UbFluor: A Fluorescent Thioester to Monitor HECT E3 Ligase Catalysis

David T. Krist1, Peter K. Foote1, Alexander V. Statsyuk1

1 Northwestern University, Department of Chemistry, Chemistry of Life Processes Institute, Evanston, Illinois
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/cpch.17
Online Posting Date:  March, 2017
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HECT E3 ubiquitin ligases (∼28 are known) are associated with many phenotypes in eukaryotes and are important drug targets. However, assays used to screen for small molecule inhibitors of HECT E3s are complex and require ATP, Ub, E1, E2, and HECT E3 enzymes, producing three covalent thioester enzyme intermediates E1∼Ub, E2∼Ub, and HECT E3∼Ub (where ∼ indicates a thioester bond), and mixtures of polyubiquitin chains. To reduce the complexity of the assay, we developed a novel class of fluorescent probes, UbFluor, that act as mechanistically relevant pseudosubstrates of HECT E3s. These probes undergo a direct transthiolation reaction with the catalytic cysteine of HECT E3s, producing the catalytically active HECT E3∼Ub thioester accompanied by fluorophore release. Thus, a fluorescence polarization assay can continuously monitor UbFluor consumption by HECT E3s, and changes in UbFluor consumption rendered by biochemical point mutations or small molecule modulation of HECT E3 activity. © 2017 by John Wiley & Sons, Inc.

Keywords: fluorescence polarization; HECT ligase; thioester; ubiquitin

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: E1 Enzyme UBA1 Expression and Purification
  • Basic Protocol 2: Synthesis of Ubfluor
  • Basic Protocol 3: Fluorescence Polarization Assay with Ubfluor
  • Alternate Protocol 1: Fluorescence Polarization Assay with UbFluor for Fast Reactions
  • Basic Protocol 4: Data Analysis for Ubfluor FP Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: E1 Enzyme UBA1 Expression and Purification

  • E. coli BL21 (DE3) cells (Thermo Fisher Scientific)
  • E1 enzyme UBA1 plasmid for expression in E. coli, (pET‐28b mE1) available from addgene (plasmid #32534)
  • Kanamycin sulfate (Sigma‐Aldrich)
  • LB agar (Thermo Fisher Scientific)
  • LB broth (Thermo Fisher Scientific)
  • 0.5 M isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) in water
  • Resuspension buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Triton X‐100, 1 mM DTT, with cOmplete, EDTA‐free Protease Inhibitor Cocktail [Roche]; see recipe)
  • cOmplete Protease EDTA‐free Inhibitor Cocktail (Roche)
  • Ni‐NTA agarose (Qiagen)
  • Ni‐NTA wash buffer (50 mM Na 2HPO 4/NaH 2PO 4, pH 8.0, 150 mM NaCl; see recipe)
  • Ni‐NTA elution buffer (50 mM Na 2HPO 4/NaH 2PO 4, pH 8.0, 150 mM NaCl, 300 mM imidazole; see recipe)
  • E1 storage buffer (20 mM HEPES, pH 8.0, 100 mM NaCl, 1 mM DTT; see recipe)
  • Lab benchtop scale
  • Petri dishes (Thermo Fisher Scientific)
  • 250‐ml and 2‐liter baffled Erlenmeyer flask (Thermo Fisher Scientific)
  • Incubator or shaker, 37°C
  • Absorbance spectrophotometer (to measure optical density of E. coli solution)
  • Polycarbonate centrifuge bottles (40 and 500 ml)
  • High‐speed centrifuge (for E1 expression steps; Thermo Scientific Sorvall RC 6 Plus or equivalent)
  • Centrifuge rotor (for pelleting cells in 500‐ml bottles; PTI/Thermo Scientific F10S‐6 × 500y centrifuge rotor or equivalent)
  • Centrifuge rotor (for clearing lysate; Thermo Scientific F21S‐8 × 50y centrifuge rotor or equivalent)
  • Sonicator/sonic dismembrator (Fisher Scientific FB505 or equivalent)
  • 0.45‐μm syringe filter (Thermo Fisher Scientific)
  • Econo column (10 × 2.5 cm; BioRad)
  • Rocking platform (BioRad)
  • Centrifuge (for 50‐ml conical tubes; Eppendorf 5430 R or equivalent)
  • FPLC system (Akta)
  • HiLoad Superdex 200 (GE Healthcare)
  • Amicon 30 kDa MWCO spin filters
  • −80°C freezer

Basic Protocol 2: Synthesis of Ubfluor

  • 500 mM sodium phosphate dibasic/sodium phosphate monobasic (Na 2HPO 4/NaH 2PO 4), pH 8.0 (see recipe)
  • Adenosine triphosphate disodium salt hydrate (ATP, Sigma‐Aldrich)
  • E1 enzyme UBE1 (see protocol 1)
  • 2‐Mercaptoethanesulfonic acid sodium salt (MESNa, Sigma‐Aldrich)
  • Magnesium chloride
  • Ubiquitin from bovine erythrocytes (Sigma‐Aldrich)
  • Storage buffer A (25 mM NaCl, 12.5 mM HEPES, pH 6.7; see recipe)
  • Cysteamine hydrochloride
  • Trifluoroacetic acid (TFA)
  • Methylene chloride
  • Triphenylmethyl (trityl) chloride
  • 1 M NaOH aqueous solution
  • Saturated aqueous sodium chloride solution
  • Magnesium sulfate (anhydrous)
  • 1:4 diethyl ether/n‐pentane
  • Dimethylformamide (DMF)
  • Fluorescein isothiocyanate (Sigma‐Aldrich, isomer 1)
  • N,N‐Diisopropylethylamine
  • 50 mM HEPES, pH 6.5 (see recipe)
  • Silica gel, standard grade (Sorbent Technologies, 60 Å, 40 to 63 µm particle size or equivalent)
  • Ethyl acetate
  • Sand
  • Methanol
  • Triethylsilane
  • Diethyl ether
  • 3:7 acetonitrile/water with 0.1% trifluoroacetic acid (TFA; see recipe)
  • Triethylamine
  • 1:1 DMSO/H 2O (see recipe)
  • Saturated sodium bicarbonate (NaHCO 3) solution
  • 1 M HEPES, pH 7.5 (see recipe)
  • Tris(2‐carboxyethyl)phosphine hydrochloride (Sigma‐Aldrich)
  • Guanidine hydrochloride
  • Storage buffer B (250 mM NaCl, 12.5 mM HEPES, pH 6.0; see recipe)
  • 20 mM β‐mercaptoethanol (BME) in 1× PBS (see recipe)
  • Lab benchtop scale
  • 50‐ml Falcon conical tubes
  • Incubator, 37°C
  • Amicon 3 kDa MWCO spin filters
  • Centrifuge (for 50‐ml conical tubes; Eppendorf 5430 R or equivalent)
  • FPLC system (Akta)
  • HiLoad Superdex 75 FPLC column (GE Healthcare)
  • Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific or equivalent)
  • Chemistry fume hood equipped with nitrogen and vacuum sources
  • Stir bar with standard stir plate
  • Glass round‐bottomed flasks (single 24/40 neck, 100 and 250 ml; single 14/20 neck, 25 ml)
  • Glass separatory funnels (30 and 250 ml)
  • Glass cone funnel (for 24/40 joint)
  • Whatman filter paper (70 mm)
  • Fritted filter funnel (150 ml)
  • High‐vacuum pump (Welch DuoSeal, 1 × 10‐4 torr)
  • Glass elbow joint for N 2(g) on glass chromatography column
  • Tygon tubing for N 2(g)
  • Rotary evaporator
  • 20‐ml glass scintillation vial
  • Aluminum foil
  • Standard lab vortex mixer (up to 3000 rpm)
  • Symphony ultrasonic cleaner (VWR) or equivalent sonication bath
  • HPLC system
  • HPLC column (Restek Pinnacle DB C18)
  • Lyophilizer
  • Liquid chromatography/time‐of‐flight mass spectrometer (LC/TOF‐MS; for intact protein analysis)
  • HiTrap desalting columns (5 ml size, GE Healthcare)
  • −80°C freezer

Basic Protocol 3: Fluorescence Polarization Assay with Ubfluor

  • UbFluor (from protocol 2)
  • HECT ligase (either the catalytic domain or full length enzyme; Boston Biochem and obtainable from E.coli expression)
  • 240 μM Tween 20
  • 24.4 mM TCEP
  • UbFluor assay buffer (1.5 M NaCl, 500 mM HEPES, pH 7.5; see recipe)
  • Centrifuge (for 1.5‐ml microcentrifuge tubes, Thermo Scientific Sorvall Legend Micro 21R or equivalent)
  • 384‐well plate (low volume, low binding; Corning, cat. no. 3820)
  • Centrifuge (for 384‐well plates; Jouan RC1022 or equivalent)
  • Synergy 4 fluorescence plate reader running Gen5 software (BioTek)

Alternate Protocol 1: Fluorescence Polarization Assay with UbFluor for Fast Reactions

  • See protocol 3

Basic Protocol 4: Data Analysis for Ubfluor FP Assay

  • UbFluor (from protocol 2)
  • HECT ligase (either the catalytic domain or full length enzyme; Boston Biochem and obtainable from E.coli expression)
  • 240 μM Tween 20
  • 24.4 mM TCEP
  • UbFluor assay buffer (1.5 M NaCl, 500 mM HEPES, pH 7.5; see recipe)
  • 1 mM β‐mercaptoethanol (BME) in 1× PBS
  • Centrifuge (for 1.5‐ml microcentrifuge tubes, Thermo Scientific Sorvall Legend Micro 21R or equivalent)
  • 384‐well plate (low volume, low binding; Corning, cat. no. 3820)
  • Centrifuge (for 384‐well plates; Jouan RC1022 or equivalent)
  • Synergy 4 fluorescence plate reader running Gen5 software (BioTek)
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