Overview of the Quantitation of Protein Interactions

John S. Philo1

1 Alliance Protein Laboratories, Thousand Oaks
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 20.1
DOI:  10.1002/0471140864.ps2001s17
Online Posting Date:  May, 2001
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Abstract

The biological function of many proteins involves reversible interactions with other proteins, nucleic acids, or other non‐protein ligands. Such interactions play many different roles in a wide range of cellular processes. A few examples are: (1) storing or transporting key metabolites (e.g., O2 storage by myoglobin); (2) forming and maintaining the quaternary structure of multi‐subunit enzymes; (3) specific binding and recognition events (antigen‐antibody, hormone‐receptor, transcription factor‐promoter); and (4) self‐assembly of large structures (microtubules, chromatin). Thus, the quantitative characterization of such interactions represents an important part of understanding the function of such proteins and their role in these cellular events. This unit sets the tone for the rest of the chapter, and gives important information necessary to understand some of the topics that will be covered in future supplements, such as sedimentation equilibrium (analytical and micro‐preparative), surface plasmon resonance (SPR), size‐exclusion chromatography (SEC) with on‐line light scattering, and chemical cross‐linking.

     
 
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Table of Contents

  • Some Comments on Techniques
  • The Importance of Data Analysis Methods
  • Sensitivity to Contaminants and Protein Quality
  • The Advent of Recombinant Proteins Enhances Our Capabilities for Studying Protein Interactions
  • The Virtues of Using Multiple Techniques
  • Some Thoughts on Choosing Methods
  • Obtaining Additional Thermodynamic Information
  • Thermodynamic Linkage Relationships
  • Figures
  • Tables
     
 
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Materials

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Figures

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Literature Cited

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Key References
   Cantor, C.R. and Schimmel, P.R. 1980. Biophysical chemistry. Part II: Techniques for the study of biological structure and function. W.H. Freeman, San Francisco.
  More advanced texts; coverage of binding equilibria and thermodynamics, spectroscopy, and centrifugation methods.
   Freifelder, D. 1982. Physical Biochemistry: Applications to Biochemistry and Molecular Biology. W.H. Freeman, New York.
  Good introductory text; strong on spectroscopy and centrifugation methods
   Johnson, M.L. and Frasier, S.G. 1985. Nonlinear least‐squares analysis. Methods Enzymol. 117:301‐342.
  Good overview of the fitting of experimental data (a key part of all protein interaction studies), with some examples for ligand binding.
   van Holde, K.E. et al. 1998. See above.
  More advanced texts; coverage of binding equilibria and thermodynamics, spectroscopy, and centrifugation methods.
   Winsor, D.J. and Sawyer, W.H. 1995. Quantitative characterization of ligand binding. John Wiley & Sons, New York.
  An excellent but heavily mathematical text covering theory, data analysis, and interpretation. Methods based on phase separation, spectral changes, affinity and gel chromatography, and competitive binding are discussed in some detail, with some coverage of sedimentation, calorimetry, and other techniques. An entire chapter is devoted to DNA‐ligand interactions (also see APPENDIX ).
Internet Resources
   rasmb-manager@bbri.org (subscriptions)
  Reversible Associations in Structural and Molecular Biology (RASMB) e‐mail list server. This e‐mail group list is for discussions and questions regarding the study of reversible interactions. The dominant focus is analytical ultracentrifugation (and nearly all the experts participate), but tremendous expertise in many other relevant areas is represented as well.
   rasmb@rasmb.bbri.org (to post questions)
  Reversible Associations in Structural and Molecular Biology (RASMB) Web site. Distributes public domain software for analytical ultracentrifugation.
   http://www.bbri.org/RASMB/rasmb.html
  Distributes public domain software for analytical ultracentrifugation. Also maintains an archive of the RASMB discussions and tutorials on sedimentation methodology.
   http://www.cauma.uthscsa.edu
  This site by the Protein Interaction Facility at the Huntsman Cancer Institute nicely describes the basics of SPR, analytical ultracentrifugation, and titration calorimetry, and is the distribution point for the CLAMP package of public‐domain SPR analysis software.
   http://www.hci.utah.edu/groups/interaction/index.htm
  Analytical ultracentrifugation products from Beckman Coulter. Useful application notes and bibliographies.
   http://www.beckmancoulter.com/beckman/biorsrch/prodinfo/xla/xlaprod.asp
  Calorimetry Sciences Web site. Useful calorimetry application notes and bibliographies.
   http://www.calscorp.com
  Microcal Web site. Useful calorimetry application notes and bibliographies.
   http://www.microcalorimetry.com
  Panvera Web site (fluorescence polarization). Useful application notes and bibliographies.
   http://www.panvera.com/ls/fpabout.html
  Wyatt Technologies Web site (light scattering). Useful application notes and bibliographies.
   http://www.wyatt.com
  BIAcore Web site (SPR). Useful application notes and bibliographies.
   http://www.biacore.com
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