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Identifying Protein Interactions by Hydroxyl‐Radical Protein Footprinting

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  • Abstract
  • Table of Contents
  • Materials
  • Figures
  • Literature Cited

Abstract

 

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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Materials

Basic Protocol 1: Generation of an Active Recombinant Protein That is End‐Labeled

  Materials
  • 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‐32 P]ATP or 2000 Ci/mmol [γ‐33 P]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

  Materials
  • 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

  Materials
  • 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
  • TEMED
  • 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

  Materials
  • 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
GO TO THE FULL PROTOCOL:
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Figures

  •   Figure 19.9.1 Flow chart of hydroxyl‐radical protein footprinting protocol.
    View Image
  •   Figure 19.9.2 Schematic drawings of (A ) PK‐pET16b and (B ) pET1529‐PK expression vectors. Features of these vectors include the ampicillin (AMP) resistance gene, lacIq repressor gene, pBR322 origin of DNA replication, and T7 promoter and terminator. PK‐pET16b was kindly provided by Dr. O'Donnell (Kelman et al., ). Plasmid pET1529‐PK was constructed to contain a pET15b background and pET29a multiple cloning site—i.e., the multiple cloning site of pET29a (sequences between the Xba I to Bpu 1102I sites) was exchanged with the multiple cloning site of pET15b (sequences between the Xba I to Bpu 1102I sites). The HMK coding sequence was then inserted by recombinant PCR directly downstream of and in frame with the His tag.
    View Image
  •   Figure 19.9.3 Hydroxyl radical footprint of core RNAP (ligand) on GreB (test protein). (A ) phosphorimaged gel of hydroxyl‐radical cleaved GreB and GreB‐Core complex. Enhanced and protected areas are marked with arrows and brackets, respectively. (B ) Top part of phosphorimaged gel from experiment in which gel run time was extended and GreB was labeled with 33 P. Asterisk marks additional protected area not seen in (A) due to masking by the intensity of the uncleaved product. (C ) Difference plot showing normalized intensity difference, ( I complexI GreB )/( I complex + I GreB ), plotted against residue number (Icomplex and IGreB are the corrected intensities for the GreB‐core complex and GreB, respectively). Statistically significant differences according to a Student's t test (confidence level of 0.99999) are denoted by black bars.
    View Image

Videos

Literature Cited

Literature Cited
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   Borukhov, S., Lee, J., and Goldfarb, A. 1991. Mapping of a contact for the RNA 3′ terminus in the largest subunit of RNA polymerase. J. Biol. Chem. 266:23932‐23935.
   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.
   Casaz, P. and Buck, M. 1999. Region I modifies DNA‐binding domain conformation of sigma 54 within the holoenzyme. J. Mol. Biol. 285:507‐514.
   Colland, F., Orsini, G., Brody, E.N., Buc, H., and Kolb, A. 1998. The bacteriophage T4 AsiA protein: A molecular switch for sigma 70‐dependent promoters. Mol. Microbiol. 27:819‐829.
   Heyduk, E. and Heyduk, T. 1994. Mapping protein domains involved in macromolecular interactions: A novel protein footprinting approach. Biochemistry 33:9643‐9650.
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   Loizos, N. and Darst, S.A. 1999. Mapping interactions of Escherichia coli GreB with RNA polymerase and ternary elongation complexes. J. Biol. Chem. 274:23378‐23386.
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   Mossessova, E., Gulbis, J.M., and Goldberg, J. 1998. Structure of the guanine nucleotide exchange factor Sec7 domain of human arno and analysis of the interaction with ARF GTPase. Cell 92:415‐423.
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   Schagger, H. and von Jagow, G. 1987. Tricine‐sodium dodecyl sulfate‐polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166:368‐379.
   Wang, Y., Severinov, K., Loizos, N., Fenyo, D., Heyduk, E., Heyduk, T., Chait, B.T., and Darst, S.A. 1997. Determinants for Escherichia coli RNA polymerase assembly within the beta subunit. J. Mol. Biol. 270:648‐662.
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