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Target Identification by Diazirine Photo‐Cross‐Linking and Click Chemistry

Andrew L. MacKinnon1,  Jack Taunton2,1

1Program in Chemistry and Chemical Biology and Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California
2Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California


Publication Name: 
Current Protocols in Chemical Biology
Unit Number: 
 
DOI: 
10.1002/9780470559277.ch090167
Online Posting Date: 
December, 2009
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Abstract

Target identification is often the rate-determining step in deciphering the mechanism of action of biologically active small molecules. Photo-affinity labeling (PAL) represents a useful biochemical strategy for target identification in complex protein mixtures. This unit describes the use of alkyl diazirine-based photo-affinity probes and Cu(I)-catalyzed click chemistry to covalently label and visualize the targets of biologically active small molecules. A general method for affinity purification of probe-modified proteins, useful for identification of protein targets, is also described. Curr. Protoc. Chem Biol. 1:55-73. © 2009 by John Wiley & Sons, Inc.

Keywords: photo-affinity labeling; diazirine; click chemistry; target identification; affinity purification

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol: Diazirine Photoactivation and Cu(I)-Catalyzed Click Chemistry for Covalent Labeling and Detection of Protein Targets
  • Support Protocol: Affinity Purification of Probe-Modified Proteins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol: Diazirine Photoactivation and Cu(I)-Catalyzed Click Chemistry for Covalent Labeling and Detection of Protein Targets

 Materials
  • Endoplasmic reticulum (ER) microsomes (~1 mg/ml total protein) or other soluble or membrane protein lysate containing the unknown macromolecular target, in PBS (see recipe for PBS)
  • 0.8 mM stock solution of photostable competitor compound (labeled 3 in Fig. 1)
  • Dimethylsulfoxide (DMSO)
  • 20 µM stock solution of photo-affinity probe (labeled 2 in Fig. 1) in DMSO
  • 10% (w/v) sodium dodecyl sulfate (SDS) in H2O
  • 5 mM TAMRA-azide (labeled 4 in Fig. 2) or biotin-azide (labeled 5 in Fig. 2), synthesized by published methods (Speers and Cravatt, 2004; Weerapana et al., 2007); similar reagents are available commercially from Invitrogen, e.g., PEG4 carboxamide-6-azidohexanyl biotin (Fig. 2)
  • 1.7 mM TBTA in 80% t-butanol/20% DMSO (see recipe)
  • 50 mM CuSO4 in H2O
  • 50 mM Tris(2-carboxyethyl)phosphine (TCEP) in H2O, adjusted to pH ~7 with 1 M NaOH; prepare immediately before use
  • 6× Laemmli sample buffer (see recipe)
  • Fluorescent molecular weight markers (Pierce)
  • 96-well plate or other open, shallow container
  • 1000 W Hg(Xe) lamp (Oriel Instruments, model 66923) with band-pass filter for irradiation at ~355 nm (Oriel Instruments, cat. no. 59810) and a filter to absorb heat (Oriel Instruments, cat. no. 59044); http://www.oriel.com/
  • 0.5-ml polypropylene microcentrifuge tubes
  • Typhoon 9400 phosphor imager (Amersham)
  • Additional reagents and equipment for SDS-PAGE (e.g., Gallagher, 2006) and immunoblotting (western blotting ; e.g., Gallagher et al., 2008)

Support Protocol: Affinity Purification of Probe-Modified Proteins

 Additional Materials (also see Basic Protocol)
  • Protein mixture labeled with photo-affinity probe (Basic Protocol, steps 1 to 5)
  • Liquid N2
  • Acetone cooled to –20°C
  • 1% SDS in PBS (see recipe for PBS)
  • Affinity purification buffer (see recipe)
  • Protein A–Sepharose beads (GE Healthcare)
  • Anti-TAMRA antibody (Invitrogen, cat. no. A6397)
  • Monomeric avidin–agarose beads (Pierce)
  • Wash buffer (see recipe)
  • Elution buffer (see recipe)
  • Polyallomer 1.5-ml microcentrifuge tubes (Beckman-Coulter)
  • Benchtop ultracentrifuge
  • Sonicating water bath
  • Refrigerated microcentrifuge
  • Rotating tube mixer
Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure 1.
    (A) Generalized scheme for photo-affinity labeling and detection using a diazirine and alkyne-containing photo-affinity probe (I). UV irradiation of the diazirine generates a carbene intermediate (II) that covalently cross-links to the protein target (III). The adduct is then detected by conjugation with an azide-containing reporter group under click chemistry conditions (IV). (B) Structures of the natural product HUN7293 (1), photo-affinity probe (2), and the photostable control compound (3).

    View Image
  • Figure 2.
    Structures of TAMRA-azide (4) and biotin-azide (5) used in the protocols in this unit, and the structure of a commercially available biotin-azide reagent (Invitrogen, cat. no. B10184).

    View Image
  • Figure 3.
    Structures of compounds 6, 7, and 8, which have a fluorescent reporter group (TAMRA) directly incorporated into the natural product scaffold.

    View Image
  • Figure 4.
    (A) Photo-cross-linking in ER microsomes with 2 (see Fig. 1) followed by click chemistry with TAMRA-azide (4; see Fig. 2) as described in the Basic Protocol. Sec61 is marked with an asterisk (figure adapted with permission from MacKinnon et al., 2007). (B) Photo-cross-linking in ER microsomes with 2 followed by click chemistry with biotin-azide (5) and affinity purification using monomeric avidin as described in the Support Protocol. Samples representing the click reaction (lane 1), the resolubilized protein pellet following acetone precipitation (lane 2), the post-monomeric avidin supernatant (lane 3), and the eluent (lane 4) were resolved by SDS-PAGE, transferred to nitrocellulose, and probed for biotinylated proteins with streptavidin-HRP (Strep-HRP). Percentages indicate the fraction of the total sample that was loaded in each lane. The position of Sec61 is marked with an asterisk. The biotinylated protein at ~21 kDa is a background band.

    View Image

Literature Cited

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    MacKinnon, A.L., Garrison, J.L., Hegde, R.S., and Taunton, J. 2007. Photo-leucine incorporation reveals the target of a cyclodepsipeptide inhibitor of cotranslational translocation. J. Am. Chem. Soc. 129:14560-14561.
    Okerberg, E.S., Wu, J., Zhang, B., Samii, B., Blackford, K., Winn, D.T., Shreder, K.R., Burbaum, J.J., and Patricelli, M.P. 2005. High-resolution functional proteomics by active-site peptide profiling. Proc. Natl. Acad. Sci. U.S.A. 102:4996-5001.
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    Osborne, A.R., Rapoport, T.A., and van den Berg, B. 2005. Protein translocation by the Sec61/SecY channel. Annu. Rev. Cell Dev. Biol. 21:529-550.
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    Sadakane, Y. and Hatanaka, Y. 2006. Photochemical fishing approaches for identifying target proteins and elucidating the structure of a ligand-binding region using carbene-generating photoreactive probes. Anal. Sci. 22:209-218.
    Saghatelian, A., Jessani, N., Joseph, A., Humphrey, M., and Cravatt, B.F. 2004. Activity-based probes for the proteomic profiling of metaloproteases. Proc. Natl. Acad. Sci. U.S.A. 101:1000-1005.
    Sin, N., Meng, L., Wang, M.Q.W., Wen, J.J., Bornmann, W.G., and Crews, C.M. 1997. The anti-angiogenic agent fumagillin covalently binds and inhibits the methionine aminopeptidase, MetAP-2. Proc. Natl. Acad. Sci. U.S.A. 94:6099-6103.
    Speers, A.E. and Cravatt, B.F. 2004. Profiling enzyme activities in vivo using click chemistry methods. Chem. Biol. 11:535-546.
    Speers, A.E. and Cravatt, B.F. 2005. A tandem orthogonal proteolysis strategy for high-content chemical proteomics. J. Am. Chem. Soc. 127:10018-10019.
    Strable, E., Prasuhn, D.E. Jr., Udit, A.K., Brown, S., Link, A.J., Ngo, J.T., Lander, G., Quispe, J., Potter, C.S., Carragher, B., Tirrell, D.A., and Finn, M.G. 2008. Unnatural amino acid incorporation into virus-like particles. Bioconjug. Chem. 19:866-875.
    Suchanek, M., Radzikowska, A., and Thiele, C. 2005. Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells. Nat. Methods 2:261-268.
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    Weerapana, E., Speers, A.E., and Cravatt, B.F. 2007. Tandem orthogonal proteolysis-activity-based protein profiling (TOP-ABPP)—A general method for mapping sites of probe modification in proteomes. Nat. Protoc. 2:1414-1425.
    Wittelsberger, A., Thomas, B.E., Mierke, D.F., and Rosenblatt, M. 2006. Methionine acts as a “magnet” in photoaffinity crosslinking experiments. FEBS Lett. 580:1872-1876.
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 Key References
    Best, M.D. 2009. See above.

A recent review of bio-orthogonal click chemistry methods.

    Brunner, 1993. See above.

An excellent introduction to the structure and chemistry of photoreactive groups and their use in photo-affinity labeling in biological systems.

    Colca et al., 2004. See above.

An excellent example of PAL for identifying a novel integral membrane target of a therapeutically relevant small molecule.

     
 
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