In Vitro GEF and GAP Assays

Alexander Eberth1, Mohammad Reza Ahmadian1

1 Institut für Biochemie und Molekularbiologie II, Klinikum der Heinrich‐Heine‐Universität, Düsseldorf, Germany
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 14.9
DOI:  10.1002/0471143030.cb1409s43
Online Posting Date:  June, 2009
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Abstract

Small GTPases act as tightly regulated molecular switches governing a large variety of critical cellular functions. Their activity is controlled by two different biochemical reactions, GDP/GTP exchange and GTP hydrolysis. These very slow reactions require catalysis in cells by two kinds of regulatory proteins. While the guanine nucleotide exchange factors (GEFs) activate small GTPases by stimulating the slow exchange of bound GDP for the cellularly abundant GTP, GTPase‐activating proteins (GAPs) accelerate the slow intrinsic rate of GTP hydrolysis by several orders of magnitude, leading to inactivation. There are a number of methods that can be used to characterize the specificity and activity of such regulators, to understand the effect of binding on the protein structure, and, ultimately, to obtain insights into their biological functions. This unit describes (1) detailed protocols for the expression and the purification of small GTPases and the catalytic domains of GEFs and GAPs; (2) preparation of nucleotide‐free and fluorescent nucleotide‐bound small GTPases; and (3) methods for monitoring of the intrinsic and GEF‐catalyzed nucleotide exchange as well as intrinsic and GAP‐stimulated GTP hydrolysis. Curr. Protoc. Cell Biol. 43:14.9.1‐14.9.25. © 2009 by John Wiley & Sons, Inc.

Keywords: fluorescence spectroscopy; guanine nucleotide; mant; tamra

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of Intrinsic and Slow Guanine Nucleotide Exchange Factor (GEF)–Catalyzed Nucleotide Exchange Reactions
  • Support Protocol 1: Preparation of mantGDP‐Bound GTPases
  • Support Protocol 2: Preparation of Nucleotide‐Free Forms of Small GTPases
  • Support Protocol 3: Determining Nucleotide Concentration Using HPLC
  • Alternate Protocol 1: Measurement of Fast GEF‐Catalyzed Nucleotide Exchange Reactions
  • Basic Protocol 2: Measurement of GTPase‐Activating Protein (GAP)‐Stimulated GTP Hydrolysis by HPLC
  • Alternate Protocol 2: Measurement of Slow GAP‐Stimulated GTP Hydrolysis Using mantGTP
  • Alternate Protocol 3: Measurement of Slow GAP‐Stimulated GTP Hydrolysis Using tamraGTP
  • Alternate Protocol 4: Measurement of Fast GAP‐Catalyzed GTP Hydrolysis with tamraGTP
  • Support Protocol 4: GENE Expression and Bacterial Culture Conditions
  • Support Protocol 5: Bacterial Lysis by Sonication
  • Support Protocol 6: Bacterial Lysis Using a Microfluidizer
  • Support Protocol 7: Protein Purification for GST Fusion Proteins
  • Support Protocol 8: Determining Protein Concentration Using the Bradford Assay
  • Support Protocol 9: Determining Protein Concentration Using the Ehresmann Assay
  • Support Protocol 10: Concentrating a Dilute Protein Solution
  • Support Protocol 11: Thrombin Proteolytic Cleavage of GST Fusion Proteins
  • Support Protocol 12: GEL‐Filtration Chromatography
  • Support Protocol 13: Freezing and Thawing Proteins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Intrinsic and Slow Guanine Nucleotide Exchange Factor (GEF)–Catalyzed Nucleotide Exchange Reactions

  Materials
  • >5 µM mantGDP‐bound GTPase ( protocol 2)
  • GEF buffer (see recipe), store at 25°C
  • >50 µM GEF protein including the catalytic domains (recombinant protein, expressed and purified as described in Support Protocols protocol 104 to protocol 1913)
  • 10 mM GDP (Pharma Waldhof, http://www.pharmawaldhof.de/), pH 7.5
  • 0.5 M EDTA, pH 8.0 ( appendix 2A)
  • Fluorescence cuvettes (Suprasil quartz glass; Hellma, cat. no. 108.002F‐QS)
  • Fluorescence spectrometer (Perkin‐Elmer, Spex Instruments)
  • Grafit program (Erithacus Software) or alternative program packages for evaluation of the data

Support Protocol 1: Preparation of mantGDP‐Bound GTPases

  Materials
  • Standard buffer (see recipe)
  • Nucleotide‐free GTPase (see protocol 3)
  • mantGDP (synthesized as described in Hemsath and Ahmadian, , or purchased from Jena Bioscience, http://www.jenabioscience.com/)
  • Ponceau S (unit 6.2)
  • HPLC buffer (see recipe) containing 20% to 25% (v/v) acetonitrile
  • NAP‐5 column (GE Healthcare)
  • Nitrocellulose membrane (unit 6.2)
  • Additional reagents and equipment for Ponceau S staining of proteins on nitrocellulose membrane (unit 6.2) and HPLC ( protocol 4)

Support Protocol 2: Preparation of Nucleotide‐Free Forms of Small GTPases

  Materials
  • Alkaline phosphatase, agarose bead‐coupled (Sigma‐Aldrich)
  • Nonhydrolyzable GTP analog Gpp(CH 2)p (Sigma‐Aldrich)
  • GDP‐bound GTPase (expressed and purified from E. coli; as described in Support Protocols protocol 104 to protocol 1913)
  • 10× exchange buffer (see recipe)
  • HPLC buffer (see recipe) containing 7.5% (v/v) acetonitrile
  • Snake venom phosphodiesterase (Sigma‐Aldrich, cat. no. P3134)
  • Liquid nitrogen
  • Additional reagents and equipment for HPLC ( protocol 4 and protocol 6)

Support Protocol 3: Determining Nucleotide Concentration Using HPLC

  Materials
  • HPLC buffer (see recipe)
  • 50 to 100 µM GTPase (recombinant protein, expressed and purified as described in Support Protocols protocol 104 to protocol 1913)
  • Nucleotide standard solutions: e.g., 20 to 400 µM GDP
  • Beckman Gold HPLC instrument (Beckman Coulter)
  • Reversed‐phase C18 HPLC column: Ultrasphere ODS, 5‐µM; 250 × 4, 6‐mm (Beckman Coulter)
  • Guard column: Nucleosil 100‐5‐C18, 5 µM (Bischoff Chromatography; http://www.bischoff‐chrom.de/)
  • 20‐ or 50‐µl sample loop (Bischoff Chromatography; http://www.bischoff‐chrom.de/)

Alternate Protocol 1: Measurement of Fast GEF‐Catalyzed Nucleotide Exchange Reactions

  • Stopped‐flow instrument (Applied Photophysics SX18MV or Hi‐Tech SF‐61 DX2; http://www.photophysics.com)
NOTE: Because the samples are mixed 1:1, all stock solutions for components of the samples should be 2×.

Basic Protocol 2: Measurement of GTPase‐Activating Protein (GAP)‐Stimulated GTP Hydrolysis by HPLC

  Materials
  • 500 µM nucleotide‐free GTPase ( protocol 3)
  • GAP buffer (see recipe)
  • 10 mM GTP (Pharma Waldhof; http://www.pharmawaldhof.de/), pH 7.5
  • >50 µM GAP protein including the catalytic domains (recombinant protein, expressed and purified as described in Support Protocols protocol 104 to protocol 1913)
  • Liquid nitrogen
  • HPLC buffer (see recipe) containing 7.5% (v/v) acetonitrile
  • Thermomixer/thermoblock
  • Beckman Gold HPLC instrument (Beckman Coulter)
  • Reversed‐phase C18 HPLC column: Ultrasphere ODS, 5‐µM; 250 × 4.6–mm (Beckman Coulter)
  • Guard column: Nucleosil 100‐5‐C18, 5‐µM (Bischoff Chromatography)

Alternate Protocol 2: Measurement of Slow GAP‐Stimulated GTP Hydrolysis Using mantGTP

  • 10 mM mantGTP (synthesized as described in Hemsath and Ahmadian, or purchased from Jena Biosciences) bound to Ras, pH 7.5
  • 20 µM catalytic domains of RasGAP protein (e.g., p120RasGAP) or any other GAP protein specific for Ras (expressed and purified as in Support Protocols protocol 104 to protocol 1913)
  • Stopped‐flow instrument (Applied Photophysics SX18MV or Hi‐Tech SF‐61 DX2)
NOTE: Because the samples are mixed 1:1, all stock solutions for components of the samples should be 2×.

Alternate Protocol 3: Measurement of Slow GAP‐Stimulated GTP Hydrolysis Using tamraGTP

  • 2 mM tamraGTP (synthesized as described in Eberth et al., ), pH 7.5
  • 50 µM nucleotide‐free GTPase ( protocol 3) in GAP buffer (see recipe for buffer)
  • Fluorescence cuvettes, Suprasil quartz glass; Hellma, cat. no. 108.002F‐QS
  • Fluorescence spectrometer (Perkin Elmer, Spex Instruments)
  • Grafit program (Erithacus Software) or alternative program packages for evaluation of the data

Alternate Protocol 4: Measurement of Fast GAP‐Catalyzed GTP Hydrolysis with tamraGTP

  • tamraGTP (synthesized as described in Eberth et al., ; 2 mM in deionized H 2O, pH 7.5)
  • Stopped‐flow instrument (Applied Photophysics SX18MV or Hi‐Tech SF‐61 DX2)
NOTE: Because the samples are mixed 1:1, all stock solutions for components of the samples should be 2×.

Support Protocol 4: GENE Expression and Bacterial Culture Conditions

  Materials
  • Escherichia coli strain: BL21(DE3), BL21(DE3) Codon plus RIL, BL21(DE3) pLysS, or Rosetta (DE3) (Novagen) containing prokaryotic expression plasmid carrying gene for protein of interest
  • Terrific broth medium (TB medium; see recipe)
  • Appropriate selection antibiotics
  • Isopropyl‐β‐D‐thiogalactopyranoside (IPTG; Gerbu Biochemicals, http://www.gerbu.de)
  • Wash buffer (see recipe)
  • 150‐ to 1000‐ml and 5‐liter sterilized Erlenmeyer flasks
  • Horizontal environmental shaker incubator (Infors HT, http://www.infors‐ht.com/)
  • 1000‐ml and 30‐ to 250‐ml centrifuge bottles
  • Centrifuge: Avanti J‐20 XP (Beckman Coulter) or equivalent
  • 6‐liter rotor: JLA‐8.1000 (Beckman Coulter) or equivalent
  • 50‐ml plastic tubes

Support Protocol 5: Bacterial Lysis by Sonication

  Materials
  • 70% ethanol
  • Bacterial sample ( protocol 10)
  • Pefabloc (ICN Biochemicals)
  • Lysozyme (Sigma‐Aldrich)
  • DNase I (Sigma‐Aldrich)
  • Sonicator: Branson Sonifier S‐450A and 3‐ to 19‐mm titanium probe
NOTE: The protease inhibitor Pefabloc (0.02% w/v) and lysozyme (2 µg/ml suspension), as well as DNase I (10 µg/ml suspension) are added to the bacterial suspension before lysis.

Support Protocol 6: Bacterial Lysis Using a Microfluidizer

  Materials
  • Bacterial sample ( protocol 10)
  • Buffer to be used for protein purification
  • Pefabloc (ICN Biochemicals)
  • Lysozyme (Sigma‐Aldrich)
  • DNase I (Sigma‐Aldrich)
  • 100% 2‐propanol
  • Microfluidizer (Microfluidics Corp., http://www.microfluidicscorp.com)
NOTE: The protease inhibitor Pefabloc (0.02% w/v) and lysozyme (2 µg/ml suspension) as well as DNase I (10 µg/ml suspension) are added to the bacterial suspension before lysis.

Support Protocol 7: Protein Purification for GST Fusion Proteins

  Materials
  • Bacterial lysate with an overexpressed GST‐fusion protein
  • Glutathione‐Sepharose 4B FF (GE Healthcare)
  • Standard buffer (see recipe)
  • Standard buffer (see recipe) containing 500 mM KCl and 1 mM ATP
  • Standard buffer containing 20 mM glutathione (adjusted to pH 7.5 with NaOH again after addition of glutathione)
  • 0.01% (w/v) sodium azide or 20% (v/v) ethanol
  • Centrifuge: Avanti J‐30I (Beckman Coulter) or equivalent
  • Rotor: JA‐30.50 or JA‐17 (Beckman Coulter) or equivalent
  • Äkta Sytem, e.g. Äkta Prime (GE Healthcare)
  • XK 26/20 chromatography column chassis (GE Healthcare)
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1) and Coomassie staining (unit 6.6)

Support Protocol 8: Determining Protein Concentration Using the Bradford Assay

  Materials
  • Protein solutions: standards (e.g., BSA or γ‐globulin) and test sample
  • Bradford reagent (Coomassie dye reagent; Sigma, Pierce, Bio‐Rad, or see appendix 3H)
  • Spectrophotometer

Support Protocol 9: Determining Protein Concentration Using the Ehresmann Assay

  Materials
  • Protein solution
  • Quartz cuvettes
  • UV/VIS spectrophotometer

Support Protocol 10: Concentrating a Dilute Protein Solution

  Materials
  • Protein solution
  • Refrigerated centrifuge
  • Amicon filter, MWCO 5 to 100 kDa

Support Protocol 11: Thrombin Proteolytic Cleavage of GST Fusion Proteins

  Materials
  • GST‐fusion protein
  • Thrombin (Serva)
  • End‐over‐end rotator
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1) and Coomassie staining (unit 6.6)

Support Protocol 12: GEL‐Filtration Chromatography

  Materials
  • Concentrated protein sample (after digestion of the fusion protein with the respective protease; see protocol 20 for concentration and protocol 21 for digestion)
  • Standard buffer (see recipe)
  • 16/600 or 26/600 columns prepacked with Superdex 75 or Superdex 200 (GE Healthcare)
  • Äkta System, e.g. Äkta Prime (GE Healthcare)
  • Prepacked 16/600 or 26/600 columns with Superdex 75 or Superdex 200 resin (GE Healthcare)
  • 1‐ to 5‐ml sample loop (GE Healthcare)
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1), Coomassie staining (unit 6.6), and concentration of protein samples ( protocol 20)

Support Protocol 13: Freezing and Thawing Proteins

  Materials
  • 50‐ to 500‐µl aliquots of purified protein (10 to 20 mg/ml)
  • Liquid nitrogen
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Figures

Videos

Literature Cited

Literature Cited
   Ahmadian, M.R., Hoffmann, U., Goody, R.S., and Wittinghofer, A. 1997a. Individual rate constants for the interaction of Ras proteins with GTPase‐activating proteins determined by fluorescence spectroscopy. Biochemistry 36:4535‐4541.
   Ahmadian, M.R., Stege, P., Scheffzek, K., and Wittinghofer, A. 1997b. Confirmation of the arginine‐finger hypothesis for the GAP‐stimulated GTP‐hydrolysis reaction of Ras. Nat. Struc. Biol. 4:686‐689.
   Ahmadian, M.R., Zor, T., Vogt, D., Kabsch, W., Selinger, Z., Wittinghofer, A., and Scheffzek, K. 1999. Guanosine triphosphatase stimulation of oncogenic Ras mutants. Proc. Natl. Acad. Sci. U.S.A. 96:7065‐7070.
   Ahmadian, M.R., Wittinghofer, A., and Herrmann, C. 2002. Fluorescence methods in the study of small GTP‐binding proteins. Methods Mol. Biol. 189:45‐63.
   Ahmadian, M.R., Kiel, C., Stege, P., and Scheffzek, K. 2003. Structural fingerprints of the Ras‐GTPase activating proteins neurofibromin and p120GAP. J. Mol. Biol. 329:699‐710.
   Alexandrov, K., Scheidig, A.J., and Goody, R.S. 2001. Fluorescence methods for monitoring interactions of Rab proteins with nucleotides, Rab escort protein, and geranylgeranyltransferase. Methods Enzymol. 329:14‐31.
   Eberth, A., Dvorsky, R., Becker, C., Beste, A., Goody, R.S., and Ahmadian, M.R. 2005. Monitoring the real‐time kinetics of the hydrolysis reaction of guanine nucleotide binding proteins. Biol. Chem. 386:1105‐1114.
   Guo, Z., Ahmadian, M.R., and Goody, R.S. 2005. Guanine nucleotide exchange factors operate by a simple allosteric competitive mechanism. Biochemistry 44:15423‐15429.
   Haeusler, L.C., Blumenstein, L., Stege, P., Dvorsky, R., and Ahmadian, M.R. 2003. Comparative functional analysis of the Rac GTPases. FEBS Lett. 555:556‐560.
   Hemsath, L. and Ahmadian, M.R. 2005. Fluorescence approaches for monitoring interactions of RhoGTPases with nucleotides, regulators and effectors. Methods 37:173‐182.
   Hiratsuka, T. 2003. Fluorescent and colored trinitrophenylated analogs of ATP and GTP. Eur. J. Biochem. 270:3479‐85.
   John, J., Sohmen, R., Feuerstein, J., Linke, R., Wittinghofer, A., and Goody, R.S. 1990. Kinetics of interaction with nucleotide‐free H‐ras p21. Biochemistry 29:6058‐6065.
   Scheffzek, K. and Ahmadian, M.R. 2005. GTPase activating proteins: Structural and functional insights 18 years after discovery. Cell. Mol. Life Sci. 62:3014‐3038.
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