Chemical Methods and Approaches to the Regioselective Formation of Multiple Disulfide Bonds

Shigeru Shimamoto1, Hidekazu Katayama2, Masaki Okumura3, Yuji Hidaka1

1 Faculty of Science and Engineering, Kinki University, Osaka, 2 Department of Applied Biochemistry, School of Engineering, Tokai University, Kanagawa, 3 Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Miyagi
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 28.8
DOI:  10.1002/0471140864.ps2808s76
Online Posting Date:  April, 2014
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Abstract

Disulfide‐bond formation plays an important role in the stabilization of the native conformation of peptides and proteins. In the case of multidisulfide‐containing peptides and proteins, numerous folding intermediates are produced, including molecules that contain non‐native and native disulfide bonds during in vitro folding. These intermediates can frequently be trapped covalently during folding and subsequently analyzed. The structural characterization of these kinetically trapped disulfide intermediates provides a clue to understanding the oxidative folding pathway. To investigate the folding of disulfide‐containing peptides and proteins, in this unit, chemical methods are described for regulating regioselective disulfide formation (1) by using a combination of several types of thiol protecting groups, (2) by incorporating unique SeCys residues into a protein or peptide molecule, and (3) by combining with post‐translational modification. Curr. Protoc. Protein Sci. 76:28.8.1‐28.8.28. © 2014 by John Wiley & Sons, Inc.

Keywords: folding; intermediate; disulfide; diselenide; cysteine; selenocysteine; post‐translation; native chemical ligation; topological isomer

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Regioselective Formation of Two Intra‐Molecular Disulfide Bonds
  • Basic Protocol 2: Formation of Three or More Inter‐/Intra‐Molecular Disulfide Bonds
  • Alternate Protocol 1: Regulation of Only One Disulfide Bond Among Three (Multiple) Disulfide Bonds
  • Basic Protocol 3: Regioselective Formation of Intramolecular Diselenide and Disulfide Bonds by the pH‐Jumping Method
  • Alternate Protocol 2: Regioselective Diselenide Bond Formation Prior to Disulfide‐Bond Formation
  • Support Protocol 1: Incorporation of Selenocysteine into Proteins
  • Support Protocol 2: Preparation of Reduced Peptides or Proteins Containing Selenocysteines
  • Support Protocol 3: Preparation of Peptides Carrying 2xCysteine(MeBzl) and 2xSelenocysteine(MeBzl)
  • Support Protocol 4: Preparation of Peptide Carrying 2xCysteine(Trt) and 2xSelenocysteine(Mob)
  • Basic Protocol 4: Regioselective Formation of Disulfide Bonds in Proteins Carrying Post‐Translational Modifications
  • Alternate Protocol 3: Peptide Condensation Reaction by the Thioester Method
  • Support Protocol 5: Chemical Synthesis of Glycosylated Polypeptides by SPPS
  • Support Protocol 6: Chemical Synthesis of Glycosylated Polypeptide α‐Thioesters
  • Support Protocol 7: Semi‐Synthetic Preparation of α‐Thioester by Bacterial Expression System
  • Support Protocol 8: Chemical Synthesis of Partially Protected Polypeptides
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Regioselective Formation of Two Intra‐Molecular Disulfide Bonds

  Materials
  • Peptide with two Cys(Acm) and two Cys residues with a free thiol group (units & )
  • Acetic acid (AcOH)
  • Ammonium hydroxide (NH 4OH), aqueous
  • Methanol (MeOH)
  • Iodine (I 2)
  • 1 M HCl
  • L‐ascorbic acid (vitamin C)
  • 0.2‐µm filter units
  • Additional reagents and equipment for peptide synthesis (unit ) and native chemical ligation of polypeptides (unit ), RP‐HPLC (unit ), disulfide‐bond formation in peptides (unit ), Ellman test (unit ), gel filtration chromatography (unit ), and ion‐exchange chromatography (unit )

Basic Protocol 2: Formation of Three or More Inter‐/Intra‐Molecular Disulfide Bonds

  Materials
  • Protected peptide with 2xCys(tBu), 2xCys(Acm), and 2xCys(Trt) (units & )
  • Diethyl ether or hexane
  • Acetic acid (AcOH)
  • Ammonium hydroxide (NH 4OH), aqueous
  • Methanol (MeOH)
  • Iodine
  • 1 M HCl
  • L‐ascorbic acid (vitamin C)
  • Trifluoroacetic acid (TFA)
  • Trichloromethylsilane (CH 3SiCl 3; see unit )
  • Diphenylsulfoxide (see unit )
  • Anisole (see unit )
  • Diethyl ether
  • Vacuum source
  • Additional reagents and equipment for peptide synthesis (unit ), native chemical ligation of polypeptides (unit ), RP‐HPLC (unit ), disulfide‐bond formation in peptides (unit ), gel filtration chromatography (unit ), and ion‐exchange chromatography (unit )

Alternate Protocol 1: Regulation of Only One Disulfide Bond Among Three (Multiple) Disulfide Bonds

  Materials
  • Peptide with two Cys(Acm) and four Cys(MeBzl) residues (units , , & )
  • Acetic acid (AcOH)
  • Ammonium hydroxide (NH 4OH), aqueous
  • Methanol (MeOH)
  • Iodine
  • 1 M HCl
  • L‐ascorbic acid (vitamin C)
  • Additional reagents and equipment for peptide synthesis (unit ), native chemical ligation of polypeptides (unit ), RP‐HPLC (unit ), disulfide‐bond formation in peptides (unit ), gel filtration chromatography (unit ), and ion‐exchange chromatography (unit )

Basic Protocol 3: Regioselective Formation of Intramolecular Diselenide and Disulfide Bonds by the pH‐Jumping Method

  Materials
  • Nitrogen gas
  • Reduced/denatured proteins (see Support Protocols protocol 61 to protocol 94)
  • Oxidation buffer 1 (weakly acidic buffer; pH 4 to 6):
    • 0.1 M ammonium formate, pH 4.2
  • Oxidation buffer 2 (weakly alkaline buffer; pH 7 to 9):
    • 0.1 M Tris/HCl, pH 8
  • Thiol agent, selected from among:
    • 0.1 to 10 mM GSH
  • Disulfide agent, selected from among:
    • 0.1 to 10 mM GSSG
  • Additional reagents and equipment for RP‐HPLC (unit ), gel filtration chromatography (unit ), ion‐exchange chromatography (unit ), CD (unit ), or NMR (unit )

Alternate Protocol 2: Regioselective Diselenide Bond Formation Prior to Disulfide‐Bond Formation

  Materials
  • Reduced/denatured proteins (see Support Protocols protocol 61 to protocol 94)
  • Weakly alkaline buffer: 0.1 M Tris·Cl, pH 7 to 9 ( ) containing 50 mM DTT
  • Weakly alkaline buffer: 0.1 M Tris·Cl, pH 7 to 9 ( ) without DTT
  • Thiol agent, selected from among:
    • 0.1 to 10 mM GSH
  • Disulfide agent, selected from among:
    • 0.1 to 10 mM GSSG
  • Dialysis membrane
  • Additional reagents and equipment for dialysis ( ) and RP‐HPLC (unit ), CD (unit ), or NMR (unit )

Support Protocol 1: Incorporation of Selenocysteine into Proteins

  Materials
  • E. coli co‐transformed with the expression vector for target protein and the plasmid pSUABC (Arnér et al., ) which directs expression of the selA‐C gene (unit )
  • Culture medium: TB (Terrific broth) medium (see recipe) with antibiotic (unit )
  • L‐cysteine
  • Sodium selenite (Na 2SeO 3)
  • Additional reagents and equipment for spectrophotometric monitoring of bacterial growth ( ), SDS‐PAGE (unit ), and purification of recombinant protein (unit , unit , & unit )

Support Protocol 2: Preparation of Reduced Peptides or Proteins Containing Selenocysteines

  Materials
  • Peptides or proteins containing SeCys residues ( protocol 6)
  • Alkaline buffer: 0.1 M Tris·Cl, pH 7 to 9 ( ), containing excess DTT
  • 1 M HCl
  • Additional reagents and equipment for dialysis ( )

Support Protocol 3: Preparation of Peptides Carrying 2xCysteine(MeBzl) and 2xSelenocysteine(MeBzl)

  Materials
  • Peptide containing 2xCys(MeBzl) and 2xSeCys(MeBzl) residues (units , , & )
  • HF/m‐cresol (unit )
  • Acetic acid (AcOH)
  • Ethyl ether
  • Vacuum source
  • Additional reagents and equipment for peptide synthesis (unit ), native chemical ligation of polypeptides (unit ), and RP‐HPLC (unit )

Support Protocol 4: Preparation of Peptide Carrying 2xCysteine(Trt) and 2xSelenocysteine(Mob)

  Materials
  • Peptide bearing 2xCys(Trt) and 2xSeCys(Mob) residues (units & )
  • Reagent K: TFA/thioanisole/phenol/water (90:2.5:7.5:5, v/v/v/v)) in the presence of 1.3 equivalents of DTNP (2,2′‐dithiobis(5‐nitropyridine))
  • Methyl t‐butyl ether
  • Alkaline buffers: 0.1 M Tris·Cl, pH 7 to 9 ( ) with and without 50 mM DTT
  • 8% formic acid
  • Separatory funnel
  • Additional reagents and equipment for peptide synthesis (unit ) and RP‐HPLC (unit )

Basic Protocol 4: Regioselective Formation of Disulfide Bonds in Proteins Carrying Post‐Translational Modifications

  Materials
  • Peptide segment with N‐terminal Cys residue with/without carbohydrate moieties, purified (see protocol 12)
  • Peptide α‐thioester with/without carbohydrate moieties, purified (see Support Protocols protocol 66 protocol 77)
  • Ligation buffer (see recipe)
  • Urea
  • Glutathione, reduced form
  • Dilution buffer (see recipe)
  • Lyophilizer
  • Additional reagents and equipment for reversed‐phase HPLC (unit ) and MALDI‐TOF mass spectrometry (units & )

Alternate Protocol 3: Peptide Condensation Reaction by the Thioester Method

  Materials
  • Partially protected C‐terminal glycopeptide segment, previously purified (see protocol 15)
  • Partially protected glycopeptide α‐thioester, previously purified (see protocol 15)
  • 3,4‐dihydro‐3‐hydroxy‐4‐oxo‐1,2,3‐benzotriazine (HOObt)
  • N,N‐diisopropylethylamine (DIEA)
  • Piperidine
  • Diethyl ether
  • Acetic acid (AcOH)
  • Dimethyl sulfoxide (DMSO)
  • Zn (powder, <150 µm)
  • Silver nitrate solution (see recipe)
  • Dithiothreitol (DTT)
  • 0.5 M HCl aqueous solution
  • Centrifuge
  • Filter paper
  • Lyophilizer
  • Additional reagents and equipment for reversed‐phase HPLC (unit ) and MALDI‐TOF mass spectrometry (units & )

Support Protocol 5: Chemical Synthesis of Glycosylated Polypeptides by SPPS

  Materials
  • Protected glycopeptide resin after Fmoc SPPS chain assembly
  • TFA cleavage cocktail (see recipe)
  • Diethyl ether
  • Low‐acidity TfOH cocktail (see recipe)
  • 0.1% (v/v) trifluoroacetic acid (TFA)
  • Hydrazine buffer (see recipe)
  • Acetonitrile
  • Acetic acid (AcOH)
  • Centrifuge
  • Lyophilizer
  • Additional reagents and equipment for reversed‐phase HPLC (unit ) and mass spectrometry (units & )

Support Protocol 6: Chemical Synthesis of Glycosylated Polypeptide α‐Thioesters

  Materials
  • Inclusion bodies (see units & )
  • 8 M urea
  • Cleavage buffer (see recipe)
  • 1 mg/ml (100% w/v) trichloroacetic acid
  • Acetone
  • Acetic acid
  • Lyophilizer
  • Centrifuge
  • Additional reagents and equipment for reversed‐phase (unit ) or gel‐filtration (UNIT HPLC (unit )
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Figures

Videos

Literature Cited

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Key References
   Okumura, M. , Shimamoto, S. , and Hidaka, Y. 2012. A chemical method for investigating disulfide‐coupled peptide and protein folding. FEBS J. 279:2283‐2295.
  Relatively recent coverage on the regulation of disulfide formation of peptides and proteins.
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