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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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 MgCl₂ ( 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‐His₆ ‐TP or TP‐His₆ ‐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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Figures

Figure 19.9.1 Flow chart of hydroxyl‐radical protein footprinting protocol.

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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.

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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 _complex − I _GreB )/( I _complex + I _GreB ), plotted against residue number (I_complex and I_GreB 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.

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Videos

Literature Cited

Literature Cited
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Identifying Protein Interactions by Hydroxyl‐Radical Protein Footprinting

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited