Spectroscopic Methods for the Determination of Protein Interactions

Yvonne Groemping1, Nadja Hellmann2

1 Department of Biomolecular Mechanisms Max Planck Institute for Medical Research, Heidelberg, 2 Institute for Molecular Biophysics University of Mainz, Mainz
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 20.8
DOI:  10.1002/0471140864.ps2008s39
Online Posting Date:  March, 2005
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Abstract

This unit provides guidelines on how to use steady‐state fluorescence spectroscopy for the quantification of protein‐protein interactions. The fluorescence of a protein is characterized by its excitation and emission spectra, quantum yield, and anisotropy. These parameters can change upon interaction with another protein and can be used to measure the extent of complex formation. The source of fluorescence can be an intrinsic fluorophore, such as tryptophan or tyrosine; a covalently attached fluorescent dye; or a fluorescent binding partner, such as a nucleotide or cofactor, that interacts specifically with the complex. Protocols are provided in this unit for determining affinity constants and stoichiometry values for protein‐protein interactions using equilibrium titration experiments. In addition, fluorescent labeling of proteins is discussed, and an introduction to data analysis is provided. Most of the topics addressed in this unit can easily be applied to other spectroscopic methods or to the analysis of protein‐ligand interactions.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Fluorescence Titration to Monitor Complex Formation and Determine Binding Constants
  • Basic Protocol 2: Introduction of a Fluorescent Label into a Protein and Determination of the Degree of Labeling
  • Basic Protocol 3: Analysis of Titration Experiments: Determination of Binding Affinity and Stoichiometry
  • Support Protocol 1: Determination of Optimal Emission and Excitation Wavelengths for Fluorescence Titration Experiments
  • Support Protocol 2: Determination of the Stability and Propensity for Photobleaching of a Fluorescent Protein
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Fluorescence Titration to Monitor Complex Formation and Determine Binding Constants

  Materials
  • Filtered and degassed buffer solution for diluting protein samples (see unit 7.7)
  • Protein “A” and compound “S” solutions, purified and of known concentration
  • Calibrated fluorescence spectrometer (see unit 7.7)
  • Cleaned cuvettes (see unit 7.7)
  • Stirring devices
NOTE: Always make sure that the solution has reached the required temperature before starting the measurement. Extra time must be allowed for temperature equilibration if the protein solution has been on ice before the experiment, as fluorescence is strongly temperature dependent. Samples in plastic cuvettes tend to require more time to reach their final temperature than do samples in glass or quartz cuvettes.NOTE: All of the experiments mentioned in this unit are equilibrium experiments. Thus, it is very important to ensure that samples are at equilibrium (i.e., that the fluorescence reaches a constant value) after every titration step. Depending on the speed of complex formation at the concentrations chosen, it may be necessary to wait for some time (i.e., several minutes) before recording data.

Basic Protocol 2: Introduction of a Fluorescent Label into a Protein and Determination of the Degree of Labeling

  Materials
  • Protein, purified and of known concentration (5 to 20 mg/ml), to be labeled
  • Reaction buffer (as recommended by supplier of labeling compound)
  • 10 mg/ml fluorescent dye in DMSO or DMF: Use succinimidyl esters (or sulfonyl chlorides) for the modification of amine groups, and use maleimides (or iodoacetamide) for the modification of thiol groups (in cysteine residues)
  • Filtered and degassed buffer solution (see unit 7.7)
  • Dialysis membranes or Slide‐A‐Lyzer dialysis kits (Pierce)
  • PD‐10 or NAP‐5 columns (Amersham Biosciences)
  • Cuvettes (see unit 7.7)
  • UV‐vis spectrometer
  • Additional reagents and equipment for dialysis ( appendix 3B) or buffer exchange using a desalting column (unit 8.3)
CAUTION: Dimethylsulfoxide (DMSO) and dimethylformamide (DMF) should be handled with caution, as they are known to facilitate the entry of organic molecules into tissues. It is strongly recommended that double gloves be used when handling these solvents.NOTE: The amount of protein used for labeling depends mainly on the availability of the protein. Typically, 5 to 10 mg is used.NOTE: Some fluorescent dyes are very photosensitive; thus, the labeling procedure should be allowed to take place under minimal light. Ideally, samples should always be kept in the dark or wrapped in aluminum foil and, if possible, kept on ice during and between experiments.

Basic Protocol 3: Analysis of Titration Experiments: Determination of Binding Affinity and Stoichiometry

  Materials
  • Filtered and degassed buffer solution for diluting protein samples (see unit 7.7)
  • Protein “A” and compound “S” solutions, purified and of known concentration
  • Calibrated fluorescence spectrometer (see unit 7.7)
  • Cleaned cuvettes (see unit 7.7)

Support Protocol 1: Determination of Optimal Emission and Excitation Wavelengths for Fluorescence Titration Experiments

  Materials
  • Filtered and degassed buffer solution for diluting protein samples (see unit 7.7)
  • Protein “A” and compound “S” solutions, purified and of known concentration
  • Calibrated fluorescence spectrometer (see unit 7.7)
  • Cleaned cuvettes (see unit 7.7)
NOTE: In the protocol that follows, it is assumed that protein A contains the fluorescent label.
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Figures

Videos

Literature Cited

   Solomon, M. and Stansbie, D. 1984. A coupled fluorometric rate assay for pyruvate dehydrogenase in cultured human fibroblasts. Anal. Biochem. 141:337‐343.
   Bai, Y., Perez, G.M., Beechem, J.M., and Weil, P.A. 1997. Structure‐function analysis of TAF130: Identification and characterization of a high‐affinity TATA‐binding protein interaction domain in the N terminus of yeast TAF(II)130. Mol. Cell. Biol. 17:3081‐3093.
   Becker, C.F., Hunter, C.L., Seidel, R., Kent, S.B., Goody, R.S., and Engelhard, M. 2003. Total chemical synthesis of a functional interacting protein pair: The protooncogene H‐Ras and the Ras‐binding domain of its effector c‐Raf1. Proc. Natl. Acad. Sci. U.S.A. 100:5075‐5080.
   Cornea, A. and Conn, P.M. 2002. Measurement of changes in fluorescence resonance energy transfer between gonadotropin‐releasing hormone receptors in response to agonists. Methods 27:333‐339.
   Hiratsuka, T. 1998. Prodan fluorescence reflects differences in nucleotide‐induced conformational states in the myosin head and allows continuous visualization of the ATPase reactions. Biochemistry 37:7167‐7176.
   Hutnik, C.M., MacManus, J.P., Banville, D., and Szabo, A.G. 1991. Metal‐induced changes in the fluorescence properties of tyrosine and tryptophan site‐specific mutants of oncomodulin. Biochemistry 30:7652‐7660.
   Jezewska, M.J. and Bujalowski, W. 1997. Quantitative analysis of ligand‐macromolecule interactions using differential dynamic quenching of the ligand fluorescence to monitor the binding. Biophys. Chem. 64:253‐269.
   Johnson, D.A., Leathers, V.L., Martinez, A.M., Walsh, D.A., and Fletcher, W.H. 1993. Fluorescence resonance energy transfer within a heterochromatic cAMP‐dependent protein kinase holoenzyme under equilibrium conditions: New insights into the conformational changes that result in cAMP‐dependent activation. Biochemistry 32:6402‐6410.
   Kelly, R.C. and von Hippel, P.H. 1976. DNA “melting” proteins: III: Fluorescence “mapping” of the nucleic acid binding site of bacteriophage T4 gene 32‐protein. J. Biol. Chem. 251:7229‐7239.
   Lakowitz, J.R. 1999. Principles of Fluorescence Spectroscopy. Kluwer Academic/Plenum Publishers, New York.
   LeTilly, V. and Royer, C.A. 1993. Fluorescence anisotropy assays implicate protein‐protein interactions in regulating trp repressor DNA binding. Biochemistry 32:7753‐7758.
   Li, Q., Du, H.N., and Hu, H.Y. 2003. Study of protein‐protein interactions by fluorescence of tryptophan analogs: Application to immunoglobulin G binding domain of streptococcal protein G. Biopolymers 72:116‐122.
   Lukas, T.J., Burgess, W.H., Prendergast, F.G., Lau, W., and Watterson, D.M. 1986. Calmodulin binding domains: Characterization of a phosphorylation and calmodulin binding site from myosin light chain kinase. Biochemistry 25:1458‐1464.
   Wagner, A., Simon, I., Sprinzl, M., and Goody, R.S. 1995. Interaction of guanosine nucleotides and their analogs with elongation factor Tu from Thermus thermophilus. Biochemistry 34:12535‐12542.
   Xia, X., Lin, J.T., and Kinne, R.K. 2003. Binding of phlorizin to the isolated C‐terminal extramembranous loop of the Na+/glucose cotransporter assessed by intrinsic tryptophan fluorescence. Biochemistry 42:6115‐6120.
Key References
   Gore, M.G. 2000. Spectrophotometry and Spectrofluorimetry: A Practical Approach. Oxford University Press, Oxford.
  Describes the theory and practical applications of many spectroscopic techniques, including fluorescence and absorption spectroscopy and circular dichroism. Emphasis is placed on the practical aspects of these techniques, and protocols for test experiments as well as lists of background literature are provided. A very good introduction to spectrometry.
   Lakowitz, 1999. See above.
  Provides basic and in‐depth information on fluorescence spectroscopy, including an overview of extrinsic fluorophores. Two chapters discuss time‐resolved measurements (advanced).
   Creighton, T.E. 1993. Proteins: Structures and Molecular Properties. W.H. Freeman, New York.
  A basic introduction to the use of spectroscopic methods for detecting conformational changes.
Internet Resources
   http://www.probes.com
  Molecular Probes Web site. Includes an online version of the Handbook of Fluorescent Probes and Research Products, with up‐to‐date background and technical information. An e‐mail newsletter is also available.
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