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Multistep protein unfolding during nanopore translocation

Nature Nanotechnology volume 8, pages 288–295 (2013)Cite this article

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Abstract

Cells are divided into compartments and separated from the environment by lipid bilayer membranes. Essential molecules are transported back and forth across the membranes. We have investigated how folded proteins use narrow transmembrane pores to move between compartments. During this process, the proteins must unfold. To examine co-translocational unfolding of individual molecules, we tagged protein substrates with oligonucleotides to enable potential-driven unidirectional movement through a model protein nanopore, a process that differs fundamentally from extension during force spectroscopy measurements. Our findings support a four-step translocation mechanism for model thioredoxin substrates. First, the DNA tag is captured by the pore. Second, the oligonucleotide is pulled through the pore, causing local unfolding of the C terminus of the thioredoxin adjacent to the pore entrance. Third, the remainder of the protein unfolds spontaneously. Finally, the unfolded polypeptide diffuses through the pore into the recipient compartment. The unfolding pathway elucidated here differs from those revealed by denaturation experiments in solution, for which two-state mechanisms have been proposed.

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Figure 1: Interaction of V5-C109–oligo(dC)30 with the αHL pore.
Figure 2: Voltage dependences of the rate constants for transitions between current levels.
Figure 3: Oligonucleotide insertion in step 1 → 2.
Figure 4: Voltage dependences of the rate constants for transitions between current levels in the presence of urea.
Figure 5: Effect of mutations on the rate constants for transitions between current levels.
Figure 6: Detection of events from the unfolded population in urea solution.

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References

  1. Van der Laan, M. et al. Motor-free mitochondrial presequence translocase drives membrane integration of preproteins. Nature Cell Biol. 9, 1152–1159 (2007).

    Article  CAS  Google Scholar 

  2. Krayl, M., Lim, J. H., Martin, F., Guiard, B. & Voos, W. A cooperative action of the ATP-dependent import motor complex and the inner membrane potential drives mitochondrial preprotein import. Mol. Cell Biol. 27, 411–425 (2007).

    Article  CAS  Google Scholar 

  3. Huang, S., Ratliff, K. S. & Matouschek, A. Protein unfolding by the mitochondrial membrane potential. Nature Struct. Biol. 9, 301–307 (2002).

    Article  CAS  Google Scholar 

  4. Collier, R. J. Membrane translocation by anthrax toxin. Mol. Aspects Med. 30, 413–422 (2009).

    Article  CAS  Google Scholar 

  5. Papadakos, G., Wojdyla, J. A. & Kleanthous, C. Nuclease colicins and their immunity proteins. Q. Rev. Biophys. 45, 57–103 (2012).

    Article  CAS  Google Scholar 

  6. Martin, A., Baker, T. A. & Sauer, R. T. Pore loops of the AAA+ ClpX machine grip substrates to drive translocation and unfolding. Nature Struct. Mol. Biol. 15, 1147–1151 (2008).

    Article  CAS  Google Scholar 

  7. Maillard, R. A. et al. ClpX(P) generates mechanical force to unfold and translocate its protein substrates. Cell 145, 459–469 (2011).

    Article  CAS  Google Scholar 

  8. Tian, P. & Andricioaei, I. Repetitive pulling catalyzes co-translocational unfolding of barnase during import through a mitochondrial pore. J. Mol. Biol. 350, 1017–1034 (2005).

    Article  CAS  Google Scholar 

  9. Makarov, D. E. Computer simulations and theory of protein translocation. Acc. Chem. Res. 42, 281–289 (2009).

    Article  CAS  Google Scholar 

  10. Muthukumar, M. Polymer Translocation (CRC, 2011).

  11. Rincon-Restrepo, M., Mikhailova, E., Bayley, H. & Maglia, G. Controlled translocation of individual DNA molecules through protein nanopores with engineered molecular brakes. Nano Lett. 11, 746–750 (2011).

    Article  CAS  Google Scholar 

  12. Krantz, B. A. et al. A phenylalanine clamp catalyzes protein translocation through the anthrax toxin pore. Science 309, 777–781 (2005).

    Article  CAS  Google Scholar 

  13. Basilio, D., Juris, S. J., Collier, R. J. & Finkelstein, A. Evidence for a proton–protein symport mechanism in the anthrax toxin channel. J. Gen. Physiol. 133, 307–314 (2009).

    Article  CAS  Google Scholar 

  14. Thoren, K. L., Worden, E. J., Yassif, J. M. & Krantz, B. A. Lethal factor unfolding is the most force-dependent step of anthrax toxin translocation. Proc. Natl Acad. Sci. USA 106, 21555–21560 (2009).

    Article  CAS  Google Scholar 

  15. Basilio, D., Jennings-Antipov, L. D., Jakes, K. S. & Finkelstein, A. Trapping a translocating protein within the anthrax toxin channel: implications for the secondary structure of permeating proteins. J. Gen. Physiol. 137, 343–356 (2011).

    Article  CAS  Google Scholar 

  16. Basilio, D., Kienker, P. K., Briggs, S. W. & Finkelstein, A. A kinetic analysis of protein transport through the anthrax toxin channel. J. Gen. Physiol. 137, 521–531 (2011).

    Article  CAS  Google Scholar 

  17. Simon, S. M., Peskin, C. S. & Oster, G. F. What drives the translocation of proteins? Proc. Natl Acad. Sci. USA 89, 3770–3774 (1992).

    Article  CAS  Google Scholar 

  18. Krantz, B. A., Finkelstein, A. & Collier, R. J. Protein translocation through the anthrax toxin transmembrane pore is driven by a proton gradient. J. Mol. Biol. 355, 968–979 (2006).

    Article  CAS  Google Scholar 

  19. Pentelute, B. L., Sharma, O. & Collier, R. J. Chemical dissection of protein translocation through the anthrax toxin pore. Angew Chem. Int. Ed. 50, 2294–2296 (2011).

    Article  CAS  Google Scholar 

  20. Wigelsworth, D. J. et al. Binding stoichiometry and kinetics of the interaction of a human anthrax toxin receptor, CMG2, with protective antigen. J. Biol. Chem. 279, 23349–23356 (2004).

    Article  CAS  Google Scholar 

  21. Chacinska, A., Koehler, C. M., Milenkovic, D., Lithgow, T. & Pfanner, N. Importing mitochondrial proteins: machineries and mechanisms. Cell 138, 628–644 (2009).

    Article  CAS  Google Scholar 

  22. Oukhaled, G. et al. Unfolding of proteins and long transient conformations detected by single nanopore recording. Phys. Rev. Lett. 98, 158101 (2007).

    Article  CAS  Google Scholar 

  23. Talaga, D. S. & Li, J. Single-molecule protein unfolding in solid state nanopores. J. Am. Chem. Soc. 131, 9287–9297 (2009).

    Article  CAS  Google Scholar 

  24. Payet, L. et al. Thermal unfolding of proteins probed at the single molecule level using nanopores. Anal. Chem. 84, 4071–4076 (2012).

    Article  CAS  Google Scholar 

  25. Pastoriza-Gallego, M. et al. Dynamics of unfolded protein transport through an aerolysin pore. J. Am. Chem. Soc. 133, 2923–2931 (2011).

    Article  CAS  Google Scholar 

  26. Merstorf, C. et al. Wild type, mutant protein unfolding and phase transition detected by single-nanopore recording. ACS Chem. Biol. 7, 652–658 (2012).

    Article  CAS  Google Scholar 

  27. Stefureac, R. I., Waldner, L., Howard, P. & Lee, J. S. Nanopore analysis of a small 86-residue protein. Small 4, 59–63 (2008).

    Article  CAS  Google Scholar 

  28. Pey, A. L. et al. Engineering proteins with tunable thermodynamic and kinetics stabilities. Proteins 71, 165–174 (2008).

    Article  CAS  Google Scholar 

  29. Rodriguez-Larrea, D. et al. Role of conservative mutations in protein multi-property adaption. Biochem. J. 429, 243–249 (2010).

    Article  CAS  Google Scholar 

  30. Henrickson, S. E., Misakian, M., Robertson, B. & Kasianowicz, J. J. Driven DNA transport into an asymmetric nanometer-scale pore. Phys. Rev. Lett. 85, 3057–3060 (2000).

    Article  CAS  Google Scholar 

  31. Rabin, Y. & Tanaka, M. DNA in nanopores: counterion condensation and coion depletion. Phys. Rev. Lett. 94, 148103 (2005).

    Article  Google Scholar 

  32. Zhang, J. & Shklovskii, B. I. Effective charge and free energy of DNA inside an ion channel. Phys. Rev. E 75, 021906 (2007).

    Article  Google Scholar 

  33. Dyson, H. J., Jeng, M-F., Model, P. & Holmgren, A. Characterization by 1H NMR of a C32S, C35S double mutant of Escherichia coli thioredoxin demonstrates its resemblance to the reduced wild type protein. FEBS Lett. 339, 11–17 (1994).

    Article  CAS  Google Scholar 

  34. Holmgren, A. Thioredoxin structure and mechanism: conformational changes on oxidation of the active site sulfhydryls to a disulfide. Structure 3, 239–343 (1995).

    Article  CAS  Google Scholar 

  35. Howorka, S., Cheley, S. & Bayley, H. Sequence-specific detection of individual DNA strands using engineered nanopores. Nature Biotechnol. 19, 636–639 (2001).

    Article  CAS  Google Scholar 

  36. Maglia, G., Heron, A. J., Stoddart, D., Japrung, D. & Bayley, H. Analysis of single nucleic acid molecules with protein nanopores. Methods Enzymol. 475, 591–623 (2010).

    Article  CAS  Google Scholar 

  37. Maglia, G., Restrepo, M. R., Mikhailova, E. & Bayley, H. Enhanced translocation of single DNA molecules through α-hemolysin nanopores by manipulation of internal charge. Proc. Natl Acad. Sci. USA 105, 19720–19725 (2008).

    Article  CAS  Google Scholar 

  38. Stoddart, D., Heron, A. J., Mikhailova, E., Maglia, G. & Bayley, H. Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore. Proc. Natl Acad. Sci. USA 106, 7702–7707 (2009).

    Article  CAS  Google Scholar 

  39. Japrung, D., Henricus, M., Li, Q., Maglia, G. & Bayley, H. Urea facilitates the translocation of single-stranded DNA and RNA through the α-hemolysin nanopore. Biophys. J. 98, 1856–1863 (2010).

    Article  CAS  Google Scholar 

  40. Bennion, B. J. & Daggett, V. The molecular basis for the chemical denaturation of proteins by urea. Proc. Natl Acad. Sci. USA 100, 5142–5147 (2003).

    Article  CAS  Google Scholar 

  41. Myers, J. K., Pace, C. N. & Scholtz, J. M. Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding. Protein Sci. 4, 2138–2148 (1995).

    Article  CAS  Google Scholar 

  42. Rodriguez-Larrea, D., Ibarra-Molero, B. & Sanchez-Ruiz, J. M. Energetic and structural consequences of desolvation/solvation barriers to protein folding/unfolding assessed from experimental unfolding rates. Biophys. J. 91, L48–L50 (2006).

    Article  CAS  Google Scholar 

  43. Muñoz, V. & Sanchez-Ruiz, J. M. Exploring protein-folding ensembles: a variable-barrier model for the analysis of equilibrium unfolding experiments. Proc. Natl Acad. Sci. USA 101, 17646–17651 (2004).

    Article  Google Scholar 

  44. Bayley, H. et al. in Single Molecules and Nanotechnology (eds Rigler, R. & Vogel, H.) Ch. 10, 251–277 (Springer, 2008).

    Google Scholar 

  45. Schlierf, M., Li, H. & Fernandez, J. M. The unfolding kinetics of ubiquitin captured with single-molecule force-clamp techniques. Proc. Natl Acad. Sci. USA 101, 7299–7304 (2004).

    Article  CAS  Google Scholar 

  46. Mathé, J., Visram, H., Viasnoff, V., Rabin, Y. & Meller, A. Nanopore unzipping of individual DNA hairpin molecules. Biophys. J. 87, 3205–3212 (2004).

    Article  Google Scholar 

  47. Wiggin, M., Tropini, C., Tabard-Cossa, V., Jetha, N. N. & Marziali, A. Nonexponential kinetics of DNA escape from α-hemolysin nanopores. Biophys. J. 95, 5317–5323 (2008).

    Article  CAS  Google Scholar 

  48. Matouschek, A., Pfanner, N. & Voos, W. Protein unfolding by mitochondria. The Hsp70 motor. EMBO Rep. 1, 404–410 (2000).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank E. Mikhailova for the αHL protein prepared by in vitro transcription and translation. D.R-L. is a recipient of an EMBO Long-Term Fellowship. This work was also supported by a grant from Oxford Nanopore Technologies.

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  1. Department of Chemistry, University of Oxford, Oxford, OX1 3TA, UK

    David Rodriguez-Larrea & Hagan Bayley

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D.R-L. and H.B. planned the research. D.R-L. perfomed the experiments and data analysis. D.R-L. and H.B. wrote the paper.

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Correspondence to Hagan Bayley.

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Rodriguez-Larrea, D., Bayley, H. Multistep protein unfolding during nanopore translocation. Nature Nanotech 8, 288–295 (2013). https://doi.org/10.1038/nnano.2013.22

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