Specificity Profiling of Protein‐Binding Domains Using One‐Bead‐One‐Compound Peptide Libraries

Andrew R. Kunys1, Wenlong Lian1, Dehua Pei1

1 Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch120125
Online Posting Date:  December, 2012
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Abstract

One‐bead‐one‐compound (OBOC) libraries consist of structurally related compounds (e.g., peptides) covalently attached to a solid support, with each resin bead carrying a unique compound. OBOC libraries of high structural diversity can be rapidly synthesized and screened without the need for any special equipment, and therefore can be employed in any chemical or biochemical laboratory. OBOC peptide libraries have been widely used to map the ligand specificity of proteins, to determine the substrate specificity of enzymes, and to develop inhibitors against macromolecular targets. They have proven particularly useful in profiling the binding specificity of protein modular domains (e.g., SH2 domains, BIR domains, and PDZ domains); subsequently, the specificity information can be used to predict the protein targets of these domains. The protocols outlined in this article describe the methodologies for synthesizing and screening OBOC peptide libraries against SH2 and PDZ domains, and the related data analysis. Curr. Protoc. Chem. Biol. 4:331‐355 © 2012 by John Wiley & Sons, Inc.

Keywords: protein‐protein interaction; protein binding domains; sequence specificity; peptide libraries; one‐bead‐one‐compound libraries; SH2 domains

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Split‐and‐Pool Synthesis of One‐Bead‐One‐Compound Libraries
  • Alternate Protocol 1: Synthesis of an Inverted Peptide Library
  • Basic Protocol 2: Chemical Labeling of Proteins with NHS‐Biotin
  • Alternate Protocol 2: Enzymatic Labeling of Proteins with Biotin‐CoA
  • Basic Protocol 3: On‐Bead Screening of One‐Bead‐One‐Compound Libraries
  • Alternate Protocol 3: On‐Bead Screening Against Fluorescently Labeled Protein
  • Basic Protocol 4: Partial Edman Degradation for Sequencing Support‐Bound Peptides
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Split‐and‐Pool Synthesis of One‐Bead‐One‐Compound Libraries

  Materials
  • TentaGel S NH 2 resin (90 µm, 0.3 mmol/g)
  • Dichloromethane (DCM; Sigma Aldrich)
  • Diethyl ether
  • Fmoc‐Met‐OSu (Aapptech, http://www.aapptec.com)
  • Boc‐Phe‐OSu (Aapptech, http://www.aapptec.com)
  • N′,N′‐dimethylformamide (DMF; Sigma‐Aldrich)
  • Acetaldehyde
  • Chloranil (Sigma‐Aldrich, cat. no. 45374)
  • Fmoc‐Met‐OH (Aapptech, http://www.aapptec.com)
  • O‐Benzotriazole‐N,N,N′,N′‐tetramethyluronium hexafluorophosphate (HBTU; Aapptech, http://www.aapptec.com)
  • N‐Hydroxybenzotriazole (HOBt; Aapptech, http://www.aapptec.com)
  • Diisopropylethylamine (DIPEA)
  • Piperidine
  • All desired Fmoc‐amino acids with acid‐liable sidechain protecting groups (tBu, Boc, etc.; Aapptech, http://www.aapptec.com)
  • Ninhydrin
  • Ethanol
  • Phenol
  • Potassium cyanide (KCN)
  • CD 3CO 2D (Sigma‐Aldrich)
  • CH 3CD 2CO 2D (Sigma‐Aldrich)
  • Fmoc‐pY‐OH (Aapptech)
  • Acetic anhydride
  • Modified reagent K (see recipe)
  • Chemglass solid‐phase peptide‐synthesis vessel with inner luer joint, >25 ml; Disc O.D: 20 mm; GL size: 25 (CG186202, Chemglass, cat. no. CG‐1862‐02)
  • Rotary shaker
  • Spatula
  • Watch glass
  • Vacuum source
  • 50‐ml conical centrifuge tubes (BD Falcon)

Alternate Protocol 1: Synthesis of an Inverted Peptide Library

  • Nα‐Fmoc‐Glu(δ‐N‐hydroxysuccinimidyl)‐O‐CH 2CH=CH 2 (Aapptech, http://www.aapptec.com)
  • Boc‐Gly‐OH (Aapptech, http://www.aapptec.com)
  • p‐Hydroxymethylbenzoic acid (HMBA; Sigma‐Aldrich)
  • Trifluoroacetic acid (TFA)
  • Fmoc‐Arg(Pbf)‐OH (Aapptech, http://www.aapptec.com)
  • N,N‐dicyclohexylcarbodiimide (DCC; Sigma‐Aldrich)
  • 4‐Dimethylaminopyridine (DMAP)
  • Tetrakis(triphenylphosphine)palladium (Sigma‐Aldrich)
  • Triphenylphosphine
  • N‐Methylaniline (Sigma‐Aldrich)
  • 0.5% sodium dimethyldithiocarbamate hydrate (Sigma‐Aldrich)
  • Benzotriazole‐1‐yl‐oxy‐tris‐pyrrolidino‐phosphonium hexafluorophosphate (PyBOP; Aapptech, http://www.aapptec.com)
  • 1 M NaOH

Basic Protocol 2: Chemical Labeling of Proteins with NHS‐Biotin

  Materials
  • Purified protein of interest dissolved in any buffer without strong nucleophiles (avoid Tris)
  • Bradford assay kit (BioRad)
  • Bicarbonate buffer: 100 mM sodium bicarbonate/100 mM NaCl
  • NHS‐biotin stock solution: dissolve NHS‐biotin (Thermo Scientific, cat. no. 21312) in DMSO for a final concentration of 10 mg/ml (store in small aliquots up to 1 year at −20 or −80°C
  • Sephadex G‐25 resin (GE Healthcare; optional)
  • Protein purification buffer: 50 mM HEPES/100 mM NaCl; adjust pH to 7.5 with HCl or NaOH
  • 30% or 50% (v/v) glycerol
  • Amicon ultracentrifugation unit (Millipore)
  • Bio‐Spin disposable chromatography columns (BioRad, cat. no. 732‐6008; optional)

Alternate Protocol 2: Enzymatic Labeling of Proteins with Biotin‐CoA

  Materials
  • 50 to 500 µM protein to be labeled in any common buffer (avoid metal chelators such as EDTA)
  • 10× Sfp reaction buffer (see recipe)
  • Purified Sfp enzyme (Yin et al., )
  • Coenzyme A–biotin: Dissolve biotin‐CoA (New England Biolabs, cat. no. S59351S) in DMSO to a final concentration of 10 mg/ml and store it in small aliquots at −20° or −80°C.
  • Protein purification buffer: 50 mM HEPES/100 mM NaCl; adjust pH to 7.5 with HCl or NaOH
  • Sephadex G‐25 column (GE Healthcare)

Basic Protocol 3: On‐Bead Screening of One‐Bead‐One‐Compound Libraries

  Materials
  • Library resin (synthesized in protocol 1)
  • N′,N′‐dimethylformamide (DMF; Sigma Aldrich)
  • Dichloromethane (DCM; Sigma Aldrich)
  • HBST blocking buffer (see recipe)
  • Biotinylated protein ( protocol 3)
  • SAAP buffer (see recipe)
  • 1 mg/ml SAAP (streptavidin–alkaline phosphatase; Prozyme, http://www.prozyme.com/)
  • Staining buffer (see recipe)
  • 5 mg/ml BCIP in H 2O (Sigma‐Aldrich)
  • 1 N HCl
  • Micro‐BioSpin columns (0.8 ml; BioRad)
  • Rotary shaker
  • 35‐mm Petri dish (nonsterile, non‐culture‐treated polystyrene dishes work fine)
  • Dissection microscope (10× to 40× magnification)

Alternate Protocol 3: On‐Bead Screening Against Fluorescently Labeled Protein

  Materials
  • Library resin (synthesized on PEGA resin in protocol 1)
  • N′,N′‐dimethylformamide (DMF; Sigma Aldrich)
  • HBST blocking buffer (see recipe)
  • Fluorescently labeled protein (e.g., protocol 3)
  • Micro‐BioSpin columns (0.8 ml; BioRad
  • Rotary shaker
  • Fluorescence microscope

Basic Protocol 4: Partial Edman Degradation for Sequencing Support‐Bound Peptides

  Materials
  • Positive beads ( protocol 5 or protocol 6)
  • Pyridine
  • Triethylamine
  • 8.8 to 11.0 mM fluorenylmethyoxylcarbonyl‐N‐hydroxysuccinimidyl ester (Fmoc‐OSu; Sigma‐Aldrich) in pyridine
  • Phenylisothiocyanate (PITC; can be purchased in 1‐ml sealed ampules from Sigma‐Aldrich)
  • Dichloromethane (DCM)
  • 20% (v/v) piperidine in dimethylformamide (DMF)
  • Trifluoroacetic acid (TFA; Sigma‐Aldrich)
  • Dimethyl sulfide (Sigma‐Aldrich)
  • Ammonium iodide
  • 40 mg/ml cyanogen bromide in 70% (v/v) TFA
  • Acetonitrile
  • Methanol
  • 4‐hydroxy‐α‐cyanocinnamic (Sigma‐Aldrich)
  • Custom‐designed reaction vessel (12‐mm diameter, 20 mm height, a 10‐ 20‐µm glass frit, and 1 mm luer tip at the bottom; see Fig. )
  • Speedvac evaporator and vacuum source
  • Additional reagents and equipment for MALDI‐TOF mass spectrometry (Henzel and Stults, )
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Figures

Videos

Literature Cited

Literature Cited
   Chen, X., Tan, P.H., Zhang, Y., and Pei, D. 2009. On‐bead screening of combinatorial libraries: Reduction of nonspecific binding by decreasing surface ligand density. J. Comb. Chem. 11:604‐611.
   Chen, X.W., Ren, L., Kim, S., Carpino, N., Daniel, J.L., Kunapuli, S.P., Tsygankov, A.Y., and Pei, D.H. 2010. Determination of the substrate specificity of protein‐tyrosine phosphatase TULA‐2 and identification of Syk as a TULA‐2 substrate. J. Biol. Chem. 285:31268‐31276.
   Ekici, O.D., Karla, A., Paetzel, M., Lively, M.O., Pei, D.H., and Dalbey, R.E. 2007. Altered‐3 substrate specificity of Escherichia coli signal peptidase 1 mutants as revealed by screening a combinatorial peptide library. J. Biol. Chem. 282:417‐425.
   Henzel, W. J. and Stults, J. T. 1996. Matrix‐assisted laser desorption/ionization time‐of‐flight mass analysis of peptides. Curr. Protoc. Protein Sci. 16.2.1‐16.2.11.
   Hoffmuller, U., Russwurm, M., Kleinjung, F., Ashurst, J., Oschkinat, H., Volkmer‐Engert, R., Koesling, D., and Schneider‐Mergener, J. 1999. Interaction of a PDZ protein domain with a synthetic library of all human protein C termini. Angew. Chem. Int. Ed. 38:2000‐2004.
   Joo, S.H. and Pei, D. 2008. Synthesis and screening of support‐bound combinatorial peptide libraries with free C‐termini: Determination of the sequence specificity of PDZ domains. Biochemistry 47:3061‐3072.
   Kessels, H.W., Ward, A.C., and Schumacher, T.N. 2002. Specificity and affinity motifs for Grb2 SH2‐ligand interactions. Proc. Natl. Acad. Sci. U.S.A. 99:8524‐8529.
   Kodadek, T. and Bachhawat‐Sikder, K. 2006. Optimized protocols for the isolation of specific protein‐binding peptides or peptoids from combinatorial libraries displayed on beads. Mol. Biosyst. 2:25‐35.
   Kritzer, J.A., Luedtke, N.W., Harker, E.A., and Schepartz, A. 2005. A rapid library screen for tailoring beta‐peptide structure and function. J. Am. Chem. Soc. 127:14584‐14585.
   Lam, K.S., Salmon, S.E., Hersh, E.M., Hruby, V.J., Kazmierski, W.M., and Knapp, R.J. 1991. A new type of synthetic peptide library for identifying ligand‐binding activity. Nature 354:82‐84.
   Liu, R., Marik, J., and Lam, K.S. 2002. A novel peptide‐based encoding system for “one‐bead one‐compound” peptidomimetic and small molecule combinatorial libraries. J. Am. Chem. Soc. 124:7678‐7680.
   Liu, T., Qian, Z., Xiao, Q., and Pei, D. 2011. High‐throughput screening of one‐bead‐one‐compound libraries: Identification of cyclic peptidyl inhibitors against calcineurin/NFAT interaction. ACS Comb. Sci. 13:537‐546.
   Manke, I.A., Lowery, D.M., Nguyen, A., and Yaffe, M.B. 2003. BRCT repeats as phosphopeptide‐binding modules involved in protein targeting. Science 302:636‐639.
   Moerke, N.J. 2009. Fluorescence polarization (FP) assays for monitoring peptide‐protein or nucleic acid‐protein binding. Curr. Protoc. Chem. Biol. 1:1‐15.
   Olivos, H.I., Bachhawat‐Sikder, K., and Kodadek, T. 2003. Quantum dots as a visual aid for screening bead‐bound combinatorial libraries. Chembiochem 4:1242‐1245.
   Ren, L., Chen, X., Luechapanichkul, R., Selner, N.G., Meyer, T.M., Wavreille, A.S., Chan, R., Iorio, C., Zhou, X., Neel, B.G., and Pei, D. 2011. Substrate specificity of protein tyrosine phosphatases 1B, RPTPalpha, SHP‐1, and SHP‐2. Biochemistry 50:2339‐2356.
   Rickles, R.J., Botfield, M.C., Zhou, X.M., Henry, P.A., Brugge, J.S., and Zoller, M.J. 1995. Phage display selection of ligand residues important for Src homology 3 domain binding specificity. Proc. Natl. Acad. Sci. U.S.A. 92:10909‐10913.
   Rodriguez, M., Yu, X., Chen, J., and Songyang, Z. 2003. Phosphopeptide binding specificities of BRCA1 COOH‐terminal (BRCT) domains. J. Biol. Chem. 278:52914‐52918.
   Rodriguez, M., Li, S.S., Harper, J.W., and Songyang, Z. 2004. An oriented peptide array library (OPAL) strategy to study protein‐protein interactions. J. Biol. Chem. 279:8802‐8807.
   Sadowski, I., Stone, J.C., and Pawson, T. 1986. A noncatalytic domain conserved among cytoplasmic protein‐tyrosine kinases modifies the kinase function and transforming activity of Fujinami sarcoma‐virus P130gag‐Fps. Mol. Cell. Biol. 6:4396‐4408.
   Seet, B.T., Dikic, I., Zhou, M.M., and Pawson, T. 2006. Reading protein modifications with interaction domains. Nat. Rev. Mol. Cell. Biol. 7:473‐483.
   Songyang, Z., Shoelson, S.E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W.G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R.J. et al. 1993. SH2 domains recognize specific phosphopeptide sequences. Cell 72:767‐778.
   Sweeney, M.C., Wavreille, A.S., Park, J., Butchar, J.P., Tridandapani, S., and Pei, D. 2005. Decoding protein‐protein interactions through combinatorial chemistry: Sequence specificity of SHP‐1, SHP‐2, and SHIP SH2 domains. Biochemistry 44:14932‐14947.
   Thakkar, A., Wavreille, A.S., and Pei, D.H. 2006. Traceless capping agent for peptide sequencing by partial Edman degradation and mass spectrometry. Anal. Chem. 78:5935‐5939.
   Tonikian, R., Zhang, Y.N., Sazinsky, S.L., Currell, B., Yeh, J.H., Reva, B., Held, H.A., Appleton, B.A., Evangelista, M., Wu, Y., Xin, X.F., Chan, A.C., Seshagiri, S., Lasky, L.A., Sander, C., Boone, C., Bader, G.D., and Sidhu, S.S. 2008. A specificity map for the PDZ domain family. Plos Biol. 6:2043‐2059.
   Waksman, G., Kominos, D., Robertson, S.C., Pant, N., Baltimore, D., Birge, R.B., Cowburn, D., Hanafusa, H., Mayer, B.J., Overduin, M., Resh, M.D., Rios, C.B., Silverman, L., and Kuriyan, J. 1992. Crystal‐structure of the phosphotyrosine recognition domain Sh2 of V‐Src complexed with tyrosine‐phosphorylated peptides. Nature 358:646‐653.
   Wang, X.B., Zhang, J.H., Song, A.M., Lebrilla, C.B., and Lam, K.S. 2004. Encoding method for OBOC small molecule libraries using a biphasic approach for ladder‐synthesis of coding tags. J. Am. Chem. Soc. 126:5740‐5749.
   Wang, X., Peng, L., Liu, R., Xu, B., and Lam, K.S. 2005. Applications of topologically segregated bilayer beads in ‘one‐bead one‐compound’ combinatorial libraries. J. Pept. Res. 65:130‐138.
   Wu, J., Ma, Q.N., and Lam, K.S. 1994. Identifying substrate motifs of protein kinases by a random library approach. Biochemistry 33:14825‐14833.
   Xiao, Q., Zhang, F., Nacev, B.A., Liu, J.O., and Pei, D. 2010. Protein N‐terminal processing: Substrate specificity of Escherichia coli and human methionine aminopeptidases. Biochemistry 49:5588‐5599.
   Yin, J., Straight, P.D., McLoughlin, S.M., Zhou, Z., Lin, A.J., Golan, D.E., Kelleher, N.L., Kolter, R., and Walsh, C.T. 2005. Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferase. Proc. Natl. Acad. Sci. U.S.A. 102:15815‐15820.
   Yu, X.C., Chini, C.C.S., He, M., Mer, G., and Chen, J.J. 2003. The BRCT domain is a phospho‐protein binding domain. Science 302:639‐642.
   Zhang, X., Morera, S., Bates, P.A., Whitehead, P.C., Coffer, A.I., Hainbucher, K., Nash, R.A., Sternberg, M.J., Lindahl, T., and Freemont, P.S. 1998. Structure of an XRCC1 BRCT domain: A new protein‐protein interaction module. EMBO J. 17:6404‐6411.
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