Skip to main content
SpringerLink
Log in

Mechanically Watching the ClpXP Proteolytic Machinery

  • Protocol
  • First Online:
Optical Tweezers

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1486))

Abstract

Energy-dependent protein degradation is studied through the dual bead ClpXP motility assay. Processing of folded proteins involves recognition, unfolding, translocation, and degradation stages. A dual optical trap, in a passive force-clamp geometry, exhibits bead-to-bead displacements that directly follow subprocesses underlying protein degradation. Discrete nanometer-scale displacements of the bead position reveal steps, dwells and pauses during the unfolding and translocation substeps. With a few structural modifications to the protease machinery and an engineered substrate, the assay represents a “chassis” for the measurement of a wide range of substrates and related machinery. The methods described faithfully record our assay as implemented, including substrate design, wet assay preparation, and the motility assay experiment protocol. The strategies herein permit adaptation of the ClpXP mechanical assay to a wide range of protein degradation systems.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 179.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Olivares AO, Baker TA, Sauer RT (2016) Mechanistic insights into bacterial AAA+ proteases and protein-remodelling machines. Nat Rev Microbiol 14:33–44

    Article  CAS  Google Scholar 

  2. Sauer RT, Baker TA (2011) AAA+ proteases: ATP-fueled machines of protein destruction. Annu Rev Biochem 80:587–612

    Article  CAS  Google Scholar 

  3. Striebel F, Kress W, Weber-Ban E (2009) Controlled destruction: AAA+ ATPases in protein degradation from bacteria to eukaryotes. Curr Opin Struct Biol 19:209–217

    Article  CAS  Google Scholar 

  4. Wendler P, Ciniawsky S, Kock M, Kube S (2012) Structure and function of the AAA+ nucleotide binding pocket. Biochim Biophys Acta 1823:2–14

    Article  CAS  Google Scholar 

  5. Erdmann R (2012) AAA ATPases: structure and function. Biochim Biophys Acta 1823:1

    Article  CAS  Google Scholar 

  6. Gennerich A, Carter AP, Reck-Peterson SL, Vale RD (2007) Force-induced bidirectional stepping of cytoplasmic dynein. Cell 131:952–965

    Article  CAS  Google Scholar 

  7. Mallik R, Carter BC, Lex SA, King SJ, Gross SP (2004) Cytoplasmic dynein functions as a gear in response to load. Nature 427:649–652

    Article  CAS  Google Scholar 

  8. Smith DE, Tans SJ, Smith SB, Grimes S, Anderson DL, Bustamante C (2001) The bacteriophage straight phi29 portal motor can package DNA against a large internal force. Nature 413:748–752

    Article  CAS  Google Scholar 

  9. Moffitt JR, Chemla YR, Aathavan K, Grimes S, Jardine PJ, Anderson DL, Bustamante C (2009) Intersubunit coordination in a homomeric ring ATPase. Nature 457:446–450

    Article  CAS  Google Scholar 

  10. Rao VB, Feiss M (2008) The bacteriophage DNA packaging motor. Annu Rev Genet 42:647–681

    Article  CAS  Google Scholar 

  11. Ogura T, Wilkinson AJ (2001) AAA+ superfamily ATPases: common structure--diverse function. Genes Cells 6:575–597

    Article  CAS  Google Scholar 

  12. Egli M, Johnson CH (2013) A circadian clock nanomachine that runs without transcription or translation. Curr Opin Neurobiol 23:732–740

    Article  CAS  Google Scholar 

  13. Diaz-Valencia JD, Morelli MM, Bailey M, Zhang D, Sharp DJ, Ross JL (2011) Drosophila katanin-60 depolymerizes and severs at microtubule defects. Biophys J 100:2440–2449

    Article  CAS  Google Scholar 

  14. Desantis ME, Shorter J (2012) The elusive middle domain of Hsp104 and ClpB: location and function. Biochim Biophys Acta 1823:29–39

    Article  CAS  Google Scholar 

  15. Hwang W, Lang MJ (2013) Nucleotide-dependent control of internal strains in ring-shaped AAA+ motors. Cell Mol Bioeng 6:65–73

    Article  CAS  Google Scholar 

  16. Erzberger JP, Berger JM (2006) Evolutionary relationships and structural mechanisms of AAA+ proteins. Annu Rev Biophys Biomol Struct 35:93–114

    Article  CAS  Google Scholar 

  17. Baker TA, Sauer RT (2012) ClpXP, an ATP-powered unfolding and protein-degradation machine. Biochim Biophys Acta 1823:15–28

    Article  CAS  Google Scholar 

  18. Martin A, Baker TA, Sauer RT (2005) Rebuilt AAA + motors reveal operating principles for ATP-fuelled machines. Nature 437:1115–1120

    Article  CAS  Google Scholar 

  19. Shin Y, Davis JH, Brau RR, Martin A, Kenniston JA, Baker TA, Sauer RT, Lang MJ (2009) Single-molecule denaturation and degradation of proteins by the AAA+ ClpXP protease. Proc Natl Acad Sci U S A 106:19340–19345

    Article  CAS  Google Scholar 

  20. Martin A, Baker TA, Sauer RT (2008) Protein unfolding by a AAA+ protease is dependent on ATP-hydrolysis rates and substrate energy landscapes. Nat Struct Mol Biol 15:139–145

    Article  CAS  Google Scholar 

  21. Aubin-Tam ME, Olivares AO, Sauer RT, Baker TA, Lang MJ (2011) Single-molecule protein unfolding and translocation by an ATP-fueled proteolytic machine. Cell 145:257–267

    Article  CAS  Google Scholar 

  22. Maillard RA, Chistol G, Sen M, Righini M, Tan J, Kaiser CM, Hodges C, Martin A, Bustamante C (2011) ClpX(P) generates mechanical force to unfold and translocate its protein substrates. Cell 145:459–469

    Article  CAS  Google Scholar 

  23. Sen M, Maillard RA, Nyquist K, Rodriguez-Aliaga P, Presse S, Martin A, Bustamante C (2013) The ClpXP protease unfolds substrates using a constant rate of pulling but different gears. Cell 155:636–646

    Article  CAS  Google Scholar 

  24. Cordova JC, Olivares AO, Shin Y, Stinson BM, Calmat S, Schmitz KR, Aubin-Tam ME, Baker TA, Lang MJ, Sauer RT (2014) Stochastic but highly coordinated protein unfolding and translocation by the ClpXP proteolytic machine. Cell 158:647–658

    Article  CAS  Google Scholar 

  25. Olivares AO, Nager AR, Iosefson O, Sauer RT, Baker TA (2014) Mechanochemical basis of protein degradation by a double-ring AAA+ machine. Nat Struct Mol Biol 21:871–875

    Article  CAS  Google Scholar 

  26. Iosefson O, Olivares AO, Baker TA, Sauer RT (2015) Dissection of axial-pore loop function during unfolding and translocation by a AAA+ proteolytic machine. Cell Rep 12:1032–1041

    Article  CAS  Google Scholar 

  27. Stinson BM, Baytshtok V, Schmitz KR, Baker TA, Sauer RT (2015) Subunit asymmetry and roles of conformational switching in the hexameric AAA+ ring of ClpX. Nat Struct Mol Biol 22:411–416

    CAS  Google Scholar 

  28. Kenniston JA, Baker TA, Fernandez JM, Sauer RT (2003) Linkage between ATP consumption and mechanical unfolding during the protein processing reactions of an AAA+ degradation machine. Cell 114:511–520

    Article  CAS  Google Scholar 

  29. Karzai AW, Roche ED, Sauer RT (2000) The SsrA-SmpB system for protein tagging, directed degradation and ribosome rescue. Nat Struct Biol 7:449–455

    Article  CAS  Google Scholar 

  30. Lang MJ, Asbury CL, Shaevitz JW, Block SM (2002) An automated two-dimensional optical force clamp for single molecule studies. Biophys J 83:491–501

    Article  CAS  Google Scholar 

  31. Visscher K, Gross SP, Block SM (1996) Construction of multiple-beam optical traps with nanometer-resolution position sensing. IEEE J Sel Top Quantum Electron 2:1066–1076

    Article  CAS  Google Scholar 

  32. Kenniston JA, Baker TA, Sauer RT (2005) Partitioning between unfolding and release of native domains during ClpXP degradation determines substrate selectivity and partial processing. Proc Natl Acad Sci U S A 102:1390–1395

    Article  CAS  Google Scholar 

  33. Howarth M, Takao K, Hayashi Y, Ting AY (2005) Targeting quantum dots to surface proteins in living cells with biotin ligase. Proc Natl Acad Sci U S A 102:7583–7588

    Article  CAS  Google Scholar 

  34. Bewley MC, Graziano V, Griffin K, Flanagan JM (2006) The asymmetry in the mature amino-terminus of ClpP facilitates a local symmetry match in ClpAP and ClpXP complexes. J Struct Biol 153:113–128

    Article  CAS  Google Scholar 

  35. Kapust RB, Tozser J, Fox JD, Anderson DE, Cherry S, Copeland TD, Waugh DS (2001) Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng 14:993–1000

    Article  CAS  Google Scholar 

  36. Greenleaf WJ, Woodside MT, Abbondanzieri EA, Block SM (2005) Passive all-optical force clamp for high-resolution laser trapping. Phys Rev Lett 95:208102

    Article  Google Scholar 

Download references

Acknowledgements

We are grateful to Bob Sauer, Tania Baker and members of the Sauer, Baker and Lang Labs who over the years developed and refined the protocols and methods detailed herein. This work was supported by grants from the National Science Foundation (MCB-1330792) and the NIH (GM-101988). J.C.C was supported in part by a GAANN fellowship from the US Department of Education under grant no. P200A090323.

Author information

Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, Vanderbilt University, 308-A Olin Hall, VU Mailbox: PMB 351604, Nashville, TN, 37235, USA

    Juan Carlos Cordova

  2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA

    Adrian O. Olivares

  3. Department of Chemical and Biomolecular Engineering and Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 308-A Olin Hall, VU Mailbox: PMB 351604, Nashville, TN, 37235, USA

    Matthew J. Lang

Authors
  1. Juan Carlos Cordova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Adrian O. Olivares
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Matthew J. Lang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Matthew J. Lang .

Editor information

Editors and Affiliations

  1. Department of Anatomy and Structural Biology & Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, USA

    Arne Gennerich

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media New York

About this protocol

Cite this protocol

Cordova, J.C., Olivares, A.O., Lang, M.J. (2017). Mechanically Watching the ClpXP Proteolytic Machinery. In: Gennerich, A. (eds) Optical Tweezers. Methods in Molecular Biology, vol 1486. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6421-5_12

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6421-5_12

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6419-2

  • Online ISBN: 978-1-4939-6421-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation