Misincorporation Proton‐Alkyl Exchange (MPAX): Engineering Cysteine Probes into Proteins

Alondra Schweizer Burguete1, Pehr B. Harbury1, Suzanne R. Pfeffer1

1 Stanford School of Medicine, Stanford, California
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 26.1
DOI:  10.1002/0471140864.ps2601s42
Online Posting Date:  December, 2005
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit describes a rapid and efficient method to screen a polypeptide for amino acid residues that contribute to protein‐protein interaction interfaces. Cysteine residues are introduced as positional probes in a protein at random by co‐expression in bacteria with specific cysteine misincorporator tRNAs. The protein is then purified as an ensemble of polypeptides containing cysteine at low frequency, at different positions in each molecule. The ability of the native protein structure to protect different cysteine residues from chemical modification by iodoacetamide is determined to obtain a protein surface map that reveals candidate surface residues that are likely to be important for protein‐protein interaction. Cysteine mutants with altered ligand binding can also be selected simultaneously by affinity chromatography.

Keywords: Protein‐protein interaction; solvent accessibility; protein structure; cysteine misincorporation; cysteine mutagenesis; cysteine alkylation

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Introduction of Cysteine Residues by Translational Misincorporation
  • Basic Protocol 2: Chemical Cleavage at Cysteine
  • Basic Protocol 3: Native Cysteine Alkylation
  • Basic Protocol 4: Intrinsic Alkylation Rate
  • Basic Protocol 5: Prediction of Binding Interfaces
  • Basic Protocol 6: Selection of Mutants with Altered Ligand or Partner Binding
  • Support Protocol 1: Tris·Tricine Gel to Resolve Small Peptides from MPAX Experiments
  • Support Protocol 2: Gene Assembly
  • Support Protocol 3: Urea‐Acrylamide Gel
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Introduction of Cysteine Residues by Translational Misincorporation

  Materials
  • pMPAX vectors (Harbury laboratory, Stanford University; )
  • Kanamycin‐resistance plasmid (pET28a, Novagen; pET28a pH6_PKA and pET28a PKA_pH6, Harbury laboratory, Stanford University)
  • BL21(DE3) E. coli cells (Novagen)
  • LB plates ( appendix 4A) containing 30 µg/ml kanamycin and 100 µg/ml carbenicillin
  • LB medium (EMD Biosciences) containing 30 µg/ml kanamycin and 100 µg/ml carbenicillin
  • 1× PBS (see recipe)
  • M63 medium containing 30 µg/ml kanamycin and 100 µg/ml carbenicillin (see recipe)
  • 1 M isopropyl‐β‐D‐thiogalactopyranoside (IPTG)
  • Incubator and shaker (37°C and 30°C)
  • Culture flasks, sterile
  • Additional reagents and equipment for electroporation (unit 5.10)

Basic Protocol 2: Chemical Cleavage at Cysteine

  Materials
  • Cysteine‐misincorporated, purified protein (see protocol 1)
  • 10× PKA buffer (see recipe)
  • 100 mM dithiothreitol (DTT)
  • 1% Triton X‐100
  • 10 mg/ml bovine serum albumin
  • 5 U/µl protein kinase A suspended in 1× protein kinase A buffer (see recipe)
  • 10 µCi/µl adenosine triphosphate‐γ‐33P (3000 Ci/mmol)
  • 10 µM adenosine triphosphate (cold)
  • 2 M Bicine, pH 8.6
  • 8 M guanidine·HCl
  • 1 M 2‐nitro‐5‐thiocyanobenzoic acid (NTCB) in dry dioxane
  • 0.05% (w/v) deoxycholic acid sodium salt
  • 50% (v/v) trichloroacetic acid
  • Acetone
  • 8 M urea
  • 0.1 M ammonium hydroxide (NH 4OH)
  • 3× Tricine loading dye (see recipe)
  • Tris·Tricine gel (see protocol 7)
  • 1.5‐ml microcentrifuge tubes
  • Vortex
  • Speed‐vacuum centrifuge

Basic Protocol 3: Native Cysteine Alkylation

  Materials
  • Labeled protein (see protocol 2, steps and )
  • 2 M Bicine, pH 8.6
  • Quench solution (see recipe)
  • 100 mM iodoacetamide
  • 8 M guanidine·HCl
  • Tris·Tricine gel (see protocol 7)

Basic Protocol 4: Intrinsic Alkylation Rate

  Materials
  • Labeled protein
  • 2 M Bicine, pH 8.6
  • 8 M guanidine·HCl
  • Quench solution (see recipe)
  • 100 and 8 mM iodoacetamide
  • Tris·Tricine gel (see protocol 7)

Basic Protocol 5: Prediction of Binding Interfaces

  Materials
  • Radiolabeled protein (see protocol 2)
  • Ligand or partner protein
  • Solid support
  • Binding buffer
  • 4 M guanidine·HCl
  • 100 mM sodium Bicine, pH 8.6
  • 1 M 2‐nitro‐5‐thiocyanobenzoic acid (NTCB) in dry dioxane
  • Tris·Tricine gel (see protocol 7)
  • Scintillation counter

Basic Protocol 6: Selection of Mutants with Altered Ligand or Partner Binding

  Materials
  • 1.5% (w/v) agarose solution (see recipe)
  • 40% (v/v) 19:1 acrylamide
  • 3× gel buffer (see recipe)
  • 3× comb buffer (see recipe)
  • Glycerol
  • 1% (w/v) Coomassie brilliant blue G250
  • 10% (w/v) ammonium persulfate
  • TEMED
  • Butanol
  • 1× cathode buffer (see recipe)
  • 10× anode buffer (see recipe)
  • Glass plates (20 × 20–cm and 18 × 20–cm)
  • Spacers (0.5‐mm)
  • Clamps
  • Whatman filter paper
  • Comb (0.5‐mm; 19 wells ∼0.5‐cm wide)
  • Gel‐running apparatus
  • Capillary pipet tips
  • Power supply
  • Plastic wrap
  • Heated vacuum gel dryer
  • Phosphor image plate and casette
  • Phosphorimager

Support Protocol 1: Tris·Tricine Gel to Resolve Small Peptides from MPAX Experiments

  Materials
  • Sense and antisense oligonucleotides (5‐nmol scale)
  • Ammonia
  • Formamide loading dye (see recipe)
  • Urea‐acrylamide gel (see protocol 9)
  • 1× TBE (see recipe)
  • Ethidium bromide solution in TBE (see recipe)
  • Sep‐Pak C18 cartridge (Vac 1 cc, 100 mg; Waters)
  • 50% (v/v) acetonitrile
  • 2 M and 100 mM triethylammonium acetate
  • EcoRI and HindIII restriction enzymes
  • pET28a pH6_PKA plasmid modified to provide an N‐terminal histidine tag and a C‐terminal protein kinase A recognition site
  • 2‐ml screw‐cap tubes
  • 50°C incubator
  • Speed vacuum centrifuge
  • Power supply
  • Metal spatula
  • Low‐intensity UV source
  • Scalpel
  • Metal spatula
  • Rocker
  • Glass fiber paper (Whatman)
  • Additional reagents and equipment for chloroform extraction and ethanol precipitation ( appendix 4E)

Support Protocol 2: Gene Assembly

  Materials
  • Urea‐acrylamide gel (see recipe)
  • TEMED
  • Glass plates, spacers, and combs (see protocol 7)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Arnold, U. and Ulbrich‐Hofmann, R. 1999. Quantitative protein precipitation from guanidine hydrochloride‐containing solutions by sodium deoxycholate/trichloroacetic acid. Anal. Biochem. 271:197‐199.
   Burguete, A.S., Harbury, P.B., and Pfeffer, S.R. 2004. In vitro selection and prediction of TIP47 protein‐interaction interfaces. Nat. Methods 1:55‐60.
   de Arruda, M.V., Bazari, H., Wallek, M., and Matsudaira, P. 1992. An actin footprint on villin. Single site substitutions in a cluster of basic residues inhibit the actin severing but not capping activity of villin. J. Biol. Chem. 267:13079‐13085.
   Doering, D.S. and Matsudaira, P. 1996. Cysteine scanning mutagenesis at 40 of 76 positions in villin headpiece maps the F‐actin binding site and structural features of the domain. Biochemistry 35:12677‐12685.
   Doonan, S. and Fahmy, H.M. 1975. Specific enzymic cleavage of polypeptides at cysteine residues. Eur. J. Biochem. 56:421‐426.
   Hanai, R. and Wang, J.C. 1994. Protein footprinting by the combined use of reversible and irreversible lysine modifications. Proc. Natl. Acad. Sci. U.S.A. 91:11904‐11908.
   Janin, J. 1979. Surface and inside volumes in globular proteins. Nature 277:491‐492.
   Jacobson, G.R., Schaffer, M.H., Stark, G.R., and Vanaman, T.C. 1973. Specific chemical cleavage in high yield at the amino peptide bonds of cysteine and cystine residues. J. Biol. Chem. 248:6583‐6591.
   Lo Conte, L., Chothia, C., and Janin, J. 1999. The atomic structure of protein‐protein recognition sites. J. Mol. Biol. 285:2177‐2198.
   Raschke, T.M. and Marqusee, S. 1998. Hydrogen exchange studies of protein structure. Curr. Opin. Biotechnol. 9:80‐86.
   Schagger, H. and von Jagow, G. 1987. Tricine‐sodium dodecyl sulfate‐polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166:368‐379.
   Silverman, J.A. and Harbury, P.B. 2002a. Rapid mapping of protein structure, interactions, and ligand binding by misincorporation proton‐alkyl exchange. J. Biol. Chem. 277:30968‐30975.
   Silverman, J.A. and Harbury, P.B. 2002b. The equilibrium unfolding pathway of a (beta/alpha)8 barrel. J. Mol. Biol. 324:1031‐1040.
   Stemmer, W.P., Crameri, A., Ha, K.D., Brennan, T.M., and Heyneker, H.L. 1995. Single‐step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164:49‐53.
   Tu, B.P. and Wang, J.C. 1999. Protein footprinting at cysteines: Probing ATP‐modulated contacts in cysteine‐substitution mutants of yeast DNA topoisomerase II. Proc. Natl. Acad. Sci. U.S.A. 96:4862‐4867.
   Wu, J. and Watson, J.T. 1998. Optimization of the cleavage reaction for cyanylated cysteinyl proteins for efficient and simplified mass mapping. Anal. Biochem. 258:268‐276.
Key References
   Burguete et al., 2004. See above.
  This is the first “blind” study where protein structure is mapped using the MPAX protocol and subsequently compared to crystallographic data. A method to select for residues that contribute to protein‐interaction interfaces is developed and MPAX technology is also used to enable prediction of residues that lie in protein‐protein interfaces. Residues that are highly likely to contribute to partner protein binding are selected by screening a population of different cysteine misincorporation mutants in a single affinity chromatography step.
   Silverman and Harbury, 2002a. See above.
  MPAX technology to footprint protein structure at a single amino acid resolution is developed and described here for the first time. The substrate‐binding site for triosephosphate isomerase (β/α)8 barrel is accurately mapped and the stability of the barrel is determined. In addition, a mass spectrometry method to measure the alkylation rate at misincorporated cysteines is described.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library