Skip to main content
SpringerLink
Log in
Book cover

Protein Structure, Stability, and Interactions pp 83–113Cite as

Protein–Protein and Ligand–Protein Interactions Studied by Analytical Ultracentrifugation

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 490))

Abstract

All biological processes involve molecular interactions that result in either binding, self-association, or hetero-associations of one form or another. It is important to understand that no interactions are completely all-or-none. Some approach all-or-none only when there is strong positive cooperativity. Examples will be given of typical biomolecular interactions and their expected dependence on concentration, in order to point out the relatively wide range of concentration over which these types of phenomena take place. This chapter is concerned both with the binding of low-molecular-weight ligands to macromolecules as well as interactions between macromolecules using analytical ultracentrifugation (AUC) as a tool for measuring association properties of these systems. The theory of sedimentation of both ideal and nonideal interacting and noninteracting systems is discussed. Examples are given of each type of system along with a discussion of how each type of system can be analyzed. Several methods of data analysis are discussed.

This is a preview of subscription content, 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   129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   169.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
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

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Svensson, H. (1954) The second order aberrations in the interferometric measurement of concentration gradients. Optica Acta 1, 25–32.

    Google Scholar 

  2. Svensson, H. (1954) The second order aberrations in the interferomentric measurement of concentration gradients. II. Experimental verification of theory. Optica Acta 1, 90–93.

    Google Scholar 

  3. Svensson, H. (1956) The third order aberrations in the interferometric measurement of concentration gradients. Optica Acta 3, 164–168.

    Google Scholar 

  4. Stafford, W. F. (unpublished observation).

    Google Scholar 

  5. Richards, E. G., Teller, D., Schachman, H. K. (1971) Alignment of Schlieren and Rayleigh optical systems in the ultracentrifuge. I. Focusing of the camera and cylindrical lenses. Anal Biochem 41, 189–214.

    Article  PubMed  CAS  Google Scholar 

  6. Richards, E. G., Teller, D. C., Hoagland, V. J., et al. (1971) Alignment of Schlieren and Rayleigh optical systems in the ultracentrifuge. II. A general procedure. Anal Biochem 41, 215–247.

    Article  PubMed  CAS  Google Scholar 

  7. Richards, E. G., Bell, C. J., Kirschner, M., et al. (1972) Alignment of Schlieren and Rayleigh optical systems in the ultracentrifuge. 3. Design, construction, and placement of Rayleigh mask. Anal Biochem 46, 295–331.

    Article  PubMed  CAS  Google Scholar 

  8. Casassa, E. F., Eisenberg, H. (1964) Thermodynamic analysis of multicomponent solutions. Adv Protein Chem 19, 287–395.

    Article  PubMed  CAS  Google Scholar 

  9. Kar, S. R., Kingsbury, J. S., Lewis, M. S., et al. (2000) Analysis of transport experiments using pseudo-absorbance data. Anal Biochem 285, 135–142.

    Article  PubMed  CAS  Google Scholar 

  10. Schuck, P., Demeler, B. (1999) Direct sedimentation analysis of interference optical data in analytical ultracentrifugation. Biophys J 76, 2288–2296.

    Article  PubMed  CAS  Google Scholar 

  11. Stafford, W. F. (1992) Boundary analysis in sedimentation transport experiments: a procedure for obtaining sedimentation coefficient distributions using the time derivative of the concentration profile. Anal Biochem 203, 295–301.

    Article  PubMed  CAS  Google Scholar 

  12. Stafford, W. F., Sherwood, P. J. (2004) Analysis of heterologous interacting systems by sedimentation velocity: Curve fitting algorithms for estimation of sedimentation coefficients, equilibrium and rate constants. Biophys Chem 108, 231–243.

    Article  PubMed  CAS  Google Scholar 

  13. Rivas, G., Stafford, W. F., Minton, A. P. (1999) Characterization of heterologous protein–protein interaction via analytical ultracentrifugation. Methods: A Companion to Methods in Enzymology.

    Google Scholar 

  14. Stafford, W. F. (1998) Time difference sedimentation velocity analysis of rapidly reversible interacting systems: determination of equilibrium constants by non-linear curve fitting procedures. Biophys J 74, A301.

    Google Scholar 

  15. Schuck, P. (1999) Sedimentation equilibrium analysis of interference optical data by systematic noise decomposition. Anal Biochem 272, 199–208.

    Article  PubMed  CAS  Google Scholar 

  16. Schuck, P. (2000) Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophys J 78, 1606–1619.

    Article  PubMed  CAS  Google Scholar 

  17. Johnson, M., Faunt, L. (1992) Parameter estimation by least-squares methods. Methods in Enzymology 210, 1–37.

    Article  PubMed  CAS  Google Scholar 

  18. Johnson, M. L. (1992) Why, when, and how biochemists should use least squares, Anal Biochem 206, 215–225.

    Article  PubMed  CAS  Google Scholar 

  19. Schuck, P., Perugini, M. A., Gonzales, N. R., et al. (2002) Size-distribution analysis of proteins by analytical ultracentrifugation: strategies and application to model systems, Biophys J 82, 1096–1111.

    Article  PubMed  CAS  Google Scholar 

  20. Svedberg, T., Pederson, K. O. (1940) The Ultracentrifuge, Oxford University Press, New York.

    Google Scholar 

  21. Schachman, H. K. (1959) . Academic Press, New York.

    Google Scholar 

  22. Williams, J. W., Van Holde, K. E., Baldwin, R. L., et al. (1958) The theory of sedimentation analysis. Chem Rev 58, 715–806.

    Article  CAS  Google Scholar 

  23. Fujita, H. (1962) Mathematical Theory of Sedimentation Analysis. Academic Press, New York.

    Google Scholar 

  24. Fujita, H. (1976) Foundations of Ultracentrifugal Analysis. John Wiley & Sons, New York.

    Google Scholar 

  25. Tanford, C. (1961) Physical Chemistry of Macromolecules, p. 710. John Wiley & Sons, New York.

    Google Scholar 

  26. Harding, S. E., Johnson, P. (1985) The concentration-dependence of macromolecular parameters, Biochem J 231, 543–547.

    PubMed  CAS  Google Scholar 

  27. Solovyova, A., Schuck, P., Costenaro, L., Ebel, C. (2001) Non-ideality by sedimentation velocity of halophilic malate dehydrogenase in complex solvents, Biophys J 81, 1868–1880.

    Article  PubMed  CAS  Google Scholar 

  28. Roark, D. E., Yphantis, D. A. (1971) Equilibrium centrifugation of nonideal systems. The Donnan effect in self-associating systems. Biochemistry 10, 3241–328149.

    Article  PubMed  CAS  Google Scholar 

  29. Lamm, O. (1929) Die Differentialgleichung der Ultrazentrifugierung. Arkiv Math. Astron. Fysik 21B, No.2, 1–4.

    Google Scholar 

  30. Claverie, J.-M. (1976) Sedimentation of generalized systems of interacting particles III. Concentration dependent sedimentation and extension to other transport methods. Biopolymers 15, 843–857.

    Article  PubMed  CAS  Google Scholar 

  31. Claverie, J. M., Dreux, H., Cohen, R. (1975) Sedimentation of generalized systems of interacting particles. I. Solution of systems of complete Lamm equations. Biopolymers 14, 1685–1700.

    Article  PubMed  CAS  Google Scholar 

  32. Todd, G. P. Haschemeyer, R. H. (1981) General solution to the inverse problem of the differentia17l equation of the ultracentrifuge. Proc Natl Acad Sci U S A 78, 6739–6743.

    Article  PubMed  CAS  Google Scholar 

  33. Todd, G., Haschemeyer, R. (1983) Generalized finite element solution to one-dimensional flux problems. Biophys Chem 17, 321–326.

    Article  PubMed  CAS  Google Scholar 

  34. Holladay, L. (1980) Simultaneous rapid estimation of sedimentation coefficient and molecular weight. Biophys Chem 11, 303–308.

    Article  PubMed  CAS  Google Scholar 

  35. Holladay, L. (1979) An approximate solution to the Lamm equation. Biophys Chem 10, 187–190.

    Article  PubMed  CAS  Google Scholar 

  36. Philo, J. S. (1997) An improved function for fitting sedimentation velocity data for low-molecular-weight solutes. Biophys J 72, 435–444.

    Article  PubMed  CAS  Google Scholar 

  37. Behlke, J., Ristau, O. (2002) A new approximate whole boundary solution of the Lamm differential equation for the analysis of sedimentation velocity experiments. Biophys Chem 95, 59–68.

    Article  PubMed  CAS  Google Scholar 

  38. Gilbert, G. A. (1955) The physical chemistry of enzymes – general discussion. Discuss. Farad Soc 20, 65–77.

    Article  Google Scholar 

  39. Gilbert, G. A. (1959) Sedimentation and electrophoresis of interacting substances .1. Idealized boundary shape for a single substance aggregating reversibly. Proc R Soc A253, 377–388.

    Google Scholar 

  40. Gilbert, G. A., Jenkins, R. C. (1959) Sedimentation and electrophoresis of interacting substances .2. Asymptotic boundary shape for 2 substances interacting reversibly, Proc. R Soc A253, 420–437.

    Google Scholar 

  41. Schuck, P. (www.analyticalultracentrifugation.com/references.htm.).

  42. Cann, J. R. (1970) 89 Interacting Macromolecules. New York: Academic Press.

    Google Scholar 

  43. Dam, J., Velikovsky, C. A., Mariuzza, R. A., et al. (2005) Sedimentation velocity analysis of heterogeneous protein–protein interactions: Lamm equation modeling and sedimentation coefficient distributions c(s), Biophys J , 619–34.

    Article  PubMed  CAS  Google Scholar 

  44. Demeler, B. (2005) UltraScan A Comprehensive Data Analysis Software Package for Analytical Ultracentrifugation Experiments, in Modern Analytical Ultracentrifugation: Techniques and Methods, S.E.H.a.A.J.R. D. J. Scott, Editor. 2005, Royal Society of Chemistry, UK. p. 210–229.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Boston Biomedical Research Institute, Watertown, MA, USA

    Walter F. Stafford III

Authors
  1. Walter F. Stafford III
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

    Rights and permissions

    Reprints and permissions

    Copyright information

    © 2009 Humana Press, a part of Springer Science+Business Media, LLC

    About this protocol

    Cite this protocol

    Stafford, W.F. (2009). Protein–Protein and Ligand–Protein Interactions Studied by Analytical Ultracentrifugation. In: Shriver, J. (eds) Protein Structure, Stability, and Interactions. Methods in Molecular Biology, vol 490. Humana Press. https://doi.org/10.1007/978-1-59745-367-7_4

    Download citation

    • DOI: https://doi.org/10.1007/978-1-59745-367-7_4

    • Published:

    • Publisher Name: Humana Press

    • Print ISBN: 978-1-58829-954-3

    • Online ISBN: 978-1-59745-367-7

    • eBook Packages: Springer Protocols

    Publish with us

    Policies and ethics

    Search

    Navigation