Chemical Methods for Producing Disulfide Bonds in Peptides and Proteins to Study Folding Regulation

Masaki Okumura1, Shigeru Shimamoto2, Yuji Hidaka2

1 Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Miyagi, 2 Faculty of Science and Engineering, Kinki University, Osaka
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 28.7
DOI:  10.1002/0471140864.ps2807s76
Online Posting Date:  April, 2014
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Abstract

Disulfide bonds play a critical role in the folding of secretory and membrane proteins. Oxidative folding reactions of disulfide bond–containing proteins typically require several hours or days, and numerous misbridged disulfide isomers are often observed as intermediates. The rate‐determining step in refolding is thought to be the disulfide‐exchange reaction from nonnative to native disulfide bonds in folding intermediates, which often precipitate during the refolding process because of their hydrophobic properties. To overcome this, chemical additives or a disulfide catalyst, protein disulfide isomerase (PDI), are generally used in refolding experiments to regulate disulfide‐coupled peptide and protein folding. This unit describes such methods in the context of the thermodynamic and kinetic control of peptide and protein folding, including (1) regulation of disulfide‐coupled peptides and protein folding assisted by chemical additives, (2) reductive unfolding of disulfide‐containing peptides and proteins, and (3) regulation of disulfide‐coupled peptide and protein folding using PDI. Curr. Protoc. Protein Sci. 76:28.7.1‐28.7.13. © 2014 by John Wiley & Sons, Inc.

Keywords: additive; disulfide; folding; protein disulfide isomerase; glutathione

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Regulation of Disulfide‐Coupled Peptide and Protein Folding in the Presence of Chemical Additives
  • Alternate Protocol 1: Disulfide‐Coupled Protein Folding at High Salt Concentrations
  • Basic Protocol 2: Reductive Unfolding of Disulfide‐Containing Peptides and Proteins
  • Alternate Protocol 2: Disulfide‐Coupled Protein Folding in Organic Solvents
  • Support Protocol 1: Fractionation of Disulfide Intermediates
  • Basic Protocol 3: Regulation of Disulfide‐Coupled Peptide and Protein Folding Using Protein Disulfide Isomerase
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Regulation of Disulfide‐Coupled Peptide and Protein Folding in the Presence of Chemical Additives

  Materials
  • Reduced, denatured proteins (e.g., see Arolas et al., ; Okumura et al., )
  • Buffer, selected from the following:
    • 0.05 to 0.2 M Tris·Cl, pH 7.3 to 9.3 ( )
    • 0.05 to 0.2 M sodium phosphate, pH 6.2 to 8.2 ( )
  • Thiol agent, selected from the following:
    • Glutathione (GSH)
    • 2‐aminoethanethiol (AET)
    • 2‐dimethylaminoethanethiol (DMAET)
    • 2‐diethylaminoethanethiol (DEAET)
    • diisopropylaminoethanethiol (DPAET; Tokyo Chemical Industry Co.)
  • Disulfide agent: glutathione disulfide (GSSG)
  • Appropriate size tubes with O‐ring seals (Sarstedt)
  • Additional reagents and equipment for purifying or analyzing products by, e.g., reversed‐phase (RP)‐HPLC (unit ), gel filtration (unit ), or ion‐exchange (unit ) chromatography

Alternate Protocol 1: Disulfide‐Coupled Protein Folding at High Salt Concentrations

  Materials
  • Reduced peptide (e.g., see Arolas et al., ; Okumura et al., )
  • Buffer, selected from the following:
    • 0.05 to 0.2 M Tris·Cl, pH 7.3 to 9.3 (e.g., see )
    • 0.05 to 0.2 M sodium phosphate, pH 6.2 to 8.2 (e.g., see )
    • 0.05 to 2 M ammonium acetate (NH 4OAc), pH 6 to 10 (e.g., see or Kubo et al., ).
  • Salt, selected from the following:
    • 0.05 to 2 M (NH 4) 2SO 4
    • 0.05 to 2 M NH 4Cl
    • 0.2 to 1 M KCl
    • 0.2 to 4 M NaCl
  • Thiol agent: glutathione (GSH)
  • Disulfide agent: glutathione disulfide (GSSG)
  • Appropriate size tubes with O‐ring seals (Sarstedt)
  • Additional regents and solutions for analyzing reaction products by circular dichroism (unit ) and RP‐HPLC (unit )

Basic Protocol 2: Reductive Unfolding of Disulfide‐Containing Peptides and Proteins

  Materials
  • Folded proteins (e.g., see Arolas et al., ; Okamura et al, )
  • Buffer, selected from the following:
    • 0.05 to 0.2 M Tris·Cl, pH 7.3 to 9.3 ( )
    • 0.05 to 0.2 M sodium phosphate, pH 6.2 to 8.2 ( )
  • Thiol agent: glutathione (GSH)
  • Disulfide agent: glutathione disulfide (GSSG)
  • Appropriate size tubes with O‐ring seals (Sarstedt)
  • Additional regents and solutions for analyzing reaction products by RP‐HPLC (unit ) and mass spectrometry (unit )

Alternate Protocol 2: Disulfide‐Coupled Protein Folding in Organic Solvents

  Materials
  • Reduced peptide (e.g., see Arolas et al., ; Okumura et al., )
  • Buffer, selected from the following:
    • 0.05 to 0.2 M Tris·Cl, pH 7.3 to 9.3 (e.g., see )
    • 0.05 to 0.2 M sodium phosphate, pH 6.2 to 8.2 (e.g., see )
  • Organic solvent, selected from the following:
    • 5% to 60% (v/v) trifluoroethanol (TFE)
    • 5% to 60% (v/v) hexafluoroisopropanol (HFIP)
    • 5% to 60% (v/v) ethanol (EtOH)
  • Redox reagents (optional), e.g., glutathione and glutathione disulfide
  • Appropriate size tubes with O‐ring seals (Sarstedt)
  • Additional regents and solutions for analyzing reaction products by RP‐HPLC (unit ) and circular dichroism (unit ; optional)

Support Protocol 1: Fractionation of Disulfide Intermediates

  Materials
  • Reduced, denatured proteins (e.g., see Arolas et al., )
  • Buffer, selected from the following:
    • 0.05 to 0.2 M Tris·Cl, pH 7.3 to 9.3 (e.g., see )
    • 0.05 to 0.2 M phosphate buffer, pH 6.2 to 8.2 (e.g., see )
  • Thiol agent: glutathione (GSH)
  • Disulfide agent: glutathione disulfide (GSSG)
  • 2‐aminoethyl methanethiosulfonate (AEMTS)
  • Triethylammonium acetate (TEAA) buffer, pH 5
  • Appropriate size tubes with O‐ring seals (Sarstedt)
  • Additional reagents and solutions for carrying out RP‐HPLC (unit ), gel filtration chromatography (unit ), or ion‐exchange chromatography (unit )

Basic Protocol 3: Regulation of Disulfide‐Coupled Peptide and Protein Folding Using Protein Disulfide Isomerase

  Materials
  • Reduced, denatured proteins (e.g., see Arolas et al., ; Okumura et al., )
  • Buffer, selected from the following:
    • 0.05 to 0.2 M Tris·Cl, pH 7.3 to 9.3 (e.g., see )
    • 0.05 to 0.2 M phosphate buffer, pH 6.2 to 8.2 (e.g., see )
  • Thiol agent: glutathione (GSH)
  • Disulfide agent: glutathione disulfide (GSSG)
  • Disulfide catalyst: protein disulfide isomerase (PDI)
  • Appropriate size tubes with O‐ring seals (Sarstedt)
  • Additional reagents and equipment for carrying out RP‐HPLC (unit ), gel filtration chromatography (unit ), or ion‐exchange chromatography (unit )
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Figures

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Literature Cited

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   Okumura, M. , Shimamoto, S. , Nakanishi, T. , Yoshida, Y. , Konogami, T. , Maeda, S. , and Hidaka, Y. 2012b. Effects of positively charged redox molecules on disulfide‐coupled protein folding. FEBS Lett. 586:3926‐3930.
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Key References
   Okumura et al., 2012a. See above.
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