Controlling the Motility of ATP-Driven Molecular Motors Using High Hydrostatic Pressure

  • Masayoshi Nishiyama


High-pressure microscopy is a powerful technique for visualizing the effects of hydrostatic pressure on research targets. It can be used for monitoring the pressure-induced changes in the structure and function of molecular machines in vitro and in vivo. This chapter focuses on the use of high-pressure microscopy to measure the dynamic properties of molecular machines. We describe a high-pressure microscope that is optimized both for the best image formation and for stability under high hydrostatic pressure. The developed system allows us to visualize the motility of ATP-driven molecular motors under high pressure. The techniques described could be extended to study the detailed mechanism by which molecular machines work efficiently in collaboration with water molecules.


High-pressure microscopy Molecular motors Kinesin F1-ATPase Single-molecule measurement 



We would like to thank Yoshifumi Kimura for developing a prototype of the high-pressure chamber for optical microscopy, Daichi Okuno and Hiroyuki Noji for the measurement of F1-ATPase, and Eiro Muneyuki, Shoichi Toyabe, Shou Furuike, Masahide Terazima, Yoshie Harada, and Akitoshi Seiyama for discussion. This work was supported by Grants-in-Aid for Scientific Research on the Innovative Area “Water plays the main role in ATP energy transfer, Group Leader; Prof. Makoto Suzuki” (Nos. 21118511 and 23118710), Grants-in-Aid for Scientific Research from MEXT (Nos. 16 K04908 and 17H05880), and the Takeda Science Foundation, Research Foundation for Opto-Science and Technology, Shimadzu Science Foundation, and Nakatani Foundation for Advancement of Measuring Technologies in Biomedical Engineering.


  1. Abe F (2015) Effects of high hydrostatic pressure on microbial cell membranes: structural and functional perspectives. Subcell Biochem 72:371–381. Scholar
  2. Adachi K, Oiwa K, Nishizaka T, Furuike S, Noji H, Itoh H, Yoshida M, Kinosita K Jr (2007) Coupling of rotation and catalysis in F1-ATPase revealed by single-molecule imaging and manipulation. Cell 130:309–321. Scholar
  3. Adachi K, Oiwa K, Yoshida M, Nishizaka T, Kinosita K Jr (2012) Controlled rotation of the F1-ATPase reveals differential and continuous binding changes for ATP synthesis. Nat Commun 3:1022.
  4. Akasaka K (2006) Probing conformational fluctuation of proteins by pressure perturbation. Chem Rev 106:1814–1835CrossRefGoogle Scholar
  5. Bartlett DH (2002) Pressure effects on in vivo microbial processes Bba-Protein Struct M 1595:367–381CrossRefGoogle Scholar
  6. Block SM (2007) Kinesin motor mechanics: binding, stepping, tracking, gating, and limping. Biophys J 92:2986–2995. Scholar
  7. Boonyaratanakornkit BB, Park CB, Clark DS (2002) Pressure effects on intra- and intermolecular interactions within proteins. Biochem Biophys Acta 1595:235–249PubMedGoogle Scholar
  8. Chaplin M (2006) Do we underestimate the importance of water in cell biology? Nat Rev Mol Cell Biol 7:861–866. Scholar
  9. Cross RA (2016) Review: Mechanochemistry of the kinesin-1 ATPase. Biopolymers 105:476–482. Scholar
  10. Dzwolak W, Kato M, Taniguchi Y (2002) Fourier transform infrared spectroscopy in high-pressure studies on proteins. Biochem Biophys Acta 1595:131–144PubMedGoogle Scholar
  11. Fourme R, Girard E, Akasaka K (2012) High-pressure macromolecular crystallography and NMR: status, achievements and prospects. Curr Opin Struct Biol 22:636–642. Scholar
  12. Fujii S, Masanari-Fujii M, Kobayashi S, Kato C, Nishiyama M, Harada Y, Wakai S, Sambongi Y (2018) Commonly stabilized cytochromes c from deep-sea Shewanella and Pseudomonas. Biosci Biotechnol Biochem.
  13. Fujisawa T (2015) High pressure small-angle X-ray scattering. Subcell Biochem 72:663–675.
  14. Furuike S, Adachi K, Sakaki N, Shimo-Kon R, Itoh H, Muneyuki E, Yoshida M, Kinosita K Jr (2008) Temperature dependence of the rotation and hydrolysis activities of F1-ATPase. Biophys J 95:761–770. Scholar
  15. Hayashi M, Nishiyama M, Kazayama Y, Toyota T, Harada Y, Takiguchi K (2016) Reversible morphological control of tubulin-encapsulating giant liposomes by hydrostatic pressure. Langmuir 32:3794–3802. Scholar
  16. Ishii Y, Nishiyama M, Yanagida T (2004) Mechano-chemical coupling of molecular motors revealed by single molecule measurements. Curr Protein Pept Sci 5:81–87. Scholar
  17. Ito Y, Ikeguchi M (2014) Molecular dynamics simulations of F1-ATPase. Adv Exp Med Biol 805:411–440. Scholar
  18. Kawaguchi K, Ishiwata S (2000) Temperature dependence of force, velocity, and processivity of single kinesin molecules. Biochem Biophys Res Commun 272:895–899. Scholar
  19. Kitahara R (2015) High-Pressure NMR spectroscopy reveals functional sub-states of ubiquitin and ubiquitin-like proteins. Subcell Biochem 72:199–214. Scholar
  20. Kojima H, Muto E, Higuchi H, Yanagida T (1997) Mechanics of single kinesin molecules measured by optical trapping nanometry. Biophys J 73:2012–2022. Scholar
  21. Kuffel A, Zielkiewicz J (2013) Properties of water in the region between a tubulin dimer and a single motor head of kinesin. Phys Chem Chem Phys 15:4527–4537. Scholar
  22. Luong TQ, Kapoor S, Winter R (2015) Pressure-A gateway to fundamental insights into protein solvation. Dyn Func Chemphyschem 16:3555–3571CrossRefGoogle Scholar
  23. Maeno A, Akasaka K (2015) High-pressure fluorescence spectroscopy. Subcell Biochem 72:687–705.
  24. Mazumdar M, Cross RA (1998) Engineering a lever into the kinesin neck. J Biol Chem 273:29352–29359. Scholar
  25. Miyamoto Y, Muto E, Mashimo T, Iwane AH, Yoshiya I, Yanagida T (2000) Direct inhibition of microtubule-based kinesin motility by local anesthetics. Biophys J 78:940–949. Scholar
  26. Mozhaev VV, Heremans K, Frank J, Masson P, Balny C (1996) High pressure effects on protein structure and function. Proteins-Struct Func Genet 24:81–91CrossRefGoogle Scholar
  27. Nishiyama M (2017) High-pressure microscopy for tracking dynamic properties of molecular machines. Biophys Chem. Scholar
  28. Nishiyama M, Higuchi H, Ishii Y, Taniguchi Y, Yanagida T (2003) Single molecule processes on the stepwise movement of ATP-driven molecular motors. Biosystems 71:145–156. Scholar
  29. Nishiyama M, Higuchi H, Yanagida T (2002) Chemomechanical coupling of the forward and backward steps of single kinesin molecules. Nat Cell Biol 4:790–797. Scholar
  30. Nishiyama M, Kimura Y, Nishiyama Y, Terazima M (2009) Pressure-induced changes in the structure and function of the kinesin-microtubule complex. Biophys J 96:1142–1150. Scholar
  31. Nishiyama M, Kojima S (2012) Bacterial motility measured by a miniature chamber for high-pressure microscopy. Int J Mol Sci 13:9225–9239. Scholar
  32. Nishiyama M, Muto E, Inoue Y, Yanagida T, Higuchi H (2001) Substeps within the 8-nm step of the ATPase cycle of single kinesin molecules. Nat Cell Biol 3:425–428. Scholar
  33. Nishiyama M, Sowa Y (2012) Microscopic analysis of bacterial motility at high pressure. Biophys J 102:1872–1880. Scholar
  34. Nishiyama M, Sowa Y (2013) Manipulation of cell motility with water molecules in living cells Kagaku. Jpn J 68:33–38Google Scholar
  35. Nishiyama M, Sowa Y, Kimura Y, Homma M, Ishijima A, Terazima M (2013) High hydrostatic pressure induces counterclockwise to clockwise reversals of the Escherichia coli flagellar motor. J Bacteriol 195:1809–1814. Scholar
  36. Noji H, Ueno H, McMillan DGG (2017) Catalytic robustness and torque generation of the F1-ATPase. Biophys Rev 9:103–118 Scholar
  37. Noji H, Yasuda R, Yoshida M, Kinosita K Jr (1997) Direct observation of the rotation of F1-ATPase. Nature 386:299–302. Scholar
  38. Okuno D, Nishiyama M, Noji H (2013) Single-molecule analysis of the rotation of F1-ATPase under high hydrostatic pressure. Biophys J 105:1635–1642. Scholar
  39. Payne VA, Matubayasi N, Murphy LR, Levy RM (1997) Monte Carlo study of the effect of pressure on hydrophobic association. J Phys Chem B 101:2054–2060. Scholar
  40. Roche J, Dellarole M, Royer CA, Roumestand C (2015) Exploring the protein folding pathway with high-pressure NMR: steady-state and kinetics studies. Subcell Biochem 72:261–278. Scholar
  41. Schnitzer MJ, Block SM (1997) Kinesin hydrolyses one ATP per 8-nm step. Nature 388:386–390. Scholar
  42. Shimabukuro K, Yasuda R, Muneyuki E, Hara KY, Kinosita K Jr, Yoshida M (2003) Catalysis and rotation of F1 motor: cleavage of ATP at the catalytic site occurs in 1 ms before 40° substep rotation. Proc Natl Acad Sci U S A 100:14731–14736. Scholar
  43. Svoboda K, Block SM (1994) Force and velocity measured for single kinesin molecules. Cell 77:773–784. Scholar
  44. Svoboda K, Schmidt CF, Schnapp BJ, Block SM (1993) Direct observation of kinesin stepping by optical trapping interferometry. Nature 365:721–727. Scholar
  45. Taniguchi Y, Nishiyama M, Ishii Y, Yanagida T (2005) Entropy rectifies the Brownian steps of kinesin. Nat Chem Biol 1:342–347. Scholar
  46. Toyabe S, Watanabe-Nakayama T, Okamoto T, Kudo S, Muneyuki E (2011) Thermodynamic efficiency and mechanochemical coupling of F1-ATPase. Proc Natl Acad Sci U S A 108:17951–17956. Scholar
  47. Vale RD, Milligan RA (2000) The way things move: looking under the hood of molecular motor proteins. Science 288:88–95CrossRefGoogle Scholar
  48. Vass H, Black SL, Herzig EM, Ward FB, Clegg PS, Allen RJ (2010) A multipurpose modular system for high-resolution microscopy at high hydrostatic pressure. Rev Sci Instrum 81 Scholar
  49. Wakai N, Takemura K, Morita T, Kitao A (2014) Mechanism of deep-sea fish alpha-actin pressure tolerance investigated by molecular dynamics simulations. PLoS ONE 9:e85852. Scholar
  50. Watanabe N (2015) High pressure macromolecular crystallography. Subcell Biochem 72:677–686
  51. Watanabe TM et al (2013) Glycine insertion makes yellow fluorescent protein sensitive to hydrostatic pressure. PLoS ONE 8
  52. Watanabe-Nakayama T, Toyabe S, Kudo S, Sugiyama S, Yoshida M, Muneyuki E (2008) Effect of external torque on the ATP-driven rotation of F1-ATPase. Biochem Biophys Res Commun 366:951–957. Scholar
  53. Webb JN, Webb SD, Cleland JL, Carpenter JF, Randolph TW (2001) Partial molar volume, surface area, and hydration changes for equilibrium unfolding and formation of aggregation transition state: high-pressure and cosolute studies on recombinant human IFN-gamma. Proc Natl Acad Sci U S A 98:7259–7264. Scholar
  54. Winter R (2002) Synchrotron X-ray and neutron small-angle scattering of lyotropic lipid mesophases, model biomembranes and proteins in solution at high pressure. Biochem Biophys Acta 1595:160–184PubMedGoogle Scholar
  55. Winter R (2015) Pressure effects on the intermolecular interaction potential of condensed protein solutions. Subcell Biochem 72:151–176. Scholar
  56. Yasuda R, Noji H, Yoshida M, Kinosita K Jr, Itoh H (2001) Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase. Nature 410:898–904. Scholar

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© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  1. 1.The HAKUBI Center for Advanced Research/Graduate School of MedicineKyoto UniversityKyotoJapan

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