Detecting the Interaction of Peptide Ligands with Plant Membrane Receptors

Sarah Refi Hind1, Jason S. Hoki2, Joshua A. Baccile2, Patrick C. Boyle1, Frank C. Schroeder2, Gregory B. Martin3

1 Boyce Thompson Institute, Ithaca, New York, 2 Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, 3 School of Integrative Plant Science, Cornell University, Ithaca, New York
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20053
Online Posting Date:  September, 2017
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The field of plant receptor biology has rapidly expanded in recent years, however the demonstration of direct interaction between receptor‐ligand pairs remains a challenge. Click chemistry has revolutionized small molecule research but lacks popularity in plant research. Here we describe a method that tests for the direct physical interaction of a candidate receptor protein and a peptide ligand. This protocol describes the generation of the ligand probe, transient expression of a receptor protein, enrichment of membrane‐bound receptors, photo‐crosslinking and click chemistry‐mediated reporter addition, and detection of the receptor‐ligand complex. Copper‐based click chemistry confers several advantages, including the versatility to use almost any azide‐containing reporter molecule for detection or visualization of the complex and enables addition of the reporter molecule after receptor‐ligand binding which reduces the need for bulky ligand modifications that could interfere with the interaction. © 2017 by John Wiley & Sons, Inc.

Keywords: click chemistry; ligand; plant receptor; photo‐crosslinking

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Generation of Ligand Probes
  • Alternate Protocol 1: Ligand Probe Synthesis on Beads
  • Support Protocol 1: HPLC‐MS Analysis and Purification
  • Basic Protocol 2: Expression and Enrichment of Candidate Receptor Proteins
  • Support Protocol 2: Preparation of PEG (Upper) and Dextran (Lower) Phase Solutions
  • Basic Protocol 3: Formation and Detection of Receptor‐Ligand Complexes
  • Support Protocol 3: Preparation of Click Chemistry Master Mix
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Generation of Ligand Probes

  • 2‐[2‐(Prop‐2‐ynyloxy)ethoxy]‐4‐[3‐(trifluoromethyl)‐3H‐diazirin‐3‐yl]benzoic acid (trifluoromethyl‐aryl diazirine alkyne acid; see Mayer & Maier, or Synthetic Chemistry Considerations section for preparation)
  • Dimethylformamide (DMF)
  • 1‐Hydroxybenzotriazole hydrate (HOBt)
  • N‐Hydroxy succinimide (NHS)
  • N‐(3‐Dimethylaminopropyl)‐N′‐ethylcarbodiimide (EDC)
  • Argon or nitrogen gas (99.999% pure)
  • N,N‐Diisopropylethylamine (DIEA)
  • 50% hexane/50% ethyl acetate solution
  • Peptide ligand, free or on‐resin, lysine residues protected (see Strategic Planning)
  • Dimethylsulfoxide (DMSO)
  • 1× PBS (see recipe)
  • 8‐ml glass reaction vials with caps
  • Pre‐coated flexible TLC sheets, silica gel (Avantor Performance Materials, cat. no. 4449‐02)
  • TLC solvent chamber
  • Glass capillary spotters
  • UV lamp with 254‐nm emitted light (VWR, cat. no. 89131‐492)
  • Rotary evaporator
  • Phase liquid chromatography Combiflash system (Teledyne, cat. no. 68‐5230‐021)
  • Lo‐bind protein 1.5‐ml tubes (Eppendorf, cat. no. 925000090)
  • Silica gel, 60‐200 mesh (Avantor Performance Materials, cat. no. 3414‐01)
  • Bench top or tray lyophilizer
  • Rotary shaker or nutator

Alternate Protocol 1: Ligand Probe Synthesis on Beads

  • Crude peptide‐loaded beads from peptide vendor
  • N‐Methyl‐2‐pyrrolidone (NMP)
  • N‐Hydroxy succinimide (NHS)‐ester probe (see protocol 1)
  • N,N‐Diisopropylethylamine (DIEA)
  • Triethylamine (TEA)
  • Dichloromethane (DCM)
  • Kaiser test reagent A: 98% pyridine, 2% of 0.1% (w/v) KCN in distilled water
  • Kaiser test reagent B: 5% (w/v) ninhydrin in n‐butanol
  • Kaiser test reagent C: 67% (w/v) phenol in n‐butanol
  • Cleavage cocktail (made fresh once per daily use; depends on solid‐phase resin provided by peptide vendor)
  • Dimethylsulfoxide (DMSO)
  • Dimethylformamide (DMF)
  • Diethyl ether
  • Argon gas
  • Glacial acetic acid
  • 5‐ml peptide reaction vessel with stopcock and cap (Peptides International, cat. no. SHG‐20205‐PI)
  • Rotary shaker or nutator
  • Glass Pasteur pipets with rubber bulb
  • 9‐mm autosampler vials, screw thread caps, and inserts
  • Bench top or tray lyophilizer
  • 15‐ml Falcon conical bottom tube
  • Benchtop centrifuge and rotor for 15‐ml tubes
  • 4‐ml glass vial

Support Protocol 1: HPLC‐MS Analysis and Purification

  • Crude peptide (from protocol 2)
  • Glacial acetic acid (HPLC grade)
  • Acetonitrile (ACN; HPLC grade)
  • 9‐mm autosampler vials, screw thread caps, and inserts
  • C12 Jupiter 250‐ × 10.0‐mm semi‐preparative HPLC column (Phenomenex, cat. no. 00 G‐4396‐N0)
  • High‐performance liquid chromatography (HPLC) instrument
  • Mass spectrometer
  • HPLC fraction collector and glass vials or 125‐ml flasks for fraction collection
  • Rotary evaporator
  • Bench top or tray lyophilizer

Basic Protocol 2: Expression and Enrichment of Candidate Receptor Proteins

  • Agrobacterium tumefaciens with plasmids expressing affinity‐tagged receptor proteins
  • Antibiotic solutions, as appropriate
  • Luria‐Bertani (LB) solid medium plates and liquid broth, both supplemented with antibiotics
  • Induction medium (see recipe)
  • 200 µM acetosyringone (3′,5′‐dimethyl‐4′‐hydroxyacetophenone) in 100% methanol (Sigma‐Aldrich, cat. no. D134406)
  • Infiltration medium (see recipe)
  • Microsome extraction buffer (see recipe)
  • Microsome suspension buffer (see recipe)
  • Phase mixture (see recipe)
  • PEG (upper) and dextran (lower) phase solutions (see protocol 5)
  • 4 to 6 week‐old Nicotiana benthamiana plants
  • Liquid nitrogen
  • Cell spreaders
  • 15‐ml culture tubes
  • Benchtop centrifuge and rotor for 15‐ml tubes
  • Spectrophotometer
  • 1‐ml needleless syringes
  • Small gauge needles (optional)
  • Foil, 50‐ml conical bottom tubes, or other containers for storing frozen leaf tissue
  • Medium‐sized mortars (11.5 cm) and pestles (18 cm)
  • Miracloth (EMD Millipore, cat. no. 475855)
  • Büchner two‐piece filter funnel
  • 500‐ml filtering flask
  • High‐speed centrifuge tubes, 30 ml capacity
  • Refrigerated floor centrifuge and appropriate rotor for holding 30‐ml tubes
  • Ultracentrifuge (Beckman‐Coulter Optima XE‐90)
  • Ultracentrifuge rotor (Beckman‐Coulter SW 32‐Ti)
  • Ultracentrifuge tubes, 38.5 ml volume (Beckman‐Coulter, cat. no. 326823)
  • Immersion tip sonicator
  • 50‐ml conical centrifuge tubes

Support Protocol 2: Preparation of PEG (Upper) and Dextran (Lower) Phase Solutions

  • Polyethylene glycol (PEG, average MW 3350; Sigma‐Aldrich, cat. no. P4338)
  • Dextran from Leuconostoc spp. (M r 450,000 to 650,000; Sigma‐Aldrich, cat. no. 31392)
  • Sucrose
  • 1 M potassium phosphate, pH 7.5 (see recipe)
  • 5 M NaCl
  • Vacuum‐driven bottle top filters, 0.22 µm
  • 500‐ml Squibb pear‐shaped separatory funnel with stopcock and stopper
  • Cold room or large refrigerator

Basic Protocol 3: Formation and Detection of Receptor‐Ligand Complexes

  • Plasma membrane‐enriched microsomes (PMEMs, from protocol 6)
  • Plasma membrane (PM) binding buffer (see recipe)
  • Affinity resin (specific type depends on the affinity tag present on the receptor protein)
  • 1× PBS buffer, pH 7.4 (see recipe)
  • RIPA buffer (see recipe)
  • Click chemistry master mix (see protocol 7)
  • Laemmli sample buffer (see recipe)
  • Polyacrylamide gels (recommended 8% resolving gel)
  • 1× PAGE running buffer (see recipe)
  • Transfer buffer (see recipe)
  • TBS‐T buffer (see recipe)
  • TBS‐T blocking buffer (see recipe)
  • Primary and secondary antibodies
  • Affinity blot detection reagent
  • Stripping buffer (see recipe)
  • Sonicating water bath
  • Glass 9‐well spot plate or other appropriate sample vessel
  • Blak‐Ray B‐100AP 100‐watt lamp with 365 nm longwave bulb (UVP Ultraviolet Products, cat. no. 76005501)
  • Rotary shaker or nutator
  • Polyacrylamide gel electrophoresis (PAGE) apparatus
  • Transfer system

Support Protocol 3: Preparation of Click Chemistry Master Mix

  • BTTAA ligand (Chemical Synthesis & Biology Core at Albert Einstein College of Medicine,
  • Copper (II) sulfate, pentahydrate (CuSO 4·5H 2O)
  • L‐Ascorbic acid, sodium salt (Sigma‐Aldrich, cat. no. A4034)
  • Azide‐reporter molecule such as azide‐PEG4‐biotin conjugate (ChemPep, cat. no. 271606)
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