Determining the Conformational Stability of a Protein from Urea and Thermal Unfolding Curves

Gerald R. Grimsley1, Saul R. Trevino2, Richard L. Thurlkill3, J. Martin Scholtz4

1 Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas, 2 College of Science and Mathematics, Houston Baptist University, Houston, Texas, 3 Chemistry and Natural Sciences, University of Louisiana Monroe, Monroe, Louisiana, 4 Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 28.4
DOI:  10.1002/0471140864.ps2804s71
Online Posting Date:  February, 2013
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Abstract

This unit contains basic protocols for determining the conformational stability of a globular protein from either urea or thermal unfolding curves. Circular dichroism is the optical spectroscopic technique most commonly used to monitor protein unfolding. The protocols describe how to analyze data from an unfolding curve to obtain the thermodynamic parameters necessary to calculate conformational stability, and how to determine differences in stability between protein variants. Curr. Protoc. Protein Sci. 71:28.4.1‐28.4.14. © 2013 by John Wiley & Sons, Inc.

Keywords: conformational stability; urea denaturation; thermal denaturation; m‐value; protein folding

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Determining Protein Conformational Stability from Urea‐Induced Unfolding Curves
  • Support Protocol 1: Preparation of Urea Stock Solution
  • Support Protocol 2: Analyzing Urea Unfolding Curves
  • Basic Protocol 2: Determining the Conformational Stability of a Protein from Thermal Unfolding Curves
  • Support Protocol 3: Analyzing Thermal Unfolding Curves
  • Basic Protocol 3: Determining Differences in Conformational Stability for Protein Variants
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Determining Protein Conformational Stability from Urea‐Induced Unfolding Curves

  Materials
  • Protein of interest
  • 30 mM diglycine buffer, pH 8.0, with 0.1 M NaCl (see protocol 2)
  • ∼10 M urea stock solution (see protocol 2)
  • Clean dry test tubes (e.g., disposable 13 × 100−mm culture tubes)
  • CD spectrometer
  • Rectangular quartz cells, 0.4 × 1−cm path length (Hellma cells, http://www.hellma‐analytics.com)

Support Protocol 1: Preparation of Urea Stock Solution

  Materials
  • Solid urea (proteomics grade, AMRESCO; solubility 10.49 M at 25°C)
  • Diglycine (Gly‐Gly)
  • NaCl
  • 1 M HCl
  • 1 M NaOH
  • Refractometer (American Optical AO Abbe model 10450, desirable but not required)

Support Protocol 2: Analyzing Urea Unfolding Curves

  Materials
  • ∼0.1 mg/ml protein solution
  • Blank solution (containing all components of the protein solution except the protein)
  • Buffer
  • CD spectrometer
  • Rectangular quartz cell, 0.4 × 1−cm pathlength (Hellma cells, www.hellma‐analytics.com)
  • Small magnetic stir bar that can fit inside the quartz cell
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Figures

Videos

Literature Cited

   Becktel, W.J. and Schellman, J.A. 1987. Protein stability curves. Biopolymers 26:1859‐1877.
   Brandts, J.F. 1964. The thermodynamics of protein denaturation. I. The denaturation of chymotrypsinogen. J. Am. Chem. Soc. 86:4291‐4301.
   Dill, K.A. 1990. Dominant forces in protein folding. Biochemistry 29:7133‐7155.
   Fersht, A. 1999. Structure and Mechanism in Protein Science. W.H. Freeman and Company, New York.
   Greene, R.F. Jr. and Pace, C.N. 1974. Urea and guanidine hydrochloride denaturation of ribonuclease, lysozyme, α‐chymotrypsin, and β‐lactoglobulin. J. Biol. Chem. 249:5388‐5393.
   Grimsley, G.R., Huyghues‐Despointes, B.M.P., Pace, C.N., and Scholtz, J.M. 2004. Measuring the conformational stability of a protein. In Purifying Proteins for Proteomics (R.J. Simpson, ed.) pp. 535‐566. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
   Kawahara, K. and Tanford, C. 1966. Viscosity and density of aqueous solutions of urea and guanidine hydrochloride. J. Biol. Chem. 241:3228‐3232.
   Makhatadze, G.I. 1999. Thermodynamics of protein interactions with urea and guanidinium hydrochloride. J. Phys. Chem. B 103:4781‐4785.
   Makhatadze, G.I. and Privalov, P.L. 1995. Energetics of protein structure. Adv. Protein Chem. 47:307‐425.
   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.
   Nicholson, E.M. and Scholtz, J.M. 1996. Conformational stability of the Escherichia coli HPr protein: Test of the linear extrapolation method and a thermodynamic characterization of cold denaturation. Biochemistry 35:11369‐11378.
   Pace, C.N. and Grimsley, G.R. 1988. Ribonuclease T1 is stabilized by cation and anion binding. Biochemistry 27:3242‐3246.
   Pace, C.N. and Laurents, D.V. 1989. A new method for determining the heat capacity change for protein folding. Biochemistry 28:2520‐2525.
   Pace, C.N., Shirley, B.A., and Thomson, J.A. 1989. Measuring the conformational stability of a protein. In Protein Structure: A Practical Approach (T. Creighton, ed.) pp. 311‐329. IRL Press, Oxford.
   Pace, C.N., Hebert, E.J., Shaw, K.L., Schell, D., Both, V., Krajcikova, D., Sevcik, J., Wilson, K.S., Dauter, Z., Hartley, R.W., and Grimsley, G.R. 1998. Conformational stability and thermodynamics of folding of ribonucleases Sa, Sa2 and Sa3. J. Mol. Biol. 279:271‐286.
   Pace, C.N., Grimsley, G.R., Thomas, S.T., and Makhatadze, G.I. 1999. Heat capacity change for ribonuclease A folding. Protein Sci. 8:1500‐1504.
   Pace, C.N., Grimsley, G.R., and Scholtz, J.M. 2005. Denaturation of proteins by urea and guanidine hydrochloride. In Protein Folding Handbook, Vol. 1 (J. Buchner and T. Kiefhaber, eds.), pp. 45‐69. Wiley‐VCH, Hamburg.
   Pace, C.N., Grimsley, G.R., and Scholtz, J.M. 2009. Protein Stability. In eLS. John Wiley & Sons, Chichester, U.K. http://www.els.net/ (doi:10.1002/9780470015902.a0020887).
   Pace, C.N., Huyghues‐Despointes, B.M., Fu, H., Takano, K., Scholtz, J.M., and Grimsley, G.R. 2010. Urea denatured state ensembles contain extensive secondary structure that is increased in hydrophobic proteins. Protein Sci. 19:929‐943.
   Pace, C.N., Fu, H., Fryar, K.L., Landua, J., Trevino, S.R., Shirley, B.A., Hendricks, M.M., Iimura, S., Gajiwala, K., Scholtz, J.M., and Grimsley, G.R. 2011. Contribution of hydrophobic interactions to protein stability. J. Mol. Biol. 408:514‐528.
   Santoro, M.M. and Bolen, D.W. 1992. A test of linear extrapolation of unfolding free energy changes over an extended denaturant concentration range. Biochemistry 31:4901‐4907.
   Schellman, J.A. and Gassner, N.C. 1996. The enthalpy of transfer of unfolded proteins into solutions of urea and guanidinium chloride. Biophys. Chem. 59:259‐275.
   Scholtz, J.M. 1995. Conformational stability of HPr: The histidine‐containing phosphocarrier protein from Bacillus subtilis. Protein Sci. 4:35‐43.
   Scholtz, J.M., Grimsley, G.R., and Pace, C.N. 2009. Solvent denaturation of proteins and interpretations of the m value. Methods Enzymol. 466:549‐565.
   Swint, L. and Robertson, A.D. 1993. Thermodynamics of unfolding for turkey ovomucoid third domain: Thermal and chemical denaturation. Protein Sci. 2:2037‐2049.
   Warren, J.R. and Gordon, J.A. 1966. On the refractive indices of aqueous solutions of urea. J. Phys. Chem. 70:297‐300.
   Yang, M., Ferreon, A.C., and Bolen, D.W. 2000. Structural thermodynamics of a random coil protein in guanidine hydrochloride. Proteins Suppl. 4:44‐49.
Internet Resources
   http://gibk26.bio.kyutech.ac.jp/jouhou/Protherm/protherm.html
  ProTherm is an online database reporting unfolding thermodynamic parameters for numerous proteins and variants. It also references primary literature with additional detail for these types of protocols.
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