Refolding of SDS‐Denatured Proteins Using Amphipathic Cosolvents and Osmolytes

Guillaume Roussel1, Emmanuel Tinti1, Eric Perpète1, Catherine Michaux1

1 Department of Chemistry, Unité de Chimie Physique Théorique et Structurale, University of Namur, Namur, Belgium
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 28.5
DOI:  10.1002/0471140864.ps2805s72
Online Posting Date:  April, 2013
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Abstract

Currently, the investigation of protein refolding processes involves several time‐consuming stages that require large amounts of protein and costly chemicals. Consequently, there is great interest in developing new approaches to the study of protein renaturation that are more technically and economically feasible. It has recently been reported that certain cosolvents are able to modulate the denaturing properties of sodium dodecyl sulfate (SDS) and induce the refolding of proteins. This unit presents a protocol to study and follow the renaturation of a protein (membrane or soluble) starting from a native or SDS‐unfolded state using a variety of candidate cosolvents and osmolytes. Curr. Protoc. Protein Sci. 72:28.5.1‐28.5.9. © 2013 by John Wiley & Sons, Inc.

Keywords: sodium dodecyl sulfate; cosolvent; osmolyte; protein refolding

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Lysozyme Inactivation and Unfolding in the Presence of SDS
  • Basic Protocol 2: Lysozyme Resistance to SDS‐Induced Denaturation and Inactivation in the Presence of Osmolytes and Cosolvents
  • Basic Protocol 3: Refolding of SDS‐Unfolded Lysozyme by Addition of Osmolytes or Cosolvents
  • Support Protocol 1: Removal of SDS and MPD by Size‐Exclusion Chromatography
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Lysozyme Inactivation and Unfolding in the Presence of SDS

  Materials
  • 50 mM sodium phosphate buffer, pH 7.4 ( appendix 2E)
  • 10 mM sodium dodecyl sulfate (SDS) in 50 mM sodium phosphate buffer, pH 7.4
  • 8 mg/ml lysozyme solution (see recipe)
  • 360 µg/ml Micrococcus suspension (see recipe)
  • 1.5‐ml microcentrifuge tubes
  • Fluorometer (excitation 280 nm, emission 350 nm)
  • Eppendorf UVette (Sigma, cat. no. Z618683)
  • Spectrophotometer (absorbance at 450 nm)
  • Eppendorf cuvette, polystyrene (Sigma, cat. no. C5291)

Basic Protocol 2: Lysozyme Resistance to SDS‐Induced Denaturation and Inactivation in the Presence of Osmolytes and Cosolvents

  Materials
  • Individual 7 M solutions of osmolytes and cosolvents in 50 mM sodium phosphate buffer, pH 7.4 ( appendix 2E):
    • Osmolytes:
    • Glycerol (Sigma, cat. no. G5516): 3.30 ml of 10.6 M stock in 1.7 ml buffer
    • Proline (Sigma, cat. no. P0380): 4.029 g in 5 ml buffer
    • Glycine (Sigma, cat. no. 410225): 2.627 g in 5 ml buffer
    • Sucrose (Sigma, cat. no. S7903): 11.981 g in 5 ml buffer
    • Cosolvents:
    • 2‐Methyl‐2,4‐pentanediol (Sigma, cat. no. 112100): 4.54 ml of 7.7 M stock in 0.45 ml buffer
    • 2,4‐Pentanediol (Sigma, cat. no. 156019): 3.79 ml of 9.24 M stock in 1.21 ml buffer
    • 1,2‐Pentanediol (Sigma, cat. no. 260282): 3.67 ml of 9.53 M stock in 1.33 ml buffer
    • 1,4‐Pentanediol (Sigma, cat. no. 194182): 3.66 ml of 9.55 M stock in 1.34 ml buffer
    • 2,5‐Hexanediol (Sigma, cat. no. H11904): 4.51 ml of 7.76 M stock in 0.49 ml buffer
    • Methanol (Sigma, cat. no. 34860): 1.46 ml of 24 M stock in 3.54 ml buffer
    • Ethanol (Sigma, cat. no. 459844): 2.06 ml of 17 M stock in 2.94 ml buffer
    • 2‐Propanol (Sigma, cat. no. W292907): 2.69 ml 13 M stock in 2.31 ml buffer
  • Additional reagents and equipment for monitoring protein unfolding (see protocol 1)

Basic Protocol 3: Refolding of SDS‐Unfolded Lysozyme by Addition of Osmolytes or Cosolvents

  Materials
  • Running buffer A: 14.3 ml of 7 M 2‐methyl‐2,4‐pentanediol (MPD; see protocol 2) in 35.7 ml of 50 mM sodium phosphate buffer, pH 7.4 ( appendix 2E)
  • Running buffer B: 50 mM sodium phosphate buffer, pH 7.4 (final 438 mg sodium)
  • Lysozyme sample containing SDS and/or MPD (see Basic Protocols protocol 11 protocol 33)
  • Superdex S75 column
  • UV spectrophotometer (absorbance at 280 nm)
  • 1.5‐ml centrifuge tubes
  • Amicon concentrator or equivalent
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Figures

Videos

Literature Cited

Literature Cited
   Andersen, K.K., Oliveira, C.L., Larsen, K.L., Poulsen, F.M., Callisen, T.H., Westh, P., and Pedersen, J.S., Otzen, D. 2009. The role of decorated SDS micelles in sub‐CMC protein denaturation and association. J. Mol. Biol. 391:207‐226.
   Anfinsen, C., Axelrod, J., Berg, P., Conney, A., Horecker, B., Kaback, R., Kornberg, A., Lederberg, J., Nirenberg, M., Palade, G., Pestka, S, Reddy, P., Shatkin, A., Skalka, A., Specter, S., and Weissbach, A. 1995. Wrong move. Nature 373:184.
   Bhuyan, A. 2010. On the mechanism of SDS‐induced protein denaturation. Biopolymers 93:186‐199.
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   Dill, K.A., Ozkan, S.B., Weikl, T.R., Chodera, J.D., and Voelz, V.A. 2007. The protein folding problem: When will it be solved? Curr. Opin. Struct. Biol. 17:342‐346.
   Dill, K.A., Ozkan, S.B., Shell, M.S., and Weikl, T.R. 2008. The protein folding problem. Annu. Rev. Biophys. 37:289‐316.
   Dutta, A., Kim, T.‐Y., Moeller, M., Wu, J., Alexiev, U., and Klein‐Seetharaman, J. 2010. Characterization of membrane protein non‐native states. 2. The SDS‐unfolded states of rhodopsin. Biochemistry 49:6329‐6340.
   Gudiksen, K.L., Gitlin, I., and Whitesides, G.M. 2006. Differentiation of proteins based on characteristic patterns of association and denaturation in solutions of SDS. Proc. Natl. Acad. Sci. U.S.A. 103:7968‐7972. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1472413&tool=pmcentrez&rendertype=abstract.
   Helenius, A. and Simons, K. 1985. Solubilization of membrane by detergents. Biochim. Biophys. Acta 415:29‐79.
   Heller, K. 2010. Targeting misfolded proteins to fight neurodegenerative diseases. PLoS Biol. 8:e100290.
   Michaux, C., Pomroy, N.C., and Privé, G.G. 2008a. Refolding SDS‐denatured proteins by the addition of amphipathic cosolvents. J. Mol. Biol. 375:1477‐1488.
   Michaux, C., Pouyez, J., Wouters, J., and Privé, G.G. 2008b. Protecting role of cosolvents in protein denaturation by SDS: A structural study. BMC Struct. Biol. 8:29.
   Nielsen, M.M., Andersen, K.K., Westh, P., and Otzen, D.E. 2007. Unfolding of beta‐sheet proteins in SDS. Biophys. J. 92:3674‐3685.
   Roussel, G., Perpète, E.A., Matagne, A., Tinti, E., and Michaux, C. 2013. Towards a universal method for protein refolding: The trimeric beta barrel membrane Omp2a as a test case. Biotechnol. Bioeng. 110:417‐423.
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