Quantitative Determination of Protein Stability and Ligand Binding by Pulse Proteolysis

Chiwook Park1, Susan Marqusee2

1 Purdue University, West Lafayette, 2 University of California, Berkeley, California
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 20.11
DOI:  10.1002/0471140864.ps2011s46
Online Posting Date:  December, 2006
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Abstract

Pulse proteolysis exploits the difference in proteolytic susceptibility between folded and unfolded proteins for facile but quantitative determination of protein stability. The method requires only common biochemistry and molecular biology lab equipment. Pulse proteolysis also can be used to determine the affinity of a ligand to its protein target by monitoring the change in protein stability upon ligand binding. The Basic Protocol describes the detailed procedure for determining protein stability using pulse proteolysis. For pulse proteolysis to be used for determining a protein's stability, the protein should not be digested significantly by pulse proteolysis when it is in the folded conformation. The Support Protocol describes a procedure for determining whether a protein satisfies this requirement. The principles of protein stability determination using denaturant and pulse proteolysis are also discussed.

Keywords: protein stability; proteolysis; ligand binding

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Determination of Global Protein Stability by Pulse Proteolysis
  • Support Protocol 1: Verification of Resistance of Folded Proteins to the Pulse of Proteolysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Determination of Global Protein Stability by Pulse Proteolysis

  Materials
  • 5 to 10 mg/ml protein solution of known concentration in an appropriate buffer
  • 8.0 M urea
  • 1.0 M CaCl 2
  • 50 mM EDTA (pH 8.0)
  • 10 mg/ml thermolysin (see recipe)
  • Sample buffer (e.g., unit 10.1)
  • Sypro Red staining solution (Molecular Probes)
  • Typhoon Scanner (GE Healthcare)
  • Image analysis software (e.g., ImageJ, a freeware available for download at http://rsbweb.nih.gov/ij; or ImageQuant, GE Healthcare)
  • Additional reagents and equipment for performing SDS‐PAGE (unit 10.1)

Support Protocol 1: Verification of Resistance of Folded Proteins to the Pulse of Proteolysis

  Materials
  • 5 to 10 mg/ml protein solution of known concentration in a desired buffer
  • 8.0 M urea
  • 1.0 M CaCl 2
  • 10 mg/ml thermolysin (see recipe)
  • 50 mM EDTA (pH 8.0)
  • Sample buffer (unit 10.1)
  • Sypro Red staining solution (Molecular Probes)
  • Typhoon Scanner (GE Healthcare)
  • Image analysis software (e.g., ImageJ, a freeware available for download at http://rsbweb.nih.gov/ij; or ImageQuant, GE Healthcare)
  • Additional reagents and equipment for performing SDS‐PAGE (unit 10.1)
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Figures

Videos

Literature Cited

Literature Cited
   Dahlquist, F.W., Long, J.W., and Bigbee, W.L. 1976. Role of Calcium in the thermal stability of thermolysin. Biochemistry‐US 15:1103‐1111.
   Gill, S.C. and von Hippel, P.H. 1989. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182:319‐326.
   Greene, R.F. and Pace, C.N. 1974. Urea and guanidine‐hydrochloride denaturation of ribonuclease, lysozyme, α‐chymotrypsin, and β‐lactoglobulin. J. Biol. Chem. 249:5388‐5393.
   Imoto, T., Yamada, H., and Ueda, T. 1986. Unfolding rates of globular proteins determined by kinetics of proteolysis. J. Mol. Biol. 190:647‐649.
   Inouye, K., Kuzuya, K., and Tonomura, B. 1998. Sodium chloride enhances markedly the thermal stability of thermolysin as well as its catalytic activity. Biochim. Biophys. Acta 1388:209‐214.
   Lim, W.A. and Sauer, R.T. 1989. Alternative packing arrangements in the hydrophobic core of lambda repressor. Nature 339:31‐36.
   Linderstrøm‐Lang, K. 1938. Peptide bonds in globular proteins. Nature 142:996.
   Matouschek, A., Kellis, J.T., Jr., Serrano, L., and Fersht, A.R. 1989. Mapping the transition state and pathway of protein folding by protein engineering. Nature 340:122‐126.
   Myers, J.K., Pace, C.N., and Scholtz, J.M. 1995. Denaturant m values and heat capacity changes: Relation to changes in accessible surface areas of protein unfolding. Protein Sci. 4:2138‐2148.
   Pace, C.N. 1986. Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 131:266‐280.
   Park, C. and Marqusee, S. 2004. Probing the high energy states in proteins by proteolysis. J. Mol. Biol. 343:1467‐1476.
   Park, C., and Marqusee, S. 2005. Pulse proteolysis: A facile method for quantitative determination of protein stability and ligand binding. Nature Methods 2:207‐212.
   Tyndall, J.D., Nall, T., and Fairlie, D.P. 2005. Proteases universally recognize beta strands in their active sites. Chem. Rev. 105:973‐999.
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