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

Andrew L. MacKinnon1, Jack Taunton2

1 Program in Chemistry and Chemical Biology and Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California, 2 Howard 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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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 1: Diazirine Photoactivation and Cu(I)‐Catalyzed Click Chemistry for Covalent Labeling and Detection of Protein Targets
  • Support Protocol 1: Affinity Purification of Probe‐Modified Proteins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Diazirine Photoactivation and Cu(I)‐Catalyzed Click Chemistry for Covalent Labeling and Detection of Protein Targets

  • 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. )
  • Dimethylsulfoxide (DMSO)
  • 20 µM stock solution of photo‐affinity probe (labeled 2 in Fig. ) in DMSO
  • 10% (w/v) sodium dodecyl sulfate (SDS) in H 2O
  • 5 mM TAMRA‐azide (labeled 4 in Fig. ) or biotin‐azide (labeled 5 in Fig. ), synthesized by published methods (Speers and Cravatt, ; Weerapana et al., ); similar reagents are available commercially from Invitrogen, e.g., PEG4 carboxamide‐6‐azidohexanyl biotin (Fig. )
  • 1.7 mM TBTA in 80% t‐butanol/20% DMSO (see recipe)
  • 50 mM CuSO 4 in H 2O
  • 50 mM Tris(2‐carboxyethyl)phosphine (TCEP) in H 2O, 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);
  • 0.5‐ml polypropylene microcentrifuge tubes
  • Typhoon 9400 phosphor imager (Amersham)
  • Additional reagents and equipment for SDS‐PAGE (e.g., Gallagher, ) and immunoblotting (western blotting ; e.g., Gallagher et al., )

Support Protocol 1: Affinity Purification of Probe‐Modified Proteins

  • Protein mixture labeled with photo‐affinity probe ( protocol 1, steps 1 to 5)
  • Liquid N 2
  • 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
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Literature Cited

   Adam, G.C., Burbaum, J., Kozarich, J.W., Patricelli, M.P., and Cravatt, B.F. 2004. Mapping enzyme active sites in complex proteomes. J. Am. Chem. Soc. 126:1363‐1368.
   Al‐Mawsawi, L.Q., Fikkert, V., Dayam, R., Witvrouw, M., Burke, T.R. Jr., Borchers, C.H., and Neamati, N. 2006. Discovery of a small‐molecule HIV‐1 integrase inhibitor‐binding site. Proc. Natl. Acad. Sci. U.S.A. 103:10080‐10085.
   Best, M.D. 2009. Click chemistry and bioorthogonal reactions: Unprecedented selectivity in the labeling of biological molecules. Biochemistry 48:6571‐6584.
   Bond, M.R., Zhang, H., Vu, P.D., and Kohler, J.J. 2009. Photocrosslinking of glycoconjugates using metabolically incorporated diazirine‐containing sugars. Nat. Protoc. 4:1044‐1063.
   Brunner, J. 1993. New photolabeling and crosslinking methods. Annu. Rev. Biochem. 62:483‐514.
   Chan, T.R., Hilgraf, R., Sharpless, K.B., and Fokin, V.V. 2004. Polytriazoles as copper(I)‐stabilizing ligands in catalysis. Org. Lett. 6:2853‐2855.
   Chen, Y., Bilban, M., Foster, C.A., and Boger, D.L. 2002. Solution‐phase parallel synthesis of a pharmacophore library of HUN‐7293 analogues: A general chemical mutagenesis approach to defining structure‐function properties of naturally occurring cyclic (depsi)peptides. J. Am. Chem. Soc. 124:5431‐5440.
   Colca, J.R., McDonald, W.G., Waldon, D.J., Leone, J.W., Lull, J.M., Bannow, C.A., Lund, E.T., and Mathews, W.R. 2004. Identification of a novel mitochondrial protein (“mitoNEET”) cross‐linked specifically by a thiazolidinedione photoprobe. Am. J. Physiol. Endocrinol. Metab. 286:E252‐E260.
   Dieterich, D.C., Lee, J.J., Link, A.J., Graumann, J., Tirrell, D.A., and Schuman, E.M. 2007. Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non‐canonical amino‐acid tagging. Nat. Protoc. 2:532‐540.
   Ding, S., Wu, T.Y.H., Brinker, A., Peters, E.C., Hur, W., Gray, N.S., and Schultz, P.G. 2004. Synthetic small molecules that control stem cell fate. Proc. Natl. Acad. Sci U.S.A. 100:856‐861.
   Dormán, G. 2000. Photoaffinity labeling in biological signal transduction. Top. Curr. Chem. 211:169‐225.
   Dormán, G. and Prestwich G.D. 1994. Benzophenone photophores in biochemistry. Biochemistry 33:5661‐5673.
   Ford, F., Yuzawa, T., Platz, M.S., Matzinger, S., and Fulscher, M. 1998. Rearrangement of dimethylcarbene to propene: Study by laser flash photolysis and ab initio molecular orbital theory. J. Am. Chem. Soc. 120:4430‐4438.
   Gallagher, S. 2006. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Mol. Biol. 75:10.2A.1‐10.2A.37.
   Gallagher, S., Winston, S.E., Fuller, S.A., and Hurrell, J.G.R. 2008. Immunoblotting and immunodetection. Curr. Protoc. Mol. Biol. 83:10.8.1‐10.8.28.
   Garrison, J.L., Kunkel, E.J., Hegde, R.S., Taunton, J. 2005. A substrate‐specific inhibitor of protein translocation into the endoplasmic reticulum. Nature 436:285‐289.
   Harding, M.W., Galat, A., Uehling, D.E., and Schreiber, S.L. 1989. A receptor for the immuno‐suppressant FK506 is a cis‐trans peptidyl‐prolyl isomerase. Nature 341:758‐760.
   Kukar, T.L., Ladd, T.B., Bann, M.A., Fraering, P.C., Narlawar, R., Maharvi, G.M., Healy, B., Chapman, R., Welzel, A.T., Price, R.W., Moore, B., Rangachari, V., Cusack, B., Eriksen, J., Jansen‐West, K., Verbeeck, C., Yager, D., Eckman, C., Ye, W., Sagi, S., Cottrell, B.A., Torpey, J., Rosenberry, T.L., Fauq, A., Wolfe, M.S., Schmidt, B., Walsh, D.M., Koo, E.H., and Golde, T.E. 2008. Substrate‐targeting γ‐secretase modulators. Nature 453:925‐930.
   Kwok, B.H.B., Koh, B., Ndubuisi, M.I., Elofsson, M., and Crews, C.M. 2001. The anti‐inflammatory natural product parthenolide from the medicinal herb Feverfew directly binds to and inhibits IκB kinase. Chem. Biol. 8:759‐766.
   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.
   Ong, S., Schenone, M., Margolin, A.A., Li, X., Do, K., Doud, M.K., Mani, D.R., Kuai, L., Wang, X., Wood, J.L., Tolliday, N.J., Koehler, A.N., Marcaurelle, L.A., Golub, T.R., Gould, R.J., Schreiber, S.L., and Carr, S.A. 2009. Identifying the proteins to which small‐molecule probes and drugs bind in cells. Proc. Natl. Acad. Sci. U.S.A. 106:4617‐4622.
   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.
   Platz, M., Admasu, A.S., Kwiatkowski, S., Crocker, P.J., Imai, N., and Watt, D.S. 1991. Photolysis of 3‐aryl‐3‐(trifluoromethyl) diazirines: A caveat regarding their use in photoaffinity probes. Bioconjug. Chem. 2:337‐341.
   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.
   Taunton, J., Hassig, C.A., and Schreiber, S.L. 1996. A mammalian histone deacetylase related to the yeast transcriptional regulator Rpd3p. Science 272:408‐411.
   Verhelst, S.H.L., Fonovic, M., and Bogyo, M. 2007. A mild chemically cleavable linker system for functional proteomic applications. Angew. Chem. Int. Ed. 46:1284‐1286.
   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.
   Xi, J., Liu, R., Rossi, M.J., Yang, J., Loll, P.J., Dailey, W.P., and Eckenhoff, R.G. 2006. Photoactive analogues of the haloether anesthetics provide high‐resolution features from low‐affinity interactions. ACS Chem. Biol. 1:377‐384.
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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