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Microcalorimetry of Proteins and Their Complexes

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Protein Structure, Stability, and Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 490))

Abstract

Ultrasensitive microcalorimetric techniques for measuring the heat capacities of proteins in dilute solutions over a broad temperature range (DSC) and the heats of protein reactions at fixed temperatures (ITC) are described and the methods of working with these instruments are considered. Particular attention is paid to analyzing the thermal properties of individual proteins, their stability, the energetics of their folding, and their association with specific macromolecular partners. Use of these calorimetric methods is illustrated with examples of small compact globular proteins, small proteins having loose noncompact structure, multidomain proteins, and protein complexes, particularly with DNA.

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References

  1. Privalov, P. L., Monaselidze, D. R., Mrevlishvili, G. M. et al. (1965) Heat of intramolecular fusion of macromolecules. Soviet Physics JETP 20 1393–1396.

    Google Scholar 

  2. Spokane, R. B., Gill, S. J. (1981) Titration microcalorimeter using nanomolar quantities of reactants. Rev Sci Instrum 52, 1728–1733.

    Article  CAS  Google Scholar 

  3. Mckinnon, R., Fall, L., Parody-Morreale, A., et al. (1984) A twin titration microcalorimeter for the study of biochemical reactions. Anal Biochem 139, 134–139.

    Article  PubMed  CAS  Google Scholar 

  4. Wiseman, T., Willisten, S., Brandts, J., et al. (1989). Rapid measurement of binding constant and heats of binding using a new titration calorimeter. Anal Biochem 179, 131–137.

    Article  PubMed  CAS  Google Scholar 

  5. Bresslauer, K. J., Freire, E., Straume, M. (1992). Calorimetry: a tool for DNA and ligand–DNA studies. Methods Enzymol. 211, 533–567.

    Article  Google Scholar 

  6. Privalov P. L., Plotnikov V. V., Filimonov, V. V. (1975). Precision scanning microcalorimeter for the study of liquids. J Chem Thermodyn 7, 41–47.

    Article  CAS  Google Scholar 

  7. Privalov P. L. (1980). Scanning microcalorimeters for studying macromolecules. Pure & Appl. Chem. 52, 479–497.

    Article  CAS  Google Scholar 

  8. Privalov P. L., Potekhin S.A. (1986) Scanning microcalorimetry in studying temperature-induced changes in proteins. Methods in Enzymology, 131, 4–51.

    Article  PubMed  CAS  Google Scholar 

  9. Privalov P. L., Plotnikov V. V. (1989) Three generations of scanning microcalorimeters for liquids. Thermochim Acta 139, 257–277.

    Article  CAS  Google Scholar 

  10. Privalov, G. P., Kavina, V., Freire, E., et al. (1995) Precise scanning calorimeter for studying thermal properties of biological macromolecules in dilute solutions. Anal Biochem 232, 79–85.

    Article  PubMed  CAS  Google Scholar 

  11. Privalov G. P., Privalov, P. L. (2000) Problems and prospects in the microcalorimetry of biological macromolecules. Methods in Enzymology, 323, 31–62.

    Article  PubMed  CAS  Google Scholar 

  12. Privalov P. L. (1979) Stability of proteins. Small globular proteins. Adv. Protein Chem 33, 167–241

    Article  PubMed  CAS  Google Scholar 

  13. Privalov P. L., Khechinashvili N. N. (1974). A thermodynamic approach to the problem of stabilization of globular protein structure: a calorimetric study. J Mol Biol 86, 665–684.

    Article  PubMed  CAS  Google Scholar 

  14. Makhatadze G. I., Privalov P. L. (1990) Heat capacity of proteins. I. Partial molar heat capacity of individual amino acid residues in aqueous solutions: Hydration effect. J Mol Biol 213, 375–384

    Article  PubMed  CAS  Google Scholar 

  15. Makhatadze G. I., Privalov P. L. (1995). Energetics of protein structure. Adv. Protein Chem., 47, 307–425.

    Article  PubMed  CAS  Google Scholar 

  16. Hackel, M., Hinz, H. J., Hedwig, G. R. (1999). A new set of peptide-based group heat capacities for use in protein stability calculations. J Mol Biol 291, 197–213.

    Article  PubMed  CAS  Google Scholar 

  17. Crane-Robinson, C., Read, C. M., Cary, P. D., et al. (1998) The energetics of HMG box interactions with DNA. Thermodynamic description of the box from mouse Sox-5. J Mol Biol 281, 705–717.

    Article  PubMed  CAS  Google Scholar 

  18. Dragan, A. I., Privalov, P. L. (2002) Unfolding of a leucine zipper is not a simple two-state transition. J Mol Biol 321, 891–908.

    Article  PubMed  CAS  Google Scholar 

  19. Dragan, A. I., Klass, J., Read, C. M., et al. (2003) DNA binding of a non-sequence-specific HMG-D protein is entropy driven with a substantial non-electrostatic contribution. J Mol Biol 327, 393–411.

    Article  PubMed  CAS  Google Scholar 

  20. Dragan, A. I., Read, C. M., Makeyeva, E. N., et al. (2004). DNA binding and bending by sequence specific HMG boxes: energetic determinants of specificity. J Mol Biol 343, 371–309.

    Article  PubMed  CAS  Google Scholar 

  21. Makhatadze, G. I., Kim, K. S., Woodward, C., et al. (1993). Thermodynamics of BPTI folding. Protein Sci 2, 2028–2036.

    Article  PubMed  CAS  Google Scholar 

  22. Hutchens, J. O., Cole, A. G., Stout, J. W. (1969). Heat capacities from 11 to 305 degrees K and entropies of hydrated and anhydrous bovine zinc insulin and bovine chymotrypsinogen A. Entropy change for formation of peptide bonds. J Biol Chem 244, 26–32.

    PubMed  CAS  Google Scholar 

  23. Privalov, P. L., Griko, Yu.V., Venyaminov, S. Yu., et al. (1986) Cold denaturation of myoglobin. J Mol Biol 190, 487–498.

    Google Scholar 

  24. Privalov, P. L. (1990) Cold denaturation of proteins. CRC Crit Rev Biochem Mol Biol 25, 281–305.

    Article  CAS  Google Scholar 

  25. Griko, Yu. V., Privalov, P. L., Venyaminov, S. Yu., et al. (1988) Thermodynamic studies of apomyoglobin structure. Biofizika (USSR) 33, 18–26.

    Google Scholar 

  26. Griko, Yu. V., Privalov P. L., Sturtevant, J. M., et al. (1988) Cold denaturation of staphylococcal nuclease. Proc Natl Acad Sci USA 85, 3343–3347.

    Google Scholar 

  27. Privalov, P. L., Makhatadze, G. I. (1992) Contribution of hydration and non-covalent interactions to the heat capacity effect on protein unfolding. J Mol Biol 224, 715–723.

    Article  PubMed  CAS  Google Scholar 

  28. Makhatadze G. I., Privalov P. L. (1993). Contribution of hydration to protein folding thermodynamics. I. The enthalpy of hydration. J Mol Biol 232, 639–659.

    Article  PubMed  CAS  Google Scholar 

  29. Spolar, R. S., Livingstone, J. R., Record, M. T. Jr. (1992). Use of liquid hydrocarbon and amide transfer data to estimate contributions to thermodynamic functions of protein folding from the removal of nonpolar and polar surface from water. Biochemistry 31, 3947–3955.

    Article  PubMed  CAS  Google Scholar 

  30. Pfeil, W., Privalov, P. L. (1976). Thermodynamic investigations of proteins. I. Standard functions for proteins with lysozyme as an example. Biophys Chem 4, 23–32.

    Article  PubMed  CAS  Google Scholar 

  31. Pfeil, W., Privalov, P. L. (1976). Thermodynamic investigations of proteins. 3. Thermodynamic description of lysozyme. Biophys Chem 4, 41–50.

    Article  PubMed  CAS  Google Scholar 

  32. Freire, E. (1994). Statistical thermodynamic analysis of differential scanning calorimetry data: structural deconvolution of heat capacity function of proteins. Methods Enzymol 240, 502–530.

    Article  PubMed  CAS  Google Scholar 

  33. Privalov, P. L. (1982). Stability of proteins: Proteins which do not present a single cooperative system. Adv Prot Chem 35, 1–104.

    Article  CAS  Google Scholar 

  34. Novokhatny, V. V., Kudinov, S. A., Privalov, P. L. (1984). Domains in human plasminogen. J .Mol Biol 179, 215–232.

    Article  PubMed  CAS  Google Scholar 

  35. Tamura, A., Privalov, P. L. (1997). The entropy cost of protein association. J Mol Biol 273, 1046–1058.

    Article  Google Scholar 

  36. Dragan, A. I., Liggins, J. R., Crane-Robinson, C., et al. (2003) The energetics of specific binding of AT-hooks from HMGA1 to target DNA. J Mol Biol 327, 393–411.

    Article  PubMed  CAS  Google Scholar 

  37. Privalov, P. L., Jelesarov, I., Read, C. M., et al. (1999). The energetics of HMG box interactions with DNA. Thermodynamics of the DNA bonding of the HMG box from mouse Sox-5. J Mol Biol 294, 997–1013.

    Article  PubMed  CAS  Google Scholar 

  38. Dragan, A. I., Frank, L., Liu, Y., et al. (2004). Thermodynamic signature of GCN4-bZIP binding to DNA indicates the role of water in discriminating between the AP-1 and ATF/CREB sites. J Mol Biol 343, 865–878.

    Article  PubMed  CAS  Google Scholar 

  39. Griko, Y. V., Makhatadze, G. I., Privalov, P. L., et al. (1994). Thermodynamics of barnase unfolding. Protein Sci 3, 669–676.

    Article  PubMed  CAS  Google Scholar 

  40. Wintrode, P.L., Makhatadze, G.I., Privalov, P.L. (1994). Thermodynamics of ubiquitin unfolding. Proteins, Structure, Function and Genetics 18, 246–253.

    Article  CAS  Google Scholar 

  41. Yu, Y., Makhatadze, G. I., Pace, C. N., et al. (1994). Energetics of ribonuclease T1 structure. Biochemistry 33, 3312–3319.

    Article  PubMed  CAS  Google Scholar 

  42. Carra, J. H., Murphy, E. C., Privalov, P. L. (1996) Thermodynamic effects of mutations on the denaturation of T4 lysozyme. Biophy J 71, 1994–2001.

    Article  CAS  Google Scholar 

  43. Dragan, A. I., Li, Z., Makeyeva, E. N., et al. (2006) Forces driving the binding of homeodomains to DNA. Biochemistry 45, 141–151.

    Article  PubMed  CAS  Google Scholar 

  44. Liggins, J. R., Privalov, P. L. (2000). Energetics of the specific binding interaction of the first three zinc fingers of the transcription factor TFIIIA with its cognate DNA sequence. Proteins Suppl 4, 50–62.

    Google Scholar 

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Acknowledgments

The author would like to thank Dr. Colyn Crane-Robinson for a critical reading of the text and Dr. Anatoly Dragan for assistance in preparing illustrations. The financial support of NSF Grant MCB 0519381 is acknowledged.

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  1. Department of Biology, Johns Hopkins University, Baltimore, MD, USA

    Peter L. Privalov

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  1. Peter L. Privalov
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    © 2009 Humana Press, a part of Springer Science+Business Media, LLC

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    Privalov, P.L. (2009). Microcalorimetry of Proteins and Their Complexes. In: Shriver, J. (eds) Protein Structure, Stability, and Interactions. Methods in Molecular Biology, vol 490. Humana Press. https://doi.org/10.1007/978-1-59745-367-7_1

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    • DOI: https://doi.org/10.1007/978-1-59745-367-7_1

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    • Publisher Name: Humana Press

    • Print ISBN: 978-1-58829-954-3

    • Online ISBN: 978-1-59745-367-7

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