Structural Analysis of Biomolecules > Folding and Conformational Change

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Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum

Edwin Francis1,  Robert Daniels1,  Daniel N. Hebert1

1University of Massachusetts, Amherst, Massachusetts

Publication Name: 
Current Protocols in Cell Biology
Unit Number: 
Unit 15.6
DOI: 
10.1002/0471143030.cb1506s14
Online Posting Date: 
May, 2002
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Abstract

Proteins that travel through the secretory pathway undergo post-translational folding and oxidation steps that lead to correct conformation of the final protein. This unit focuses on methods for the analysis of folding and oxidation events and the factors responsible for their proper execution. Alkylation and nonreducing SDS-PAGE is use to analyze disulfide bond formation in ER-derived microsomes. If proteins are synthesized under reducing conditions, it is possible to initiate folding by addition of oxidizing agents, thus allowing analysis of factors necessary for the folding process. As folding progresses, the protein of interest shows a change in sensitivity to proteolysis. Co-immunoprecipitation or crosslinking and denaturing immunoprecipitation are used to explore the role of molecular chaperones and other factors. Conformation-specific antibodies can be used to probe folding. In addition, folding can be analyzed in intact or semi-permeabilized adherent or suspension cells.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Analysis of Disulfide Bond Formation in Rough Endoplasmic Reticulum–Derived Microsomes by Alkylation and Nonreducing SDS-PAGE
  • Alternate Protocol 1: Analysis of Post-Translational Disulfide Bond Formation in Rough Endoplasmic Reticulum–Derived Microsomes
  • Basic Protocol 2: Analysis of Protein Folding by Proteolytic Sensitivity
  • Support Protocol: Isolation of Semipermeabilized Cells for Analysis of Protein Folding
  • Basic Protocol 3: Analysis of Folding-Intermediate Binding to Molecular Chaperones in Rough Endoplasmic Reticulum–Derived Microsomes
  • Alternate Protocol 2: Monitoring Transient Chaperone Interactions Using Cross-Linking and Denaturing Immunoprecipitations
  • Basic Protocol 4: Monitoring Protein Folding Using Conformation-Specific Antibodies
  • Basic Protocol 5: Analysis of Protein Folding and Disulfide Bond Formation in Cells Grown in Intact Monolayers (Adherent Cells)
  • Alternate Protocol 3: Analysis of Protein Folding and Disulfide Bond Formation in Suspended (Nonadherent) Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Analysis of Disulfide Bond Formation in Rough Endoplasmic Reticulum–Derived Microsomes by Alkylation and Nonreducing SDS-PAGE

 Materials
  • In vitro translation reagents, including:
  •     1 equivalent/µl nuclease-treated canine pancreatic microsomes (unit) 11.4) or 1 × 105 cells/µl semipermeabilized (SP) cells (see Support Protocol)
  •     Rabbit reticulocyte lysate treated with ATP-regenerating system and nucleases (e.g., Promega)
  •     1 mM amino acid mixture lacking methionine and cysteine
  •     11 mCi/ml [35S]methionine and [35S]cysteine (1175 Ci/mmol; e.g., PE Biosystems)
  •     40 U/µl RNase inhibitor (e.g., RNasin; Promega)
  •     100 mM GSSG (see recipe) or 2.5 mM FAD (see recipe)
  •     100 mM dithiothreitol (DTT; e.g., Promega or appendix 2A)
  •     2.5 M KCl
  •     1 µg/µl mRNA for the protein of interest (e.g., unit 11.2)
  •     Nuclease-free H2O
  • 50 mM cycloheximide (see recipe)
  • 120 mM N-ethylmaleimide (NEM) (see recipe)
  • 20 mM AMS, prepared fresh (see recipe)
  • 0.5% and 2% (w/v)
  •     3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) (CHAPS) in HeBS (CHAPS/HeBS, see recipe), ice cold
  • 2× nonreducing sample buffer (see recipe)
  • 10% (w/v) protein A–Sepharose (see recipe)
  • Antibodies raised against the protein of interest
  • PBS (appendix 2A), optional
  • 2% (w/v) salicylate (see recipe)
  • 27°C water bath
  • 1.5-ml microcentrifuge tubes, RNase free
  • Tube rotator (capable of end-over-end inversions), 4°C
  • Microcentrifuge with a fixed-angle rotor (Eppendorf 5415C or equivalent), 4°C and room temperature
  • Vacuum aspirator
  • Microcentrifuge tube rack vortex mixer (e.g., Tommy MT-360; Tomy Tech USA Inc.), 4°C
  • 95°C heating block
  • Whatman 3MM filter paper
  • Additional reagents and equipment for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) minigel with Laemmli buffers (unit 6.1), Coomassie blue staining and destaining (unit 6.6), and gel autoradiography (unit 6.3)

NOTE: It is important to avoid contamination by RNases that degrade the mRNA during in vitro translations. Wear gloves throughout the experiment. Treat water and salt solutions with diethylpyrocarbonate (DEPC) to chemically inactivate RNases. Treat all glass and plasticware with DEPC-treated water or otherwise to remove RNase activity.

Basic Protocol 2: Analysis of Protein Folding by Proteolytic Sensitivity

 Materials
  • 1× sucrose cushion (see recipe)
  • 1% and 10% digestion buffer: 1% and 10% (w/v)
  •     3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) (CHAPS) in HeBS (see recipe for HeBS)
  • 50 µg/ml trypsin (prepared from 1 mg/ml stock; stored at 4°C up to 6 months)
  • 50 µg/ml soybean trypsin inhibitor (prepared from 1 mg/ml stock; stored at 4°C up to 6 months)
  • 150 mM PMSF (see recipe)
  • 2× reducing sample buffer: 10% (v/v) 1 M DTT (appendix 2A; 100 mM final) in 2× nonreducing sample buffer (see recipe)
  • Beckman Airfuge, 4°C, and 5 × 20–mm ultraclear centrifuge tubes (Beckman)
  • Additional reagents and equipment for in vitro translation using either canine pancreatic microsomes or SP cells, analyzing proteins by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and gel autoradiography (see Basic Protocol 1)

NOTE: It is important to avoid contamination by RNases that degrade the mRNA during in vitro translations. Wear gloves throughout the experiment. Treat water and salt solutions with diethylpyrocarbonate (DEPC) to chemically inactivate RNases. Treat all glass and plasticware with DEPC-treated water or otherwise to remove RNase activity.

Support Protocol: Isolation of Semipermeabilized Cells for Analysis of Protein Folding

 Materials
  • Cells in 75-cm2 tissue culture flask in appropriate medium
  • PBS (appendix 2A)
  • Trypsin/EDTA solution (see recipe), room temperature
  • KHM-STI buffer (see recipe), ice cold
  • 0.4% (w/v) trypan blue solution (Sigma-Aldrich)
  • KHM buffer (see recipe), ice cold
  • 20 mg/ml digitonin in dimethyl sulfoxide (DMSO), stored in 1-ml aliquots up to 2 years at –20°C
  • HEPES/potassium acetate buffer (see recipe), ice cold
  • 100 mM CaCl2 (appendix 2A)
  • 15,000 U/ml micrococcal nuclease, from Staphylococcal aureus (Boehringer Mannheim), stored in working-volume aliquots (e.g., 5 µl or multiples of 5 µl) at –80°C
  • 250 mM ethylene glycol bis(-aminoethyl ether)-N,N,N¢,N¢-tetraacetic acid (EGTA), adjusted to pH 7.4 with NaOH (stored up to 2 years at –20°C)
  • Vacuum aspirator
  • 15-ml sterile polystyrene centrifuge tubes, prechilled on ice
  • Beckman GPR centrifuge (or equivalent), 4°C
  • Microcentrifuge, 4°C
  • Additional reagents and equipment for counting cells (unit 1.1)

NOTE: All solutions and equipment coming into contact with living cells must be sterile and aseptic technique should be used accordingly.

NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.


Basic Protocol 3: Analysis of Folding-Intermediate Binding to Molecular Chaperones in Rough Endoplasmic Reticulum–Derived Microsomes

 Materials
  • Antiserum raised against protein of interest
  • Antiserum raised against chaperone of interest
  • Additional reagents and equipment for in vitro translation using canine pancreatic microsomes or SP cells, immunoprecipitation, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and gel autoradiography (see Basic Protocol 1)

NOTE: It is important to avoid contamination by RNases that degrade the mRNA during in vitro translations. Wear gloves throughout the experiment. Treat water and salt solutions with diethylpyrocarbonate (DEPC) to chemically inactivate RNases. Treat all glass and plasticware with DEPC-treated water or otherwise to remove RNase activity.

Alternate Protocol 2: Monitoring Transient Chaperone Interactions Using Cross-Linking and Denaturing Immunoprecipitations

 Additional Materials (also see Basic Protocol 3)
  • Isotonic sucrose cushion (see recipe)
  • Isotonic resuspension buffer (see recipe)
  • 10 mM BMH (see recipe) or alternative cross-linker
  • Quenching solution: 100 mM 2-mercaptoethanol (stored up to 1 month at 4°C)
  • 20% (w/v) SDS (appendix 2A)
  • Denaturing immunoprecipitation buffer (see recipe)
  • Beckman Optima TLX ultracentrifuge and TLA 120.2 rotor, 4°C, and 7 × 20–mm ultracentrifuge tubes or Beckman Airfuge, 4°C, and 5 × 20–mm ultraclear centrifuge tubes

NOTE: It is important to avoid contamination by RNases that degrade the mRNA during in vitro translations. Wear gloves throughout the experiment. Treat water and salt solutions with diethylpyrocarbonate (DEPC) to chemically inactivate RNases. Treat all glass and plasticware with DEPC-treated water or otherwise to remove RNase activity.

Basic Protocol 4: Monitoring Protein Folding Using Conformation-Specific Antibodies

 Materials
  • 50% (w/v) protein G–Sepharose or protein A–Sepharose bead (Sigma-Aldrich) slurry in PBS/0.1% (w/v) BSA/0.01% (w/v) sodium azide
  • Conformation-specific monoclonal antibody (mAb) against protein of interest
  • Control antibodies (preimmune control and antibody control that recognizes all conformations of the protein of interest)
  • Additional reagents and equipment for in vitro translation using canine pancreatic microsomes, immunoprecipitation, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and gel autoradiography (see Basic Protocol 1)

CAUTION: Sodium azide is poisonous; follow appropriate precautions for handling, storage, and disposal.

NOTE: It is important to avoid contamination by RNases that degrade the mRNA during in vitro translations. Wear gloves throughout the experiment. Treat water and salt solutions with diethylpyrocarbonate (DEPC) to chemically inactivate RNases. Treat all glass and plasticware with DEPC-treated water or otherwise to remove RNase activity.

NOTE: All solutions should be ice cold and procedures should be carried out at 4°C or on ice unless otherwise indicated.



Basic Protocol 5: Analysis of Protein Folding and Disulfide Bond Formation in Cells Grown in Intact Monolayers (Adherent Cells)

 Materials
  • Adherent cells grown to 80% to 90% confluency in a 60-mm tissue culture dish
  • Depletion medium: cysteine- and methionine-free tissue culture medium, 37°C
  • Labeling medium: depletion medium containing 0.125 to 0.75 mCi/ml
  •     [35S]methionine and [35S]cysteine (prepared fresh), 37°C
  • Chase medium (see recipe), 37°C
  • Stop buffer: 20 mM NEM (see recipe for 1 M stock) in PBS, prepared just before use and kept on ice
  • Lysis buffer (see recipe), ice cold
  • Aspirator
  • Cell scraper
  • Additional reagents and equipment for immunoprecipitation (unit 7.2 or see Basic Protocol 1) and analysis of disulfide bond formation (see Basic Protocol 1), protein folding by proteolytic sensitivity (see Basic Protocol 2), chaperone binding (see Basic Protocol 3), or protein folding with conformation-specific antibodies (see Basic Protocol 4)

NOTE: All solutions and equipment coming into contact with living cells must be sterile and aseptic technique should be used accordingly.

NOTE: All culture incubations are performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.

NOTE: For the analysis of co-translational protein folding no reducing agents are added to either the pulse or chase medium. Chase medium should contain 1 mM cycloheximide in order to stop protein synthesis. In the case of post-translational protein folding and disulfide bond formation, reducing agent (5 mM DTT final) is added to the pulse medium. When the reducing agent is removed after pulse labeling, redox conditions are restored that support the formation of disulfide bonds.

NOTE: Separate cell culture dishes will be required for each condition and time point.




Alternate Protocol 3: Analysis of Protein Folding and Disulfide Bond Formation in Suspended (Nonadherent) Cells

 Additional Materials (also see Basic Protocol 5)
  • Cell suspension
  • Suspension labeling medium: depletion medium containing 25 to 50 µCi/ml
  •     [35S]methionine and [35S]cysteine (prepared fresh), 37°C
  • 15-ml sterile polystyrene conical centrifuge tubes
  • Beckman GPR centrifuge or equivalent
  • 37°C water bath
  • Additional reagents and equipment for immunoprecipitation (unit 7.2 or see Basic Protocol 1) and for analysis of disulfide bond formation (see Basic Protocol 1), protein folding by proteolytic sensitivity (see Basic Protocol 2), chaperone binding (see Basic Protocol 3), or protein folding with conformation-specific antibodies (see Basic Protocol 4)

NOTE: All solutions and equipment coming into contact with living cells must be sterile and aseptic technique should be used accordingly.

NOTE: All culture incubations are performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.

NOTE: Keep cells in suspension by periodically swirling the tube during incubations.



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Figures

  • Figure 15.6.1
    The effects of disulfide bond formation in proteins. (A) Schematic demonstration of the sensitivity of a reduced and oxidized polypeptide chain to protease digestion (top) and alkylation (bottom). (B) Resulting mobility changes visualized by SDS-PAGE. When a reduced or unfolded polypeptide containing free thiols is alkylated with 4-acetamido-4¢-maleimidylstilbene-2, 2¢-disulfonic acid disodium salt (AMS), a bulk alkylating agent, or N-ethylmaleimide (NEM), a shift in protein mobility on an SDS-polyacrylamide gel is observed (compare lanes 2 and 3 to lane 1). The oxidized (disulfide bonded) or folded protein is not sensitive to alkylation with either AMS or NEM as fewer or no free cysteines are available (lanes 4 to 6). The reduced or unfolded protein is more accessible to proteolytic digestion with trypsin than the oxidized or folded protein.

    View Image
  • Figure 15.6.2
    Formation of disulfide bonds. (A) Co-translational bond formation. (B) Post-translational bond formation. Under normal cellular redox conditions, the cytosol is reducing and the endoplasmic reticulum (ER) lumen is oxidizing. Disulfide bond formation and glycan addition can take place co-translationally (A) as the C terminus of the protein is being completed by the ribosome. Synchronous oxidation of the completed glycosylated and reduced chain can be initiated post-translationally (B) by synthesizing the protein under reducing conditions (e.g., DTT), inhibiting protein synthesis, and then adding oxidizing agents, such as oxidized glutathione (GSSG) or flavin adenine dinucleotide (FAD), post-translationally.

    View Image
  • Figure 15.6.3
    Disulfide bond formation and folding of hemagglutinin (HA). (A) Co-translational and post-translational formation of disulfide bonds. (B) Calnexin binding. 35S-labeled HA was in vitro translated with rabbit reticulocyte lysate at 27°C for 1 hr in the presence of canine pancreatic endoplasmic reticulum (ER)–derived microsomes under oxidizing (co-translational; Co-T) or reducing (post-translational; Post-T) conditions. Cycloheximide was added to inhibit protein synthesis and protein samples were removed after the indicated chase times and subjected to immunoprecipitation with antisera raised against the whole influenza virus (A) and resolved by nonreducing (NR) and reducing (RD) SDS-PAGE. For post-translational analysis, oxidation was initiated at time zero and followed. The same samples were used to identify interactions with calnexin by the co-immunoprecipitation of 35S-labeled HA with anti-calnexin antiserum (B). Protein forms are as follows: IT1, intermediate 1; IT2, intermediate 2; NT, native; R, reduced.

    View Image

Literature Cited

Literature Cited
    Blobel, G. and Dobberstein, B. 1975. Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components. J. Cell Biol. 67:852-862.
    Braakman, I., Hoover-Litty, H., Wagner, K.R., and Helenius, A. 1991. Folding of influenza hemagglutinin in the endoplasmic reticulum. J. Cell Biol. 114:401-411.
    Braakman, I., Helenius, J., and Helenius, A. 1992. Manipulating disulfide bond formation and protein folding in the endoplasmic reticulum. EMBO J. 11:1717-1722.
    Chen, W., Helenius, J., Braakman, I., and Helenius, A. 1995. Cotranslational folding and calnexin binding of influenza hemagglutinin in the endoplasmic reticulum. Proc. Natl. Acad. Sci. U.S.A. 92:6229-6233.
    Goldberg, M.E. 1991. Investigating protein conformation, dynamics and folding with monoclonal antibodies. Trends Biochem. Sci. 16:358-362.
    Hebert, D.N., Foellmer, B., and Helenius, A. 1995. Glucose trimming and reglucosylation determines glycoprotein association with calnexin. Cell 81:425-433.
    Hebert, D.N., Foellmer, B., and Helenius, A. 1996. Calnexin and calreticulin promote folding, delay oligomerization and suppress degradation of influenza hemagglutinin in microsomes. EMBO J. 15:2961-2968.
    Wilson, R., Allen, A.J., Oliver, J., Brookman, J.L., High, S., and Bulleid, N.J. 1995. The translocation, folding, assembly and redox-dependent degradation of secretory and membrane proteins in semi-permeabilized mammalian cells. Biochem. J. 307:679-687.
     
 
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