Skip to main content Accessibility help
×
×
Home

Thermal protein unfolding by differential scanning calorimetry and circular dichroism spectroscopy Two-state model versus sequential unfolding

  • Joachim Seelig (a1) and Hans-Joachim Schönfeld (a2)
Abstract

Thermally-induced protein unfolding is commonly described with the two-state model. This model assumes only two types of protein molecules in solution, the native (N) and the denatured, unfolded (U) protein. In reality, protein unfolding is a multistep process, even if intermediate states are only sparsely populated. As an alternative approach we explore the Zimm–Bragg theory, originally developed for the α-helix-to-random coil transition of synthetic polypeptides. The theory includes intermediate structures with concentrations determined by the cooperativity of the unfolding reaction. We illustrate the differences between the two-state model and the Zimm–Bragg theory with measurements of apolipoprotein A-1 and lysozyme by differential scanning calorimetry (DSC) and CD spectroscopy. Nine further protein examples are taken from the literature. The Zimm–Bragg theory provides a perfect fit of the calorimetric unfolding transitions for all proteins investigated. In contrast, the transition curves and enthalpies predicted by the two-state model differ considerably from the experimental results. Apolipoprotein A-1 is ~50% α-helical at ambient temperature and its unfolding follows the classical α-helix-to-random coil equilibrium. The unfolding of proteins with little α-helix content, such as lysozyme, can also be analyzed with the Zimm–Bragg theory by introducing the concept of ‘folded’ and ‘unfolded’ peptide units assuming an average unfolding enthalpy per peptide unit. DSC is the method of choice to measure the unfolding enthalpy, $\Delta H_{\rm exp} ^0 $ , but CD spectroscopy in combination with the two-state model is often used to deduce the unfolding enthalpy. This can lead to erroneous result. Not only are different enthalpies required to describe the CD and DSC transition curves but these values deviate distinctly from the experimental result. In contrast, the Zimm–Bragg theory predicts the DSC and CD unfolding transitions with the same set of parameters.

  • View HTML
    • Send article to Kindle

      To send this article to your Kindle, first ensure no-reply@cambridge.org is added to your Approved Personal Document E-mail List under your Personal Document Settings on the Manage Your Content and Devices page of your Amazon account. Then enter the ‘name’ part of your Kindle email address below. Find out more about sending to your Kindle. Find out more about sending to your Kindle.

      Note you can select to send to either the @free.kindle.com or @kindle.com variations. ‘@free.kindle.com’ emails are free but can only be sent to your device when it is connected to wi-fi. ‘@kindle.com’ emails can be delivered even when you are not connected to wi-fi, but note that service fees apply.

      Find out more about the Kindle Personal Document Service.

      Thermal protein unfolding by differential scanning calorimetry and circular dichroism spectroscopy Two-state model versus sequential unfolding
      Available formats
      ×
      Send article to Dropbox

      To send this article to your Dropbox account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Dropbox.

      Thermal protein unfolding by differential scanning calorimetry and circular dichroism spectroscopy Two-state model versus sequential unfolding
      Available formats
      ×
      Send article to Google Drive

      To send this article to your Google Drive account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Google Drive.

      Thermal protein unfolding by differential scanning calorimetry and circular dichroism spectroscopy Two-state model versus sequential unfolding
      Available formats
      ×
Copyright
This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited.
Corresponding author
*Author for correspondence: Joachim Seelig, Division of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland. Tel.: +41-61-267 2190; Fax: +41-61-267 2189; Email: joachim.seelig@unibas.ch
References
Hide All
Ahmad, F. & Bigelow, C. C. (1982). Estimation of the free energy of stabilization of ribonuclease A, lysozyme, alpha-lactalbumin, and myoglobin. The Journal of Biological Chemistry 257, 1293512938.
Arnulphi, C., Jin, L. H., Tricerri, M. A. & Jonas, A. (2004). Enthalpy-driven apolipoprotein A-I and lipid bilayer interaction indicating protein penetration upon lipid binding. Biochemistry 43, 1225812264.
Bolen, D. W. & Yang, M. (2000). Effects of guanidine hydrochloride on the proton inventory of proteins: implications on interpretations of protein stability. Biochemistry 39, 1520815216.
Carra, J. H., Murphy, E. C. & Privalov, P. L. (1996). Thermodynamic effects of mutations on the denaturation of T4 lysozyme. Biophysical Journal 71, 19942001.
Chou, P. Y. & Scheraga, H. A. (1971). Calorimetric measurement of enthalpy change in isothermal helix-coil transition of poly-L-lysine in aqueous solution. Biopolymers 10, 657680.
Davidson, N. (1962). In Statistical Mechanics, p. 385. New York: Mac Graw-Hill.
Delbaere, L. T., Vandonselaar, M., Prasad, L., Quail, J. W., Wilson, K. S. & Dauter, Z. (1993). Structures of the lectin IV of Griffonia simplicifolia and its complex with the Lewis b human blood group determinant at 2·0 A resolution. Journal of Molecular Biology 230, 950965.
Doig, A. J. (2002). Recent advances in helix-coil theory. Biophysical Chemistry 101102, 281293.
Fernandez-Vidall, M., Jayasinghe, S., Ladokhin, A. S. & White, S. H. (2007). Folding amphipathic helices into membranes: amphiphilicity trumps hydrophobicity. Journal of Molecular Biology 370, 459470.
Freire, E. & Murphy, K. P. (1991). Molecular-basis of cooperativity in protein folding. Journal of Molecular Biology 222, 687698.
Garcia-Hernandez, E., Hernandez-Arana, A., Zubillaga, R. A. & Rojo-Dominguez, A. (1998). Spectroscopic and thermodynamic evidence for a complex denaturation mechanism of bovine beta-lactoglobulin A. Biochemistry and Molecular Biology International 45, 761768.
Gursky, O. (2015). Structural stability and functional remodeling of high-density lipoproteins. Febs Letters 589, 26272639.
Gursky, O. & Atkinson, D. (1996). Thermal unfolding of human high-density apolipoprotein A-1: implications for a lipid-free molten globular state. Proceedings of the National Academy of Sciences of the United States of America 93, 29912995.
Ibarra-Molero, B., Loladze, V. V., Makhatadze, G. I. & Sanchez-Ruiz, J. M. (1999a). Thermal versus guanidine-induced unfolding of ubiquitin. An analysis in terms of the contributions from charge-charge interactions to protein stability. Biochemistry 38, 81388149.
Ibarra-Molero, B., Makhatadze, G. I. & Sanchez-Ruiz, J. M. (1999b). Cold denaturation of ubiquitin. Biochimica et biophysica acta 1429, 384390.
Jia, Z., Quail, J. W., Waygood, E. B. & Delbaere, L. T. (1993). The 2·0-A resolution structure of Escherichia coli histidine-containing phosphocarrier protein HPr. A redetermination. The Journal of Biological Chemistry 268, 2249022501.
Kiefhaber, T. (1995). Kinetic traps in lysozyme folding. Proceedings of the National Academy of Sciences of the United States of America 92, 90299033.
Konermann, L. (2012). Protein Unfolding and Denaturants. Chichester: eLSJohn Wiley & Sons, Ltd, pp. 17.
Kurapkat, G., Kruger, P., Wollmer, A., Fleischhauer, J., Kramer, B., Zobel, E., Koslowski, A., Botterweck, H. & Woody, R. W. (1997). Calculations of the CD spectrum of bovine pancreatic ribonuclease. Biopolymers 41, 267287.
Laurents, D. V. & Baldwin, R. L. (1997). Characterization of the unfolding pathway of hen egg white lysozyme. Biochemistry 36, 14961504.
Li, Y., Han, X. & Tamm, L. K. (2003). Thermodynamics of fusion peptide-membrane interactions. Biochemistry 42, 72457251.
Luo, P. & Baldwin, R. L. (1997). Mechanism of helix induction by trifluoroethanol: a framework for extrapolating the helix-forming properties of peptides from trifluoroethanol/water mixtures back to water. Biochemistry 36, 84138421.
Makhatadze, G. I. & Privalov, P. L. (1992). Protein interactions with urea and guanidinium chloride. A calorimetric study. Journal of Molecular Biology 226, 491505.
Marqusee, S., Robbins, V. H. & Baldwin, R. L. (1989). Unusually stable helix formation in short alanine-based peptides. Proceedings of the National Academy of Sciences of the United States of America 86, 52865290.
Mei, X. & Atkinson, D. (2011). Crystal structure of C-terminal truncated apolipoprotein A-I reveals the assembly of high density lipoprotein (HDL) by dimerization. The Journal of Biological Chemistry 286, 3857038582.
Meier, M. & Seelig, J. (2007). Thermodynamics of the coil <==> beta-sheet transition in a membrane environment. Journal of Molecular Biology 369, 277289.
Meier, M. & Seelig, J. (2008). Length dependence of the coil <--> beta-sheet transition in a membrane environment. Journal of the American Chemical Society 130, 10171024.
Miranker, A., Radford, S. E., Karplus, M. & Dobson, C. M. (1991). Demonstration by NMR of folding domains in lysozyme. Nature 349, 633636.
Morriset, J. D., David, J. S. K., Pownall, H. J. & Gotto, A. M. (1973). Interaction of an apolipoprotein (Apolp-Alanine) with phosphatidylcholine. Biochemistry 12, 12901299.
Myers, J. K., Pace, C. N. & Scholtz, J. M. (1995). Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding. Protein Science: a Publication of the Protein Society 4, 21382148.
Nicholson, E. M. & 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, 1136911378.
Privalov, G., Kavina, V., Freire, E. & Privalov, P. L. (1995). Precise scanning calorimeter for studying thermal-properties of biological macromolecules in dilute-solution. Analytical Biochemistry 232, 7985.
Privalov, P. L. (1997). Thermodynamics of protein folding. Journal of Chemical Thermodynamics 29, 447474.
Privalov, P. L. & Dragan, A. I. (2007). Microcalorimetry of biological macromolecules. Biophysical Chemistry 126, 1624.
Privalov, P. L. & Gill, S. J. (1988). Stability of protein-structure and hydrophobic interaction. Advances in Protein Chemistry 39, 191234.
Privalov, P. L., Griko, Y. V., Venyaminov, S. Y. & Kutyshenko, V. P. (1986). Cold denaturation of myoglobin. Journal of Molecular Biology 190, 487498.
Privalov, P. L. & Makhatadze, G. I. (1990). Heat-capacity of proteins.2. Partial molar heat-capacity of the unfolded polypeptide-chain of proteins – protein unfolding effects. Journal of Molecular Biology 213, 385391.
Privalov, P. L. & Makhatadze, G. I. (1993). Contribution of hydration to protein folding thermodynamics. II. The entropy and Gibbs energy of hydration. Journal of Molecular Biology 232, 660679.
Privalov, P. L., Tiktopulo, E. I., Venyaminov, S. Y., Griko, Y. V., Makhatadze, G. I. & Khechinashvili, N. N. (1989). Heat-capacity and conformation of proteins in the denatured state. Journal of Molecular Biology 205, 737750.
Radford, S. E., Dobson, C. M. & Evans, P. A. (1992). The folding of hen lysozyme involves partially structured intermediates and multiple pathways. Nature 358, 302307.
Reed, J. & Reed, T. A. (1997). A set of constructed type spectra for the practical estimation of peptide secondary structure from circular dichroism. Analytical Biochemistry 254, 3640.
Rialdi, G. & Hermans, J. (1966). Calorimetric heat of helix-coil transition of poly-L-glutamic acid. Journal of the American Chemical Society 88, 57195720.
Rosgen, J. & Hinz, H. J. (2000). Response functions of proteins. Biophysical Chemistry 83, 6171.
Saito, H., Dhanasekaran, P., Nguyen, D., Deridder, E., Holvoet, P., Lund-Katz, S. & Phillips, M. C. (2004). Alpha-Helix formation is required for high affinity binding of human apolipoprotein A-I to lipids. Journal of Biological Chemistry 279, 2097420981.
Saito, H., Dhanasekaran, P., Nguyen, D., Holvoet, P., Lund-Katz, S. & Phillips, M. C. (2003a). Domain structure and lipid interaction in human apolipoproteins A-I and E, a general model. Journal of Biological Chemistry 278, 2322723232.
Saito, H., Lund-Katz, S. & Phillips, M. C. (2003b). Domain structure and lipid interaction in human apolipoprotein A-I. Atherosclerosis Supplements 4, 221–221.
Santoro, M. M. & Bolen, D. W. (1988). Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants. Biochemistry 27, 80638068.
Scholtz, J. M. (1991). Correction. Proceedings of the National Academy of Sciences of the United States of America 88, 6898–6898.
Scholtz, J. M., Marqusee, S., Baldwin, R. L., York, E. J., Stewart, J. M., Santoro, M. & Bolen, D. W. (1991a). Calorimetric determination of the enthalpy change for the alpha-helix to coil transition of an alanine peptide in water. Proceedings of the National Academy of Sciences of the United States of America 88, 28542858.
Scholtz, J. M., Qian, H., York, E. J., Stewart, J. M. & Baldwin, R. L. (1991b). Parameters of helix-coil transition theory for alanine-based peptides of varying chain lengths in water. Biopolymers 31, 14631470.
Schulthess, T., Schönfeld, H. J. & Seelig, J. (2015). Thermal unfolding of apolipoprotein A-1. Evaluation of methods and models. Biochemistry 54, 30633075.
Schwarz, F. P. (1990). Biological thermodynamic data for the calibration of differential scanning calorimeters – heat-capacity data on the unfolding transition of beta-lactoglobulin in solution. Thermochimica Acta 159, 305325.
Seeley, S. K., Wittrock, G. K., Thompson, L. K. & Weis, R. M. (1996). Oligomers of the cytoplasmic fragment from the Escherichia coli aspartate receptor dissociate through an unfolded transition state. Biochemistry 35, 1633616345.
Suurkuusk, M. & Hallen, D. (1999). Denaturation of apolipoprotein A-I and the monomer form of apolipoprotein A-I-Milano. European Journal of Biochemistry 265, 346352.
Tall, A. R., Shipley, G. G. & Small, D. M. (1976). Conformational and thermodynamic properties of apo A-1 of human plasma high density lipoproteins. Journal of Biological Chemistry 251, 37493755.
Tall, A. R., Small, D. M., Shipley, G. G. & Lees, R. S. (1975). Apoprotein stability and lipid-protein interactions in human plasma high density lipoproteins. Proceedings of the National Academy of Sciences of the United States of America 72, 49404942.
Tanaka, M., Koyama, M., Dhanasekaran, P., Nguyen, D., Nickel, M., Lund-Katz, S., Saito, H. & Phillips, M. C. (2008). Influence of tertiary structure domain properties on the functionality of apolipoprotein A-I. Biochemistry 47, 21722180.
Wieprecht, T., Apostolov, O., Beyermann, M. & Seelig, J. (1999). Thermodynamics of the alpha-helix-coil transition of amphipathic peptides in a membrane environment: implications for the peptide-membrane binding equilibrium. Journal of Molecular Biology 294, 785794.
Wieprecht, T., Apostolov, O. & Seelig, J. (2000). Binding of the antibacterial peptide magainin 2 amide to small and large unilamellar vesicles. Biophysical Chemistry 85, 187198.
Wu, J. R., Long, D. G. & Weis, R. M. (1995). Reversible dissociation and unfolding of the Escherichia-coli aspartate receptor cytoplasmic fragment. Biochemistry 34, 30563065.
Yang, A. S. & Honig, B. (1995a). Free-energy determinants of secondary structure formation .2. Antiparallel beta-sheets. Journal of Molecular Biology 252, 366376.
Yang, A. S. & Honig, B. (1995b). Free energy determinants of secondary structure formation: I. alpha-Helices. Journal of Molecular Biology 252, 351365.
Zehender, F., Ziegler, A., Schönfeld, H. J. & Seelig, J. (2012). Thermodynamics of protein self-association and unfolding. The case of apolipoprotein A-I. Biochemistry 51, 12691280.
Zhou, Y., Hall, C. K. & Karplus, M. (1999). The calorimetric criterion for a two-state process revisited. Protein Science: a Publication of the Protein Society 8, 10641074.
Zimm, B. H. & Bragg, J. K. (1959). Theory of the phase transition between helix and random coil in polypeptide chains. Journal of Chemical Physics 31, 526535.
Recommend this journal

Email your librarian or administrator to recommend adding this journal to your organisation's collection.

Quarterly Reviews of Biophysics
  • ISSN: 0033-5835
  • EISSN: 1469-8994
  • URL: /core/journals/quarterly-reviews-of-biophysics
Please enter your name
Please enter a valid email address
Who would you like to send this to? *
×
Type Description Title
PDF
Supplementary materials

Seelig and Schönfeld supplementary material
Seelig and Schönfeld supplementary material 1

 PDF (382 KB)
382 KB

Metrics

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed