Fluorescence Polarization (FP) Assays for Monitoring Peptide‐Protein or Nucleic Acid‐Protein Binding

Nathan J. Moerke1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch090102
Online Posting Date:  December, 2009
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Abstract

The technique of fluorescence polarization (FP) is based on the observation that when a fluorescently labeled molecule is excited by polarized light, it emits light with a degree of polarization that is inversely proportional to the rate of molecular rotation. This property of fluorescence can be used to measure the interaction of a small labeled ligand with a larger protein and provides a basis for direct and competition binding assays. FP assays are readily adaptable to a high-throughput format, have been used successfully in screens directed against a wide range of targets, and are particularly valuable in screening for inhibitors of protein-protein and protein-nucleic acid interactions when a small binding epitope can be identified for one of the partners. The protocols in this article describe a general procedure for development of FP assays to monitor binding of such a peptide or oligonucleotide to a protein of interest. Curr. Protoc. Chem Biol. 1:1-15. © 2009 by John Wiley & Sons, Inc.

Keywords: fluorescence polarization; FP; peptides; nucleic acids; proteins; high-throughput screening

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Measurement of Direct Binding of a Fluorescently Labeled Peptide or Olignucleotide to a Protein by Fluorescence Polarization
  • Basic Protocol 2: Fluorescence Polarization Measurement of Competitive Binding to a Protein of an Unlabeled Peptide or Oligonucleotide with a Fluorescently Labeled Probe
  • Basic Protocol 3: Adaptation of a Competition Fluorescence Polarization Assay to High-Throughput Screening Format
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Measurement of Direct Binding of a Fluorescently Labeled Peptide or Olignucleotide to a Protein by Fluorescence Polarization

 Materials
  • Protein stock solution: 40 µM eIF4E (Moerke et al., 2007) in protein dilution buffer (see recipe for buffer)
  • Protein dilution buffer (see recipe)
  • Fluorescently labeled peptide stock solution: 10 µM eIF4E-fluorescein peptide KYTYDELFQLK in protein dilution buffer (see recipe for buffer)
  • Black opaque 384-well microplates (Corning, cat. no. 3820)
  • FP-capable plate reader (e.g., Analyst HT, Molecular Devices)
  • Spreadsheet or graphing software

Basic Protocol 2: Fluorescence Polarization Measurement of Competitive Binding to a Protein of an Unlabeled Peptide or Oligonucleotide with a Fluorescently Labeled Probe

 Materials
  • Unlabeled competitor peptide stock solution: 7 mM eIF4G peptide KKQYDREFLLDFQFMPA in DMSO (see recipe)
  • Dimethyl sulfoxide (DMSO)
  • Protein stock solution: 40 µM eIF4E (Moerke et al., 2007) in protein dilution buffer (see recipe for buffer)
  • Fluorescently labeled peptide stock solution: 10 µM eIF4G-fluorescein peptide KYTYDELFQLK in protein dilution buffer (see recipe)
  • Protein dilution buffer (see recipe)
  • Black opaque 384-well microplates (Corning, cat. no. 3820)
  • FP-capable plate reader (e.g., Analyst HT, Molecular Devices)
  • Spreadsheet or graphing software

Basic Protocol 3: Adaptation of a Competition Fluorescence Polarization Assay to High-Throughput Screening Format

 Materials
  • Fluorescently labeled peptide or oligonucleotide stock solution (see Basic Protocols 1 and 2)
  • Protein stock solution (see Basic Protocols 1 and 2)
  • Unlabeled competitor peptide or oligonucleotide stock solution (see Basic Protocols 1 and 2)
  • Dimethylsulfoxide (DMSO)
  • Protein dilution buffer (see recipe)
  • Polypropylene 384-well compound storage plates (Thermo Scientific, cat. no. AB-1056)
  • Automated liquid dispenser for multiwall plates (Matrix WellMate, Thermo Scientific)
  • Black opaque 384-well microplates (Corning, cat. no. 3820)
  • Pin transfer apparatus (V&P Scientific, http://www.vp-scientific.com/)
  • FP-capable plate reader (e.g., Analyst HT, Molecular Devices)
  • Spreadsheet or graphing software
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Figures

  •  FigureFigure 1. Schematic depicting the basic principle of fluorescence polarization. When a small peptide or nucleic acid ligand (dark circle) with a fluorescent label attached (white circle) is excited by polarized light at the excitation wavelength of the fluorophore, the ligand reorients to a significant degree due to molecular tumbling during the excited state lifetime of the fluorophore. This causes the emitted light to be largely depolarized. If the ligand is bound to a protein (gray ellipse), the resulting complex tumbles much slower, and the emitted light retains its polarization.
    View Image
  •  FigureFigure 2. Direct binding of a fluorescent labeled eIF4G peptide to eIF4E. Fluorescence polarization (in units of mP) is plotted as a function of eIF4E concentration using a linear (top) or logarithmic scale (bottom). The concentration of eIF4G peptide is 1 µM.
    View Image
  •  FigureFigure 3. Competitive displacement of a fluorescent labeled eIF4G peptide from eIF4E by an unlabeled eIF4G peptide. Fluorescence polarization (in units of mP) is plotted as a function of unlabeled eIF4G peptide concentration using a linear (top) or logarithmic scale (bottom). The concentration of labeled eIF4G peptide is 1 µM and the concentration of eIF4E is 5 µM.
    View Image
  •  FigureFigure 4. Flowchart describing the stages of development of an FP assay for high-throughput screening, with recommended troubleshooting procedures for each stage.
    View Image

Videos

Literature Cited

Literature Cited
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    Gingras, A.C., Raught, G., and Sonenberg, N. 1999. eIF4 initiation factors: Effectors of mRNA recruitment to ribosomes and regulators of translation. Annu. Rev. Biochem. 68:913-963.
    Huang, X. 2003. Fluorescence polarization competition assay: The range of resolvable inhibitor potency is limited by the affinity of the fluorescent ligand. J. Biomol. Scr. 8:34-38.
    Jameson, D.M. and Seifried, S.E. 1999. Quantification of protein-protein interactions using fluorescence polarization. Methods 19:222-233.
    Jameson, D.M. and Croney, J.C. 2003. Fluorescence polarization: past, present and future. Comb. Chem. High Throughput Scr. 6:167-173.
    Moerke, N.J., Aktas, H., Chen, H., Cantel, S., Reibarkh, M.Y., Fahmy, A., Gross, J.D., Degterev, A., Yuan, J., Chorev, M., Halperin, J.A., and Wagner, G. 2007. Small-molecule inhibition of the interaction between the translation initiation factors eIF4E and eIF4G. Cell 128:257-267.
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    Roehrl, M.H., Wang, J.Y., and Wagner, G. 2004. A general framework for development and data analysis of competitive high-throughput screens for small-molecule inhibitors of protein-protein interactions by fluorescence polarization. Biochemistry 43:16056-16066.
    Rusinova, E., Tretyachenko-Ladokhina, V., Vele, O.E., Senear, D.F., and Alexander Ross, J.B. 2002. Alexa and Oregon Green dyes as fluorescence anisotropy probes for measuring protein-protein and protein-nucleic acid interactions. Anal. Biochem. 308:18-25.
    Simeonov, A., Jadhav, A., Thomas, C.J., Wang, Y., Huang, R., Southall, N.T., Shinn, P., Smith, J., Austin, C.P., Auld, D.S., and Inglese, J. 2008. Fluorescence spectroscopic profiling of compound libraries. J. Med. Chem. 51:2363-2371.
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    Turek-Etienne, T.C., Small, E.C., Soh, S.C., Xin, T.A., Gaitonde, P.V., Barrabee, E.B., Hart, R.F., and Bryant, R.W. 2003. Evaluation of fluorescent compound interference in 4 fluorescence polarization assays: 2 kinases, 1 protease, and 1 phosphatase. J. Biomol. Scr. 8:176-184.
    Turek-Etienne, T.C., Lei, M., Terracciano, J.S., Langsdorf, E.F., Bryant, R.W., Hart, R.F., and Horan, A.C. 2004. Use of red-shifted dyes in a fluorescence polarization AKT kinase assay for detection of biological activity in natural product extracts. J. Biomol. Scr. 9:52-61.
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 Internet Resources
    http://www.ncgc.nih.gov/guidance/manual_toc.html

National Center for Chemical Genomics Assay Guidance Manual. This provides an overview of FP assays in high-throughput screening and a comparison to other assay methods.

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