Single-Molecule Protein Folding Experiments Using High-Precision Optical Tweezers

  • Junyi Jiao
  • Aleksander A. Rebane
  • Lu Ma
  • Yongli ZhangEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1486)


How proteins fold from linear chains of amino acids to delicate three-dimensional structures remains a fundamental biological problem. Single-molecule manipulation based on high-resolution optical tweezers (OT) provides a powerful approach to study protein folding with unprecedented spatiotemporal resolution. In this method, a single protein or protein complex is tethered between two beads confined in optical traps and pulled. Protein unfolding induced by the mechanical force is counteracted by the spontaneous folding of the protein, reaching a dynamic equilibrium at a characteristic force and rate. The transition is monitored by the accompanying extension change of the protein and used to derive conformations and energies of folding intermediates and their associated transition kinetics. Here, we provide general strategies and detailed protocols to study folding of proteins and protein complexes using optical tweezers, including sample preparation, DNA-protein conjugation and methods of data analysis to extract folding energies and rates from the single-molecule measurements.

Key words

Optical tweezers Single-molecule manipulation Protein folding gp41 SNARE proteins SNARE assembly Hidden Markov modeling Energy landscape 


  1. 1.
    Bukau B, Weissman J, Horwich A (2006) Molecular chaperones and protein quality control. Cell 125:443–451CrossRefGoogle Scholar
  2. 2.
    Knowles TP, Vendruscolo M, Dobson CM (2014) The amyloid state and its association with protein misfolding diseases. Nat Rev Mol Cell Biol 15:384–396CrossRefGoogle Scholar
  3. 3.
    Selkoe DJ (2012) Preventing Alzheimer’s disease. Science 337:1488–1492CrossRefGoogle Scholar
  4. 4.
    Morris ER, Searle MS (2012) In Current protocol protein science, vol Chapter 28, pp. Unit 28 22 21–22.Google Scholar
  5. 5.
    Englander SW, Mayne L (2014) The nature of protein folding pathways. Proc Natl Acad Sci U S A 111:15873–15880CrossRefGoogle Scholar
  6. 6.
    Fleming KG (2014) Energetics of membrane protein folding. Annu Rev Biophys 43:233–255CrossRefGoogle Scholar
  7. 7.
    Wand AJ (2001) Dynamic activation of protein function: a view emerging from NMR spectroscopy. Nat Struct Biol 8:926–931CrossRefGoogle Scholar
  8. 8.
    Fasshauer D, Antonin W, Subramaniam V, Jahn R (2002) SNARE assembly and disassembly exhibit a pronounced hysteresis. Nat Struct Biol 9:144–151CrossRefGoogle Scholar
  9. 9.
    Huang CY et al (2002) Helix formation via conformation diffusion search. Proc Natl Acad Sci U S A 99:2788–2793CrossRefGoogle Scholar
  10. 10.
    Dumont C, Emilsson T, Gruebele M (2009) Reaching the protein folding speed limit with large, sub-microsecond pressure jumps. Nat Methods 6:515–519CrossRefGoogle Scholar
  11. 11.
    Pobbati AV, Stein A, Fasshauer D (2006) N- to C-terminal SNARE complex assembly promotes rapid membrane fusion. Science 313:673–676CrossRefGoogle Scholar
  12. 12.
    Yeh SR, Takahashi S, Fan BC, Rousseau DL (1997) Ligand exchange during cytochrome c folding. Nat Struct Biol 4:51–56CrossRefGoogle Scholar
  13. 13.
    Ma L et al (2016) Munc18-1-regulated stage-wise SNARE assembly underlying synaptic exocytosis. eLIFE 4:e09580Google Scholar
  14. 14.
    Mashaghi A et al (2013) Reshaping of the conformational search of a protein by the chaperone trigger factor. Nature 500:98–U125CrossRefGoogle Scholar
  15. 15.
    Cecconi C, Shank EA, Bustamante C, Marqusee S (2005) Direct observation of the three-state folding of a single protein molecule. Science 309:2057–2060CrossRefGoogle Scholar
  16. 16.
    Kellermayer MSZ, Smith SB, Granzier HL, Bustamante C (1997) Folding-unfolding transitions in single titin molecules characterized with laser tweezers. Science 276:1112–1116CrossRefGoogle Scholar
  17. 17.
    Zoldak G, Rief M (2013) Force as a single molecule probe of multidimensional protein energy landscapes. Curr Opin Struct Biol 23:48–57CrossRefGoogle Scholar
  18. 18.
    Neuman KC, Block SM (2004) Optical trapping. Rev Sci Instrum 75:2787–2809CrossRefGoogle Scholar
  19. 19.
    Zhang XM, Ma L, Zhang YL (2013) High-resolution optical tweezers for single-molecule manipulation. Yale J Biol Med 86:367–383Google Scholar
  20. 20.
    Moffitt JR, Chemla YR, Smith SB, Bustamante C (2008) Recent advances in optical tweezers. Annu Rev Biochem 77:205–228CrossRefGoogle Scholar
  21. 21.
    Gittes F, Schmidt CF (1998) Interference model for back-focal-plane displacement detection in optical tweezers. Opt Lett 23:7–9CrossRefGoogle Scholar
  22. 22.
    Abbondanzieri EA, Greenleaf WJ, Shaevitz JW, Landick R, Block SM (2005) Direct observation of base-pair stepping by RNA polymerase. Nature 438:460–465CrossRefGoogle Scholar
  23. 23.
    Zoldak G, Stigler J, Pelz B, Li HB, Rief M (2013) Ultrafast folding kinetics and cooperativity of villin headpiece in single-molecule force spectroscopy. Proc Natl Acad Sci U S A 110:18156–18161CrossRefGoogle Scholar
  24. 24.
    Moffitt JR, Chemla YR, Izhaky D, Bustamante C (2006) Differential detection of dual traps improves the spatial resolution of optical tweezers. Proc Natl Acad Sci U S A 103:9006–9011CrossRefGoogle Scholar
  25. 25.
    Comstock MJ, Ha T, Chemla YR (2011) Ultrahigh-resolution optical trap with single-fluorophore sensitivity. Nat Methods 8:335–340CrossRefGoogle Scholar
  26. 26.
    Sirinakis G, Ren YX, Gao Y, Xi ZQ, Zhang YL (2012) Combined and versatile high-resolution optical tweezers and single-molecule fluorescence microscopy. Rev Sci Instrum 83:093708CrossRefGoogle Scholar
  27. 27.
    Gao Y et al (2012) Single reconstituted neuronal SNARE complexes zipper in three distinct stages. Science 337:1340–1343CrossRefGoogle Scholar
  28. 28.
    Liphardt J, Onoa B, Smith SB, Tinoco I, Bustamante C (2001) Reversible unfolding of single RNA molecules by mechanical force. Science 292:733–737CrossRefGoogle Scholar
  29. 29.
    Zhang XH, Halvorsen K, Zhang CZ, Wong WP, Springer TA (2009) Mechanoenzymatic cleavage of the ultralarge vascular protein von Willebrand factor. Science 324:1330–1334CrossRefGoogle Scholar
  30. 30.
    Shank EA, Cecconi C, Dill JW, Marqusee S, Bustamante C (2010) The folding cooperativity of a protein is controlled by its chain topology. Nature 465:637–U134CrossRefGoogle Scholar
  31. 31.
    Gebhardt JCM, Bornschlogla T, Rief M (2010) Full distance-resolved folding energy landscape of one single protein molecule. Proc Natl Acad Sci U S A 107:2013–2018CrossRefGoogle Scholar
  32. 32.
    Gao Y, Sirinakis G, Zhang YL (2011) Highly anisotropic stability and folding kinetics of a single coiled coil protein under mechanical tension. J Am Chem Soc 133:12749–12757CrossRefGoogle Scholar
  33. 33.
    Stigler J, Ziegler F, Gieseke A, Gebhardt JCM, Rief M (2011) The complex folding network of single calmodulin molecules. Science 334:512–516CrossRefGoogle Scholar
  34. 34.
    Stigler J, Rief M (2012) Calcium-dependent folding of single calmodulin molecules. Proc Natl Acad Sci U S A 109:17814–17819CrossRefGoogle Scholar
  35. 35.
    Yu H et al (2012) Direct observation of multiple misfolding pathways in a single prion protein molecule. Proc Natl Acad Sci U S A 109:5283–5288CrossRefGoogle Scholar
  36. 36.
    Jiao JY, Rebane AA, Ma L, Gao Y, Zhang YL (2015) Kinetically coupled folding of a single HIV-1 glycoprotein 41 complex in viral membrane fusion and inhibition. Proc Natl Acad Sci U S A 112:E2855–E2864CrossRefGoogle Scholar
  37. 37.
    Zorman S et al (2014) Common intermediates and kinetics, but different energetics, in the assembly of SNARE proteins. eLIFE 3:e03348CrossRefGoogle Scholar
  38. 38.
    Sudhof TC, Rothman JE (2009) Membrane fusion: grappling with SNARE and SM proteins. Science 323:474–477CrossRefGoogle Scholar
  39. 39.
    Pancera M et al (2014) Structure and immune recognition of trimeric pre-fusion HIV-1 Env. Nature 514:455–461CrossRefGoogle Scholar
  40. 40.
    Munro JB et al (2014) Conformational dynamics of single HIV-1 envelope trimers on the surface of native virions. Science 346:759–763CrossRefGoogle Scholar
  41. 41.
    Zhang YL, Sirinakis G, Gundersen G, Xi ZQ, Gao Y (2012) DNA translocation of ATP-dependent chromatin remodelling factors revealed by high-resolution optical tweezers. Methods Enzymol 513:3–28CrossRefGoogle Scholar
  42. 42.
    Wen JD et al (2007) Force unfolding kinetics of RNA using optical tweezers. I. Effects of experimental variables on measured results. Biophys J 92:2996–3009CrossRefGoogle Scholar
  43. 43.
    Xi ZQ, Gao Y, Sirinakis G, Guo HL, Zhang YL (2012) Direct observation of helix staggering, sliding, and coiled coil misfolding. Proc Natl Acad Sci U S A 109:5711–5716CrossRefGoogle Scholar
  44. 44.
    Chan DC, Fass D, Berger JM, Kim PS (1997) Core structure of gp41 from the HIV envelope glycoprotein. Cell 89:263–273CrossRefGoogle Scholar
  45. 45.
    Maillard RA et al (2011) ClpX(P) generates mechanical force to unfold and translocate its protein substrates. Cell 145:459–469CrossRefGoogle Scholar
  46. 46.
    Yu ZB et al (2012) Click chemistry assisted single-molecule fingerprinting reveals a 3D biomolecular folding funnel. J Am Chem Soc 134:12338–12341CrossRefGoogle Scholar
  47. 47.
    Sollner T et al (1993) SNAP receptors implicated in vesicle targeting and fusion. Nature 362:318–324CrossRefGoogle Scholar
  48. 48.
    Sutton RB, Fasshauer D, Jahn R, Brunger AT (1998) Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 angstrom resolution. Nature 395:347–353CrossRefGoogle Scholar
  49. 49.
    Stein A, Weber G, Wahl MC, Jahn R (2009) Helical extension of the neuronal SNARE complex into the membrane. Nature 460:525–528Google Scholar
  50. 50.
    Chattopadhaya S, Tan LP, Yao SQ (2006) Strategies for site-specific protein biotinylation using in vitro, in vivo and cell-free systems: toward functional protein arrays. Nat Protoc 1:2386–2398CrossRefGoogle Scholar
  51. 51.
    Woodside MT et al (2006) Direct measurement of the full, sequence-dependent folding landscape of a nucleic acid. Science 314:1001–1004CrossRefGoogle Scholar
  52. 52.
    Cecconi C, Shank EA, Dahlquist FW, Marqusee S, Bustamante C (2008) Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers. Eur Biophys J 37:729–738CrossRefGoogle Scholar
  53. 53.
    Cecconi C, Shank EA, Marqusee S, Bustamante C (2011) DNA molecular handles for single-molecule protein-folding studies by optical tweezers. DNA Nanotechnol 749:255–271CrossRefGoogle Scholar
  54. 54.
    Bustamante C, Marko JF, Siggia ED, Smith S (1994) Entropic elasticity of lambda-phage DNA. Science 265:1599–1600CrossRefGoogle Scholar
  55. 55.
    Marko JF, Siggia ED (1995) Stretching DNA. Macromolecules 28:8759–8770CrossRefGoogle Scholar
  56. 56.
    Rabiner LR (1989) A tutorial on hidden Markov-models and selected applications in speech recognition. Proc IEEE 77:257–286CrossRefGoogle Scholar
  57. 57.
    McKinney SA, Joo C, Ha T (2006) Analysis of single-molecule FRET trajectories using hidden Markov modeling. Biophys J 91:1941–1951CrossRefGoogle Scholar
  58. 58.
    Viterbi AJ (1967) Error bounds for convolutional codes and an asymptotically optimum decoding algorithm. IEEE Trans Inf Theory 13:260–269CrossRefGoogle Scholar
  59. 59.
    Baum LE, Petrie T, Soules G, Weiss N (1970) A maximization technique occurring in statistical analysis of probabilistic functions of Markov chains. Ann Math Stat 41:164CrossRefGoogle Scholar
  60. 60.
    Rebane AA, Ma L, Zhang YL (2016) Structure-based derivation of protein folding intermediates and energies from optical tweezers. Biophys J 110:441–454CrossRefGoogle Scholar
  61. 61.
    Woodside MT, Block SM (2014) Reconstructing folding energy landscapes by single-molecule force spectroscopy. Annu Rev Biophys 43:19–39CrossRefGoogle Scholar
  62. 62.
    Yu H et al (2012) Energy landscape analysis of native folding of the prion protein yields the diffusion constant, transition path time, and rates. Proc Natl Acad Sci U S A 109:14452–14457CrossRefGoogle Scholar
  63. 63.
    Popa I, Fernandez JM, Garcia-Manyes S (2011) Direct quantification of the attempt frequency determining the mechanical unfolding of ubiquitin protein. J Biol Chem 286:31072–31079CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Junyi Jiao
    • 1
  • Aleksander A. Rebane
    • 1
  • Lu Ma
    • 2
  • Yongli Zhang
    • 2
    Email author
  1. 1.Department of Cell Biology, School of Medicine and Integrated Graduate Program in Physical and Engineering BiologyYale UniversityNew HavenUSA
  2. 2.Department of Cell Biology, School of MedicineYale UniversityNew HavenUSA

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