Analysis of Protein Ligand‐Receptor Binding by Photoaffinity Cross‐Linking

Ling Wu1, Bin Xu1

1 Department of Biochemistry and Center for Drug Discovery, Virginia Polytechnic Institute and State University, Blacksburg, Virginia
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.26
DOI:  10.1002/0471140864.ps1926s79
Online Posting Date:  February, 2015
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Abstract

Photoaffinity cross‐linking is a rapidly developing technology for studying biomolecular interactions, including protein ligand‐receptor binding. This technology provides detailed binding information including receptor contact sites, active conformation of receptor‐ligand complexes, global binding surfaces, and binding modes. Advancements in genetic technology have enabled non‐natural photoactive amino acid derivatives to be incorporated into designer or target proteins, providing a host of new opportunities for manufacturing protein photo‐probes while bypassing the traditional peptide or small protein limits of classical chemical synthesis. This unit provides several protocols for performing basic photoaffinity cross‐linking and related analyses for applications in ligand‐receptor binding and protein‐protein interactions. © 2015 by John Wiley & Sons, Inc.

Keywords: photoaffinity cross‐linking; ligand‐receptor binding; protein‐protein interaction; biosynthetic photo‐probe; mapping of binding sites

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Biosynthetic Photoactive Ligands
  • Alternate Protocol 1: Preparation of Photo‐Probes using Chemical Synthesis
  • Support Protocol 1: Enrichment of the Cognate Receptor
  • Basic Protocol 2: Photoaffinity Cross‐Linking
  • Support Protocol 2: Specificity of Photoaffinity Cross‐Linking
  • Basic Protocol 3: Mapping Photo‐Probe Contacts on the Receptor
  • Support Protocol 3: Estimation of Cross‐Linking Efficiency
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Biosynthetic Photoactive Ligands

  Materials
  • Protein expression vector (such as a pET expression vector that contains a his 6‐tag in the vector; resulting expressed ligand has a his‐tag at either amino‐ or carboxy‐terminal of the protein)
  • Protein ligand expression vector with one codon mutated to TAG amber codon (for a site‐directed mutagenesis protocol, see unit 8.5; Cormack, )
  • SOC medium (Invitrogen)
  • pEVOL‐AzF‐RS vector (for expressing RNA synthetase specifically optimized for p‐azidophenylalanine [AzF]; chloramphenicol resistant; Young et al., )
  • Suitable E. coli strain for protein expression, such as BL21(DE3) cells
  • LB medium (with appropriate antibiotics, Fisher Scientific)
  • LB‐agar plates
  • p‐Azidophenylalanine (AzF; Bachem)
  • Isopropyl β‐D‐1‐thiogalactopyranoside (IPTG)
  • Arabinose
  • Cell lysis buffer (see recipe)
  • Washing buffer (see recipe)
  • Elution buffer (see recipe)
  • Ni‐NTA resin
  • SDS polyacrylamide gel
  • 17 × 100–mm sterile Falcon 2059 test tube
  • Water bath, 42°C
  • Shaker incubator (with temperature control)
  • 0.22‐μm size filter
  • Sonicator
  • Nutator
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1; Gallagher et al., 2012) and electrospray ionization mass spectrometry (ESI‐MS; Coligan et al., )

Alternate Protocol 1: Preparation of Photo‐Probes using Chemical Synthesis

  Materials
  • Protein ligand containing the Pmp side chain
  • HCl
  • Sodium nitrite (NaNO 2)
  • Sodium azide (NaN 3)
  • Saturated picric acid solution (Fisher Scientific)
  • Dry acetone (Fisher Scientific)
  • Dry ether (Fisher Scientific)
  • Sodium acetate

Support Protocol 1: Enrichment of the Cognate Receptor

  Materials
  • Cell line (such as CHO, HEK293 cells) over‐expressing membrane receptor of interest
  • Protease inhibitor cocktail (Roche)
  • Wheat germ agglutinin (WGA)‐coupled agarose (Vector Laboratories)
  • Bradford assay (see unit 3.4; Olson and Markwell, )
  • Receptor extraction buffer (see recipe)
  • Receptor washing buffer 1 (see recipe)
  • Receptor washing buffer 2 (see recipe)
  • Receptor elution buffer (see recipe)
  • Glycerol
  • N‐Acetylglucosamine
  • Nutator
  • Chromatography column
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1; Gallagher et al., 2012) and Coomassie blue staining (Sasse and Gallagher, )

Basic Protocol 2: Photoaffinity Cross‐Linking

  Materials
  • Photoactive protein ligands
  • Wheat germ agglutinin (WGA)‐agarose‐enriched cognate receptors
  • Dithiothreitol (DTT)
  • Nitrocellulose membrane
  • NeutrAvidin (Thermo Fisher Scientific)
  • Anti‐His antibody (Penta‐His antibody; Qiagen)
  • Restore Western Blot Stripping buffer (Thermo Fisher Scientific)
  • Nutator
  • Costar assay plate (Corning)
  • Mineralight lamp, model UVG‐54 (UVP)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1; Gallagher et al., 2012)

Support Protocol 2: Specificity of Photoaffinity Cross‐Linking

  Additional Materials (also see protocol 4)
  • Non‐photoactive protein ligands (ordinary ligand)

Basic Protocol 3: Mapping Photo‐Probe Contacts on the Receptor

  Materials
  • Photoactive protein ligands
  • WGA‐agarose‐enriched cognate membrane receptors
  • Proteolytic digestion buffer (see recipe)
  • Chymotrypsin (Sigma‐Aldrich)
  • Trypsin supplemented with EDTA (Cellgro)
  • Soybean trypsin inhibitor (Sigma‐Aldrich)
  • Laemmli sample buffer (Bio‐Rad)
  • Dithiothreitol (DTT)
  • Nitrocellulose membrane
  • NeutrAvidin (Thermo Fisher Scientific)
  • Anti‐His antibody (Penta‐His antibody; Qiagen)
  • Restore Western Blot Stripping buffer (Thermo Fisher Scientific)
  • Peptide‐N‐glycosidase F (N‐Glycanase; PNGase F; Prozyme)
  • Sodium phosphate
  • β‐Mercaptoethanol
  • Sodium dodecyl sulfate (SDS)
  • Costar assay plate (Corning)
  • Mineralight lamp, model UVG‐54 (UVP)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1; Gallagher et al., 2012)

Support Protocol 3: Estimation of Cross‐Linking Efficiency

  Materials
  • Photoactive ligands
  • WGA‐agarose‐enriched cognate receptors
  • Dithiothreitol (DTT)
  • Nitrocellulose membrane
  • Costar assay plate (Corning)
  • Mineralight lamp, model UVG‐54 (UVP)
  • Gel densitometry
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1; Gallagher et al., 2012)
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Figures

Videos

Literature Cited

Literature Cited
  Bayley, H. 1983. Photogenerated Reagents in Biochemistry and Molecular Biology. Elsevier, Amsterdam.
  Chin, J.W., Santoro, S.W., Martin, A.B., King, D.S., Wang, L., and Schultz, P.G. 2002. Addition of p‐azido‐l‐phenylalanine to the genetic code of Escherichia coli. J. Am. Chem. Soc. 124:9026‐9027.
  Coin, I., Katritch, V., Sun, T., Xiang, Z., Siu, F.Y., Beyermann, M., Stevens, R.C., and Wang, L. 2013. Genetically encoded chemical probes in cells reveal the binding path of urocortin‐I to CRF class B GPCR. Cell. 155:1258‐1269.
  Coligan, J.E., Dunn, B.M., Ploegh, H.L., Speicher, D.W., and Wingfield, P.T. 2014. Current Protocols in Protein Science. Chapter 16, John Wiley & Sons, Hoboken, NJ.
  Cormack, B. 2001. Directed mutagenesis using the polymerase chain reaction. Curr. Protoc. Mol. Biol. 37:8.5.1‐8.5.10.
  Creighton, T.E. 1993. Chemical properties of polypeptides. In Proteins: Structures and Molecular Properties, 2nd edition. pp. 38‐40. W. H. Freeman and Company, New York.
  Dormán, G., and Prestwich, G.D. 1994. Benzophenone photophores in biochemistry. Biochemistry 33:5661‐5673.
  Eberle, A.N. and de Graan, P.N.E. 1980. General principles for photoaffinity labeling of peptide hormone receptors. Trends Biochem. Sci. 5:320‐322.
  Fabry, M., Schaefer, E., Ellis, L., Kojro, E., Fahrenholz, F., and Brandenburg, D. 1992. Detection of a new hormone contact site within the insulin receptor ectodomain by the use of a novel photoreactive insulin. J. Biol. Chem. 267:8950‐8956.
  Gallagher, S.R. 2012. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Protein Sci. 68:10.1.1‐10.1.44.
  Grunbeck, A., Huber, T., and Sakmar, T.P. 2013. Mapping a ligand binding site using genetically encoded photoactivatable crosslinkers. Methods Enzymol. 520:307‐322.
  Grunbeck, A., Huber, T., Abrol, R., Trzaskowski, B., Goddard, W.A. III, and Sakmar, T.P. 2012. Genetically encoded photo‐cross‐linkers map the binding site of an allosteric drug on a G protein‐coupled receptor. ACS Chem. Biol. 7:967‐972.
  Hagel, L. 2001. Gel‐filtration chromatography. Curr. Protoc. Protein Sci. 14:8.3.1‐8.3.30.
  Hino, N., Okazaki, Y., Kobayashi, T., Hayashi, A., Sakamoto, K., and Yokoyama, S. 2005. Protein photo‐cross‐linking in mammalian cells by site‐specific incorporation of a photoreactive amino acid. Nat. Methods 2:201‐206.
  Huang, Y., Russell, W.K., Wan, W., Pai, P.J., Russell, D.H., and Liu, W. 2010. A convenient method for genetic incorporation of multiple noncanonical amino acids into one protein in Escherichia coli. Mol. Biosyst. 6:683‐686.
  Huang, K., Xu, B., Hu, S.Q., Chu, Y.C., Hua, Q.X., Qu, Y., Li, B., Wang, S., Wang, R.Y., Nakagawa, S.H., Theede, A.M., Whittaker, J., De Meyts, P., Katsoyannis, P.G., and Weiss, M.A. 2004. How insulin binds: The B‐chain alpha‐helix contacts the L1 beta‐helix of the insulin receptor. J. Mol. Biol. 341:529‐550.
  Kurose, T., Pashmforoush, M., Yoshimasa, Y., Carroll, R., Schwartz, G.P., Burke, G.T., Katsoyannis, P.G., and Steiner, D.F. 1994. Cross‐linking of a B25 azidophenylalanine insulin derivative to the carboxyl‐terminal region of the alpha‐subunit of the insulin receptor. Identification of a new insulin‐binding domain in the insulin receptor. J. Biol. Chem. 269:29190‐29197.
  Liu, W., Brock, A., Chen, S., Chen, S., and Schultz, P.G. 2007. The genetic incorporation of unnatural amino acids into proteins in mammalian cells. Nat. Methods. 4:239‐244.
  Marglin, A. and Merrifield, R.B. 1970. Chemical synthesis of peptides and proteins. Annu. Rev. Biochem. 39:841‐866.
  Menting, J.G., Whittaker, J., Margetts, M.B., Whittaker, L.J., Kong, G.K., Smith, B.J., Watson, C.J., Záková, L., Kletvíková E., Jiráček, J., Chan, S.J., Steiner, D.F., Dodson, G.G., Brzozowski, A.M., Weiss, M.A., Ward, C.W., and Lawrence, M.C. 2013. How insulin engages its primary binding site on the insulin receptor. Nature. 493:241‐245.
  Olson, B.J. and Markwell, J. 2007. Assays for determination of protein concentration. Curr. Protoc. Protein Sci. 48:3.4.1‐3.4.29.
  Preston, G.W. and Wilson, A.J. 2013. Photo‐induced covalent cross‐linking for the analysis of biomolecular interactions. Chem. Soc. Rev. 42:3289‐3301.
  Sasse, J. and Gallagher, S.R. 2009. Staining proteins in gels. Curr. Protoc. Mol. Biol. 85:10.6.1‐10.6.27.
  Singh, S., Zhang, M., Bertheleme, N., Kara, E., Strange, P.G. and Byrne, B. 2012. Radioligand binding analysis as a tool for quality control of GPCR production for structural characterization: adenosine A2aR as a template for study. Curr. Protoc. Protein Sci. 29:29.3.1‐29.3.22.
  Shoelson, S.E., Lee, J., Lynch, C.S., Backer, J.M., and Pilch, P.F. 1993. BpaB25 insulins. Photoactivatable analogues that quantitatively cross‐link, radiolabel, and activate the insulin receptor. J. Biol. Chem. 268:4085‐4091.
  Staros, J.V. 1980. Aryl azide photolabels in biochemistry. Trends Biochem. Sci. 5:320‐322.
  Suchanek, M., Radzikowska, A., and Thiele, C. 2005. Photo‐leucine and photo‐methionine allow identification of protein‐protein interactions in living cells. Nat. Methods. 2:261‐267.
  Wang, L., Xie, J., and Schultz, P.G. 2006. Expanding the genetic code. Annu. Rev. Biophys. Biomol. Struct. 35:225‐249.
  Wang, L., Brock, A., Herberich, B., and Schultz, P.G. 2001. Expanding the genetic code of Escherichia coli. Science. 292:498‐500.
  Xu, B. and Wu, L. 2014. Analysis of receptor‐ligand binding by photoaffinity cross‐linking. Sci. China Chem. 57:232‐242.
  Xu, B., Hu, S.Q., Chu, Y.C., Huang, K., Nakagawa, S.H., Whittaker, J., Katsoyannis, P.G., and Weiss, M.A. 2004. Diabetes‐associated mutations in insulin: Consecutive residues in the B chain contact distinct domains of the insulin receptor. Biochemistry. 43:8356‐8372.
  Xu, B., Huang, K., Chu, Y.C., Hu, S.Q., Nakagawa, S., Wang, S., Wang, R.Y., Whittaker, J., Katsoyannis, P.G., and Weiss, M.A. 2009. Decoding the cryptic active conformation of a protein by synthetic photoscanning: Insulin inserts a detachable arm between receptor domains. J. Biol. Chem. 284:14597‐14608.
  Ye, S., Köhrer, C., Huber, T., Kazmi, M., Sachdev, P., Yan, E.C., Bhagat, A., RajBhandary, U.L., and Sakmar, T.P. 2008. Site‐specific incorporation of keto amino acids into functional G protein coupled receptors using unnatural amino acid mutagenesis. J. Biol. Chem. 283:1525‐1533.
  Young, T.S., Ahmad, I., Yin, J.A., and Schultz, P.G. 2010. An enhanced system for unnatural amino acid mutagenesis in E. coli. J. Mol. Biol. 395:361‐374.
Key References
  Wang et al., 2006. See above.
  A comprehensive review on biosynthetic incorporation of unnatural amino acids (including photoactive groups) into a protein.
  Xu et al., 2004. See above.
  Detailed descriptions on experimental procedures for AzF‐based photoaffinity cross‐linking, validation, and sites mapping. A major source of information for the protocols in this unit.
  Xu and Wu, 2004. See above.
  An updated review on recent advances in the expanding utility of phtoaffinity cross‐linking in studying biomolecular interactions. The chemical mechanisms of the most commonly used photoactive probes and their respective photochemistry are summarized.
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