Identifying Protein Interactions by Hydroxyl‐Radical Protein Footprinting

Nick Loizos1

1 ImClone Systems Incorporated, New York, New York
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.9
DOI:  10.1002/0471140864.ps1909s38
Online Posting Date:  November, 2004
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Hydroxyl‐radical protein footprinting is a straightforward and direct method to map protein sites involved in macromolecular interactions. The first step is to radioactively end‐label the protein. Using hydroxyl radicals as a peptide backbone cleavage reagent, the protein is then cleaved in the absence and presence of ligand. Cleavage products are separated by high resolution gel electrophoresis. The digital image of the footprinting gel can be subjected to quantitative analysis to identify changes in the sensitivity of the protein to hydroxyl‐radical cleavage. Molecular weight markers are electrophoresed on the same gel and hydroxyl‐radical cleavage sites assigned by interpolation between the known cleavage sites of the markers. The results are presented in the form of a difference plot that shows regions of the protein that change their susceptibility to cleavage while bound to a ligand.

Keywords: hydroxyl‐radical; protein footprinting; macromolecular interactions; protein end‐labeling; Fe‐EDTA

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Table of Contents

  • Basic Protocol 1: Generation of an Active Recombinant Protein That is End‐Labeled
  • Basic Protocol 2: Hydroxyl Radical Cleavage of Protein in Absence and Presence of Ligand
  • Basic Protocol 3: Analyze Gel Data
  • Support Protocol 1: Preparation of Molecular Weight Standards
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Generation of an Active Recombinant Protein That is End‐Labeled

  • DNA insert containing test protein's open reading frame
  • Modified vectors (available from Nick Loizos on request) of the pET expression system:
    • PK‐pET‐16b
    • pET1529‐pK
  • Heart muscle kinase, bovine (HMK; Sigma): reconstitute in 40 mM dithiothreitol
  • 6000 Ci/mmol [γ‐32P]ATP or 2000 Ci/mmol [γ‐33P]ATP (Perkin Elmer)
  • 1 M Tris⋅Cl, pH 7.9 ( appendix 2E)
  • 5.0 M NaCl ( appendix 2E)
  • 1 M MgCl 2 ( appendix 2E)
  • Ni2+‐NTA agarose beads (Qiagen), preequilibrated in binding buffer
  • Binding buffer (see recipe)
  • Elution buffer (see recipe)
  • 1.5‐ml centrifuge tube
  • 30°C water bath or heatblock
  • Microcon 10 microconcentrator (Amicon)
  • Additional reagents and equipment for restriction digestion ( appendix 4I), transformation of E. coli ( appendix 4D), metal chelate affinity chromatography (MCAC; unit 9.4), and appropriate activity assay for the protein of interest

Basic Protocol 2: Hydroxyl Radical Cleavage of Protein in Absence and Presence of Ligand

  • End‐labeled protein (see protocol 1)
  • Ligand
  • Footprinting reaction buffer (see recipe)
  • Iron‐EDTA solution (see recipe)
  • 0.2 M sodium ascorbate in footprinting reaction buffer
  • 10 mM hydrogen peroxide
  • 3× loading buffer (see recipe)

Basic Protocol 3: Analyze Gel Data

  • 33%T, 3%C polyacrylamide, ultrapure (ICN)
  • 2 M Tris⋅Cl, pH 7.9 ( appendix 2E)/0.3% (w/v) SDS: store up to 6 months at room temperature
  • 80% glycerol
  • 10% (w/v) ammonium persulfate
  • Anode buffer (see recipe)
  • Cathode buffer (see recipe)
  • Cleaved test protein (TP) or test protein:ligand (TP:ligand) complex (see protocol 2)
  • Molecular weight standards (see protocol 4)
  • Vertical electrophoresis apparatus
  • PhosphorImager (Storm model from Molecular Dynamics) or equivalent
  • IMAGEQUANT software (Molecular Dynamics)
  • ALIGN software (available upon request from T. Heyduk; )
  • Excel software (Microsoft) or equivalent spreadsheet program

Support Protocol 1: Preparation of Molecular Weight Standards

  • HMK‐His 6‐TP or TP‐His 6‐HMK (see protocol 2)
  • Urea
  • 1 M Tris⋅Cl, pH 9.0 ( appendix 2E)
  • Endoproteinase Lys‐C, Glu‐C, or Asp‐N (Sigma)
  • 1× and 3× loading buffer (see recipe)
  • CNBr (Aldrich)
  • 20% SDS ( appendix 2E)
  • 1 M HCl
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Literature Cited

Literature Cited
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   Brenowitz, M., Senear, D.F., Shea, M.A., and Ackers, G.K. 1986. Quantitative DNase footprint titration: A method for studying protein‐DNA interactions. Methods Enzymol. 130:132‐181.
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