, pp 555–570. https://doi.org/10.1007/978-0-387-76497-9_19

    Book  Google Scholar 

  35. Hutter JL, Bechhoefer J (1993) Calibration of atomic-force microscope tips. Rev Sci Instrum 64(7):1868–1873

    Article  CAS  Google Scholar 

  36. Butt HJ, Jaschke M (1995) Calculation of thermal noise in atomic force microscopy. Nanotechnology 6:1–7

    Article  Google Scholar 

  37. Sader JE, Chon JWM, Mulvaney P (1999) Calibration of rectangular atomic force microscope cantilevers. Rev Sci Instrum 70(10):3967–3969. https://doi.org/10.1063/1.1150021

    Article  CAS  Google Scholar 

  38. Sader JE, Larson I, Mulvaney P, White L (1995) Method for the calibration of atomic force microscope cantilevers. Rev Sci Instrum 66(7):3789–3798

    Article  CAS  Google Scholar 

  39. Higgins MJ, Proksch R, Sader JE, Polcik M, Mc Endoo S, Cleveland JP, Jarvis SP (2006) Noninvasive determination of optical lever sensitivity in atomic force microscopy. Rev Sci Instrum 77(1):1–5. https://doi.org/10.1063/1.2162455

    Article  CAS  Google Scholar 

  40. Schillers H, Rianna C, Schäpe J, Luque T, Doschke H, Wälte M, Uriarte JJ, Campillo N, Michanetzis GPA, Bobrowska J, Dumitru A, Herruzo ET, Bovio S, Parot P, Galluzzi M, Podestà A, Puricelli L, Scheuring S, Missirlis Y, Garcia R, Odorico M, Teulon J-M, Lafont F, Lekka M, Rico F, Rigato A, Pellequer J-L, Oberleithner H, Navajas D, Radmacher M (2017) Standardized nanomechanical atomic force microscopy procedure (SNAP) for measuring soft and biological samples. Sci Rep 7(1):5117. https://doi.org/10.1038/s41598-017-05383-0

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Sader JE, Borgani R, Gibson CT, Haviland DB, Higgins MJ, Kilpatrick J, Lu, J, Mulvaney, P, Shearer, CJ, Slattery, AD, ThorÕn, P, Tran, J, Zhang H, Zheng T (2016) A virtual instrument to standardise the calibration of atomic force microscope cantilevers. Review of Scientific Instruments, 87 093711-1-093711-14

    Google Scholar 

  42. Janovjak H, Struckmeier J, Müller DJ (2005) Hydrodynamic effects in fast AFM single-molecule force measurements. Eur Biophys J 34(1):91–96. https://doi.org/10.1007/s00249-004-0430-3

    Article  CAS  PubMed  Google Scholar 

  43. Alcaraz J, Buscemi L, Puig-de-Morales M, Colchero J, Baro A, Navajas D (2002) Correction of microrheological measurements of soft samples with atomic force microscopy for the hydrodynamic drag on the cantilever. Langmuir 18(3):716–721

    Article  CAS  Google Scholar 

  44. Bell G (1978) Models for the specific adhesion of cells to cells. Science 200(4342):618–627. https://doi.org/10.1126/science.347575

    Article  PubMed  CAS  Google Scholar 

  45. Evans E, Ritchie K (1997) Dynamic strength of molecular adhesion bonds. Biophys J 72:1541–1555. https://doi.org/10.1016/S0006-3495(97)78802-7

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Dudko OK, Hummer G, Szabo A (2006) Intrinsic rates and activation free energies from single-molecule pulling experiments. Phys Rev Lett 96(10):108101. https://doi.org/10.1103/PhysRevLett.96.108101

    Article  PubMed  CAS  Google Scholar 

  47. Friddle RW, Noy a DYJJ (2012) Interpreting the widespread nonlinear force spectra of intermolecular bonds. Proc Natl Acad Sci 109(34):13573–13578. https://doi.org/10.1073/pnas.1202946109

    Article  PubMed  PubMed Central  Google Scholar 

  48. Evans E, Ritchie K (1999) Strength of a weak bond connecting flexible polymer chains. Biophys J 76(5):2439–2447. https://doi.org/10.1016/S0006-3495(99)77399-6

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Maitra A, Arya G (2010) Model accounting for the effects of pulling-device stiffness in the analyses of single-molecule force measurements. Phys Rev Lett 104(10):108301. https://doi.org/10.1103/PhysRevLett.104.108301

    Article  PubMed  Google Scholar 

  50. Bullerjahn JT, Sturm S, Kroy K (2014) Theory of rapid force spectroscopy. Nat Commun 5:4463. https://doi.org/10.1038/ncomms5463

    Article  PubMed  CAS  Google Scholar 

  51. Hummer G, Szabo A (2003) Kinetics from nonequilibrium single-molecule pulling experiments. Biophys J 85(1):5–15

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

This work was supported by Agence National de la Recherche grants BioHSFS ANR-15-CE11-0007 and ANR-11-LABX-0054 (Labex INFORM). A.M. was supported by a Long Term EMBO Fellowship (ALTF 1427-2014) and a Marie Curie Action (MSCA-IF-2014-EF-655157). We thank Michael Nash and Wolfgang Ott for sharing the XMod-dockerin-III and cohesin-III plasmids and for technical assistance.

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Authors and Affiliations

  1. LAI, Aix-Marseille Université, INSERM UMR_S 1067, CNRS UMR 7333, 13009, Marseille, France

    Fidan Sumbul, Arin Marchesi & Felix Rico

  2. Department of Anesthesiology, Department of Physiology and Biophysics, Weill Cornell Medical College, New York, NY, USA

    Hirohide Takahashi & Simon Scheuring

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  1. Fidan Sumbul
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  2. Arin Marchesi
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  3. Hirohide Takahashi
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  4. Simon Scheuring
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  5. Felix Rico
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Correspondence to Felix Rico .

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Editors and Affiliations

  1. Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska, USA

    Yuri L. Lyubchenko

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Sumbul, F., Marchesi, A., Takahashi, H., Scheuring, S., Rico, F. (2018). High-Speed Force Spectroscopy for Single Protein Unfolding. In: Lyubchenko, Y. (eds) Nanoscale Imaging. Methods in Molecular Biology, vol 1814. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8591-3_15

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  • DOI: https://doi.org/10.1007/978-1-4939-8591-3_15

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  • Print ISBN: 978-1-4939-8590-6

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