Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking

Karsten Melcher1, Hung‐Ta Chen2

1 University of Ulster, Coleraine, 2 Fred Hutchinson Cancer Research Center, Seattle, Washington
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 17.10
DOI:  10.1002/0471143030.cb1710s33
Online Posting Date:  January, 2007
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Abstract

Chemical crosslinking provides information about protein‐protein interactions in the context of intact protein complexes; therefore, it is particularly suited to the analysis of multiprotein complexes. Rather than a single distinct technique, chemical crosslinking represents a smorgasbord of techniques that differ significantly both in chemistry and in scope. This unit will attempt to guide the reader through the complexities of crosslinking to find the most suitable approach for a given biological question. Sample protocols for two crosslinking methods considered to be particularly useful for the analysis of large multiprotein complexes are provided: His6‐mediated crosslinking and photoinducible label transfer crosslinking.

Keywords: Photoinducible crosslinking; label transfer; oxidative crosslinking; His6‐mediated crosslinking; immunoblotting; MALDI‐TOF‐MS

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: His6‐Mediated Crosslinking
  • Basic Protocol 2: Bait‐Localized Photoactivatable Crosslinking
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: His6‐Mediated Crosslinking

  Materials
  • Saccharomyces cerevisiae cultures: source of multiprotein complexes, either wild‐type cells or cells in which putative target polypeptides are genomically epitope‐tagged (see Klein et al., )
  • YEPD medium (see recipe)
  • Extract buffer (see recipe)
  • 0.5‐mm glass beads, (e.g., Scientific Instruments), prechilled to −20°C
  • Siliconized glass wool, sterile (see recipe)
  • Ammonium sulfate, finely ground with mortar and pestle or with coffee grinder
  • 1 M KOH
  • Dialysis buffer (see recipe)
  • Glutathione‐Sepharose immobilized proteins (see recipe) in A50 buffer (see recipe)
    • ∼80 µg His6‐GST‐TEVcs‐bait protein
    • ∼20 µg His6‐GST‐TEVcs (control)
  • A50 buffer (see recipe)
  • Wash buffer: A50 buffer (see recipe) without DTT
  • 4× and 2× SDS sample buffer (see recipe)
  • 12 mM nickel acetate
  • 12 mM magnesium monoperphtalate (MMPP; Aldrich): prepare fresh just before use
  • TEV protease cleavage buffer (see recipe)
  • TEV protease (Invitrogen or USB)
  • Visible light spectrophotometer
  • 50‐ml centrifuge tube
  • Refrigerated centrifuge
  • 30‐ml round‐bottom Corex tube
  • 10‐ml syringe
  • Ultracentrifuge (e.g., Beckman 70.1Ti) and corresponding ∼10‐ml ultracentrifuge tubes, precooled to 4°C
  • 25‐ml beakers
  • Magnetic stir plate and stir bars
  • Dialysis tubing or dialysis cassettes (10,000 to 12,000 kDa MWCO; e.g. Slide‐A‐Lyzer cassettes from Pierce)
  • 0.5‐ml microcentrifuge tubes
  • Nutator
  • Additional reagents and equipment for determining protein concentration ( appendix 3H), performing SDS‐PAGE (unit 6.1), and detecting proteins by immunoblotting (unit 6.2)

Basic Protocol 2: Bait‐Localized Photoactivatable Crosslinking

  Materials
  • 600 mM N‐[(2‐pyridyldithio)ethyl]‐4‐azidosalicylamide (PEAS) stock solution: prepare by dissolving the solid compound (Invitrogen) in DMSO; store up to 2 years at −70°C in an amber bottle
  • 0.1 M sodium phosphate buffer, pH 7.4 ( appendix 2A)
  • Na125I in NaOH solution, pH 8.0‐12.0 (e.g., Amersham Biosciences, specific activity >0.6 TBq/mg iodide, 15 Ci/mg iodide)
  • 50 mM tyrosine in water
  • 7.5 µM purified yeast transcription factor TFIIB: pass the purified protein sample through a desalting column (Chen and Hahn, ) to remove the reducing agent in the purification buffer and elute in TFIIB conjugation buffer (see recipe)
  • TFIIB conjugation buffer
  • Dry Ice
  • Multiprotein complex: e.g., RNA polymerase II pre‐initiation complex (PIC) on beads formed using the immobilized template assay (Chen and Hahn, )
  • Acetate transcription buffer (see recipe)
  • Reducing agent: dithiothreitol or 2‐mercaptoethanol
  • IODO‐GEN precoated iodination tube (Pierce)
  • Desalting column: NAP‐5 Sephadex G‐25 column (Amersham Biosciences)
  • Scintillation counter for gamma counting
  • −70°C freezer
  • Magnet
  • UV irradiation device: ∼350‐nm wavelength
  • Additional reagents and equipment for performing SDS‐PAGE (unit 6.1) and autoradiography (unit 6.3)
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Figures

Videos

Literature Cited

Literature Cited
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   Brown, K.C., Yang, S.‐H., and Kodadek, T. 1995. Highly specific oxidative cross‐linking of proteins mediated by a nickel‐peptide complex. Biochemistry 34:4733‐4739.
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