Abstract
The practical application of scanning calorimetry and spectroscopic methods to measure the stability of multimeric proteins is described. Oligomeric proteins are stabilized by both the intrinsic folding energy of the subunits as well as interactions between the subunits. Oligomerization results in a concentration dependence for multimer stability, which increases logarithmically with increasing concentration. Since the increase in stability does not plateau at high protein concentrations, the effect of concentration must be described quantitatively. Straightforward mathematical methods are provided for deriving the appropriate models for multimer unfolding, and methods are presented for analyzing equilibrium unfolding data and stability using the models.
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Notes
- 1.
This work was supported by the National Institutes of Health (GM 49686).
References
Makhatadze, G., Privalov, P. L. (1995) Energetics of protein structure. Adv Protein Chem 47, 308–425.
Lumry, R., Biltonen, R., Brandts, J. (1966) Validity of the “two-state” hypothesis for conformational transitions of proteins. Biopolymers 4, 917–944.
Privalov, P., Khechinashvili, N. (1974) A thermodynamic approach to the problem of stabilization of globular protein structure: a calorimetric study. J Mol Biol 86, 665–684.
Pace, C. N. (1975) The stability of globular proteins. CRC Critical Rev Biochem. 3, 1–43.
Freire, E., Biltonen, R. (1978) Statistical mechanical deconvolution of thermal transitions in macromolecules. I. Theory and application to homogeneous systems. Biopolymers 17, 463–479.
Privalov, P. (1979) Stability of proteins. Small globular proteins. Adv Protein Chem 33, 167–241.
Sturtevant, J. (1987) Biochemical applications of differential scanning calorimetry. Ann Rev Phys Chem 38, 463–488.
Lewis, G. N., Randall, M., Pitzer, K. S., Brewer, L. (1961) Thermodynamics, McGraw-Hill, New York.
Eisenberg, D., Crothers, D. (1979) Physical Chemistry with Applications to the Life Sciences, Benjamin/Cummings, Menlo Park, CA.
Becktel, W., Schellman, J. (1987) Protein stability curves. Biopolymers 26, 1859–1877.
Privalov, P., Griko, Y., Venyaminov, S., et al. (1986) Cold denaturation of myoglobin. J Mol Biol 190, 487–498.
Wyman, J., Gill, S. J. (1990) Binding and Linkage: Functional Chemistry of Biological Macromolecules, University Science Books, Mill Valley, CA.
Biltonen, R. L., Freire, E. (1978) Thermodynamic characterization of conformational states of biological macromolecules using differential scanning calorimetry. CRC Crit Rev Biochem 5, 85–124.
Steif, C., Weber, P., Hinz, H.-J., et al. (1993) Subunit interactions provide a significant contribution to the stability of the dimeric four-α-helical bundle protein ROP. Biochemistry 32, 3867–3876.
Marky, L. A., Breslauer, K. J. (1987) Calculating thermodynamic data for transitions of any molecularity from equilibrium melting curves. Biopolymers 26, 1601–1620.
Hermans, J., Jr. (1965) Methods for the study of reversible denaturation of proteins and interpretation of data. Methods Biochem Anal 13, 81–111.
Privalov, P. L., Makhatadze, G. I. (1990) Heat capacity of proteins. II. Partial molar heat capacity of the unfolded polypeptide chain of proteins: protein unfolding effects. J Mol Biol 213, 385–391.
McCrary, B. S., Bedell, J., Edmondson, S. P., et al. (1998) Linkage of protonation and anion binding to the folding of Sac7d. J Mol Biol 276, 203–224.
Scholtz, J. M. (1995) Conformational stability of HPr: The histidine-containing phosphocarrier protein from Bacilus subtilis. Protein Sci. 4, 35–43.
Lumry, R., Eyring, H. (1954) Conformation Changes of Proteins. J. Phys. Chem. 58, 110.
Ahern, T., Klibanov, A. (1985) The mechanism of irreversible enzyme inactivation at 100°C, Science.
Bevington, P. R., Robinson, D. K. (1992) Data Reduction and Error Analysis for the Physical Sciences, McGraw-Hill, New York, N.Y.
Johnson, M., Faunt, L. (1992) Parameter estimation by least squares methods. Methods Enzymol 210, 1–37.
Press, W., Flannery, B., Teukolsky, S., et al. (1989) Numerical Recipes: The Art of Scientific Computing (Fortran Version), Cambridge University Press, Cambridge.
Mach, H., Volkin, D. B., Burke, C. J., et al. (1995) in (Shirley, B. A., ed.), Protein Stability and Folding: Theory and Practice, Humana Press, Inc., Totowa, N.J.
Royer, C. A. (1995) in (Shirley, B. A., ed.), Protein Stability and Folding: Theory and Practice, Humana Press, Inc., Totowa, N.J.
Kuwajima, K. (1995) in (Shirley, B. A., ed.), Protein Stability and Folding: Theory and Practice, Humana Press, Inc., Totowa, N.J.
Lopez, M. M., Chin, D. H., Baldwin, R. L., et al. (2002) The enthalpy of the alanine peptide helix measured by isothermal titration calorimetry using metal-binding to induce helix formation, Proc Natl Acad Sci USA 99, 1298–1302.
Makhatadze, G. (1998) in Current Protocols in Protein Science, John Wiley & Sons, New York.
Gordon, A., Ford, R. (1972) The Chemist's Companion: A Handbook of Practical Data, Techniques, and References, John Wiley, New York.
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Shriver, J.W., Edmondson, S.P. (2009). Defining the Stability of Multimeric Proteins. 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_3
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