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Guide to video recording of structure dynamics and dynamic processes of proteins by high-speed atomic force microscopy

Abstract

High-speed atomic force microscopy (HS-AFM) allows direct visualization of dynamic structural changes and processes of functioning biological molecules in physiological solutions, at subsecond to sub-100-ms temporal and submolecular spatial resolution. Unlike fluorescence microscopy, wherein the subset of molecular events that you see is dependent on the site where the probe is placed, dynamic molecular events unselectively appear in detail in an AFM movie, facilitating our understanding of how biological molecules function. Here we present protocols for HS-AFM imaging of proteins in action, including preparation of cantilever tips, step-by-step procedures for HS-AFM imaging, and recycling of cantilevers and sample stages, together with precautions and troubleshooting advice for successful imaging. The protocols are adaptable in general for imaging many proteins and protein–nucleic acid complexes, and examples are described for looking at walking myosin, ATP-hydrolyzing rotorless F1-ATPase and cellulose-hydrolyzing cellulase. The entire protocol takes 10–15 h, depending mainly on the substrate surface to be used.

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Figure 1
Figure 2: Flowcharts showing overview of procedures before and during HS-AFM imaging and cleanup steps for the next imaging experiments.
Figure 3
Figure 4: Pictures and schematics of a container for growing EBD tips with SEM.
Figure 5: SEM photographs of small cantilevers with and without EBD tip.
Figure 6: Storage tools used to preserve a solution droplet on the sample stage without drying it.
Figure 7: Importance of firmly mounting the cantilever base and selecting the imaging region for successful imaging.
Figure 8: Optical microscopic views of a small cantilever.
Figure 9: Clipped high-speed AFM images showing relationship between applying tapping force and image quality.
Figure 10: High-speed AFM images of proteins in action.
Figure 11: Effect of AFM tip smear and its removal on image quality.

References

  1. Müller, D.J. & Engel, A. Atomic force microscopy and spectroscopy of native membrane proteins. Nat. Protoc. 2, 2191–2197 (2007).

    PubMed  Article  CAS  Google Scholar 

  2. Viani, M.B. et al. Fast imaging and fast force spectroscopy of single biopolymers with a new atomic force microscope designed for small cantilevers. Rev. Sci. Instrum. 70, 4300–4303 (1999).

    CAS  Article  Google Scholar 

  3. Viani, M.B. Probing protein-protein interactions in real time. Nat. Struct. Biol. 7, 644–647 (2000).

    CAS  PubMed  Article  Google Scholar 

  4. Ando, T. et al. A high-speed atomic force microscope for studying biological macromolecules. Proc. Natl. Acad. Sci. USA 98, 12468–12472 (2001).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  5. Kitazawa, M., Shiotani, K. & Toda, A. Batch fabrication of sharpened silicon nitride tips. Jpn. J. Appl. Phys. 42, 4844–4847 (2003).

    CAS  Article  Google Scholar 

  6. Kodera, N., Yamashita, H. & Ando, T. Active damping of the scanner for high-speed atomic force microscopy. Rev. Sci. Instrum. 76, 053708 (2005).

    Article  CAS  Google Scholar 

  7. Ando, T. et al. High-speed atomic force microscopy for studying the dynamic behavior of protein molecules at work. e-J. Surf. Sci. Nanotechnol. 3, 384–392 (2005).

    CAS  Article  Google Scholar 

  8. Kodera, N., Sakashita, M. & Ando, T. Dynamic proportional-integral- differential controller for high-speed atomic force microscopy. Rev. Sci. Instrum. 77, 083704 (2006).

    Article  CAS  Google Scholar 

  9. Uchihashi, T., Kodera, N., Itoh, H., Yamashita, H. & Ando, T. Feed-forward control for high-speed AFM imaging of biomolecules. Jpn. J. Appl. Phys. 45, 1904–1908 (2006).

    CAS  Article  Google Scholar 

  10. Ando, T. et al. High-speed atomic force microscopy for studying the dynamic behavior of protein molecules at work. Jpn. J. Appl. Phys. 45, 1897–1903 (2006).

    CAS  Article  Google Scholar 

  11. Uchihashi, T., Yamashita, H. & Ando, T. Fast phase imaging in liquids using a rapid scan atomic force microscope. Appl. Phys. Lett. 89, 213112 (3 pp) (2006).

    Article  CAS  Google Scholar 

  12. Yamashita, H. et al. Tip-sample distance control using photo-thermal actuation of a small cantilever for high-speed atomic force microscopy. Rev. Sci. Instrum. 78, 083702 (2007).

    PubMed  Article  CAS  Google Scholar 

  13. Fukuma, T., Okazaki, Y., Kodera, N., Uchihashi, T. & Ando, T. High resonance frequency force microscopy scanner using inertia balance support. Appl. Phys. Lett. 92, 243119 (2008).

    Article  CAS  Google Scholar 

  14. Ando, T. et al. High-speed AFM and nano-visualization of biomolecular processes. Pflügers Archiv.—Eur. J. Physiol. 456, 211–225 (2008).

    CAS  Article  Google Scholar 

  15. Ando, T., Uchihashi, T. & Fukuma, T. High-speed atomic force microscopy for nano-visualization of dynamic biomolecular processes. Prog. Surf. Sci. 83, 337–437 (2008).

    CAS  Article  Google Scholar 

  16. Casuso, I., Rico, F. & Scheuring, S. High-speed atomic force microscopy: structure and dynamics of single proteins. Curr. Opin. Chem. Biol. 15, 704–709 (2011).

    CAS  PubMed  Article  Google Scholar 

  17. Katan, A.J. & Dekker, C. High-speed AFM reveals the dynamics of single biomolecules at the nanometer scale. Cell 147, 979–982 (2011).

    CAS  PubMed  Article  Google Scholar 

  18. Ando, T. High-speed atomic force microscopy coming of age. Nanotechnology 23, 062001 (27 pp) (2012).

    Article  Google Scholar 

  19. Kodera, N., Yamamoto, D., Ishikawa, R. & Ando, T. Video imaging of walking myosin V by high-speed atomic force microscopy. Nature 468, 72–76 (2010).

    CAS  PubMed  Article  Google Scholar 

  20. Shibata, M. et al. High-speed atomic force microscopy shows dynamic molecular processes in photo-activated bacteriorhodopsin. Nat. Nanotechnol. 5, 208–212 (2010).

    CAS  PubMed  Article  Google Scholar 

  21. Shibata, M., Yamashita, H., Uchihashi, T., Kandori, H. & Ando, T. Structural changes in bacteriorhodopsin in response to alternate illumination observed by high-speed atomic force microscopy. Angew. Chem. Int. ed. 50, 4410–4413 (2011).

    CAS  Article  Google Scholar 

  22. Uchihashi, T., Iino, R., Ando, T. & Noji, H. High-speed atomic force microscopy reveals rotary catalysis of rotorless F1-ATPase. Science 333, 755–758 (2011).

    CAS  PubMed  Article  Google Scholar 

  23. Igarashi, K. et al. Traffic jams reduce hydrolytic efficiency of cellulase on cellulose surface. Science 333, 1279–1282 (2011).

    CAS  Article  PubMed  Google Scholar 

  24. Yamashita, H. et al. Dynamics of bacteriorhodopsin 2D crystal observed by high-speed atomic force microscopy. J. Struct. Biol. 167, 153–158 (2009).

    CAS  PubMed  Article  Google Scholar 

  25. Milhiet, P.-E. et al. Deciphering the structure, growth and assembly of amyloid-like fibrils using high-speed atomic force microscopy. PLoS ONE 5, e13240 (8 pp) (2010).

    Article  CAS  Google Scholar 

  26. Miyagi, A. et al. Visualization of intrinsically disordered regions of proteins by high-speed atomic force microscopy. Chem. Phys. Chem. 9, 1859–1866 (2008).

    CAS  PubMed  Article  Google Scholar 

  27. Casuso, I., Sens, P., Rico, F. & Scheuring, S. Experimental evidence for membrane-mediated protein-protein interaction. Biophys. J. 99, L47–L49 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. Miyagi, A., Ando, T. & Lyubchenko, Y.L. Dynamics of nucleosomes assessed with time-lapse high-speed atomic force microscopy. Biochemistry 59, 7901–7908 (2011).

    Article  CAS  Google Scholar 

  29. Sanchez, H., Suzuki, Y., Yokokawa, M., Takeyasu, K. & Wyman, C. Protein-DNA interactions in high speed AFM: single molecule diffusion analysis of human RAD54. Integr. Biol. 3, 1127–1134 (2011).

    CAS  Article  Google Scholar 

  30. Suzuki, Y. et al. Visual analysis of concerted cleavage by type IIF restriction enzyme SfiI in subsecond time region. Biophys. J. 101, 2992–2998 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. Wickham, S.F.J. et al. Direct observation of stepwise movement of a synthetic molecular transporter. Nat. Nanotechnol. 6, 166–169 (2011).

    CAS  PubMed  Article  Google Scholar 

  32. Inoue, S., Uchihashi, T., Yamamoto, D. & Ando, T. Direct observation of surfactant aggregate behavior on a mica surface using high-speed atomic force microscopy. Chem. Commun. 47, 4974–4976 (2011).

    CAS  Article  Google Scholar 

  33. Giocondi, M.-C. et al. Surface topography of membrane domains. Biochim. Biophys. Acta.-Biomembranes 1798, 703–718 (2010).

    CAS  Article  Google Scholar 

  34. Yamamoto, D., Nagura, N., Omote, S., Taniguchi, M. & Ando, T. Streptavidin 2D crystal substrates for visualizing biomolecular processes by atomic force microscopy. Biophys. J. 97, 2358–2367 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. Yamamoto, D. et al. High-speed atomic force microscopy techniques for observing dynamic biomolecular processes. Methods Enzymol. 475 (Part B): 541–564 (2010).

    CAS  PubMed  Article  Google Scholar 

  36. Lyubchenko, Y.L., Shlyakhtenko, L.S. & Ando, T. Imaging of nucleic acids with atomic force microscopy. Methods 54, 274–283 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. Yamamoto, D., Uchihashi, T., Kodera, N. & Ando, T. Anisotropic diffusion of point defects in two-dimensional crystal of streptavidin observed by high-speed atomic force microscopy. Nanotechnol. 19, 384009 (9 pp) (2008).

    Google Scholar 

  38. Yokokawa, M et al. Fast-scanning atomic force microscopy reveals the ATP/ADP-dependent conformational changes of GroEL. EMBO J. 25, 4567–4576 (2006).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. Fantner, G.E., Barbero, R.J., Gray, D.S. & Belcher, A.M. Kinetics of antimicrobial peptide activity measured on individual bacterial cells using high-speed atomic force microscopy. Nat. Nanotechnol. 5, 280–285 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. Sakamoto, T. et al. Direct observation of processive movement by individual myosin V molecules. Biochem. Biol. Res. Commun. 272, 586–590 (2001).

    Article  CAS  Google Scholar 

  41. Koide, H. et al. Identification of the specific IQ motif of myosin V from which calmodulin dissociates in the presence of Ca2+. Biochemistry 45, 11598–11604 (2006).

    CAS  PubMed  Article  Google Scholar 

  42. Spudich, J.A. & Watt, S. The regulation of rabbit skeletal muscle contraction. J. Biol. Chem. 246, 4866–4871 (1971).

    CAS  PubMed  Article  Google Scholar 

  43. Araki, J. et al. Flow properties of microcrystalline cellulose suspension prepared by acid treatment of native cellulose. Colloid Surface A 142, 75–82 (1998).

    CAS  Article  Google Scholar 

  44. Igarashi, K. et al. Surface density of cellobiohydrolase on crystalline celluloses. A critical parameter to evaluate enzymatic kinetics at a solid-liquid interface. FEBS J. 273, 2869–2878 (2006).

    CAS  PubMed  Article  Google Scholar 

  45. Ando, T. et al. A High-speed atomic force microscope for studying biological macromolecules in action. Jpn. J. Appl. Phys. 41, 4851–4856 (2002).

    CAS  Article  Google Scholar 

  46. Ando, T. & Uchihashi, T. High-speed AFM and imaging of biomolecular processes In Nanoscale Liquid Interfaces: Wetting, Patterning and Force Microscopy at Molecular Scale. (eds. Ondarçuhu, T. & Aimé, J.P.) (Pan Stanford Publishing,) (2012).

  47. Mingeot-Leclercq, M.-P., Deleu, M., Brasseur, R. & Dufrêne, Y.F. Atomic force microscopy of supported lipid bilayers. Nat. Protoc. 3, 1654–1659 (2008).

    PubMed  Article  Google Scholar 

  48. D'Costa, N.P. & Hoh, J.H. Calibration of optical lever sensitivity for atomic force microscopy. Rev. Sci. Instrum. 66, 5096–5097 (1995).

    CAS  Article  Google Scholar 

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Acknowledgements

We thank D. Yamamoto for technical assistance. This work was supported by the Core Research for Evolutionary Science and Technology (CREST) program of the Japan Science and Technology Agency (JST); a Grant-in-Aid for Basic Research (S) from the Japan Society for the Promotion of Science (JSPS) (no. 20221006); a Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed Research Area) from the Ministry of Education, Culture, Science, Sports and Technology (MEXT)-Japan; and the Knowledge Cluster Initiative/MEXT-Japan.

Author information

Authors and Affiliations

Authors

Contributions

All the authors designed and discussed the experiments. T.U. and N.K. equally contributed to this work, conducted the experiments, prepared all figures and movies, and drafted the MATERIALS and PROCEDURE sections. T.A. wrote the introductory part of manuscript and edited the whole manuscript.

Corresponding author

Correspondence to Toshio Ando.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

Alignment of the laser position relative to the small cantilever (0 s – 12 s) and adjustment of the photodetector position (> 13 s). Inserted images on the right bottom is the 4-digit indicators for the total intensity of laser irradiated onto the four segments of the quadrant PIN photodiode (left) and difference between the laser intensities irradiated on the top two segments and the bottom two segments (right). (MOV 7097 kb)

Supplementary Video 2

Rinsing of sample or others placed on substrate disk attached to the top of a sample stage. (MOV 3561 kb)

Supplementary Video 3

Mounting of the scanner on the HS-AFM apparatus (0 s – 12 s) and position adjustment of the sample stage relative to the cantilever (> 13 s). (MOV 5127 kb)

Supplementary Video 4

HS-AFM imaging of myosin V-HMM moving on actin filament. (MOV 8683 kb)

Supplementary Video 5

Effect of dynamic PID control mode on parachuting. (MOV 8449 kb)

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Uchihashi, T., Kodera, N. & Ando, T. Guide to video recording of structure dynamics and dynamic processes of proteins by high-speed atomic force microscopy. Nat Protoc 7, 1193–1206 (2012). https://doi.org/10.1038/nprot.2012.047

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