Functional Application Areas

Protein Folding & Stability

Protein structures are stabilized by non-covalent intramolecular interactions between amino acid side chains. Protein complexes are also formed by specific non-covalent intermolecular interactions. All biological processes depend on proteins being stable and in the appropriate folded conformation. It is important to know how proteins fold into their biologically active states, and how these active states are stabilized. A primary goal of protein engineering, rational drug design and biopharmaceutical production is the development, production, and storage of stable proteins with full functionality. 

There have been rapid advances in structural biology and relating structure to biochemical function and mechanism. However, knowledge of protein structure alone does not ensure accurate prediction of stability, function and biological activity. The complete characterization of any protein requires stability determination and the forces which lead to stability and correct folding.

Differential Scanning Calorimetry (DSC) is a powerful analytical tool which directly measures the stability and unfolding of a protein.  In DSC, the protein is heated at a constant rate, and there is a detectable heat change associated with thermal denaturation

A single DSC experiment can determine:

  • Transition midpoint (Tm)
  • Enthalpy (ΔH) and heat capacity change (ΔCp) associated with unfolding
  • Presence of multiple unfolding domains

A protein in aqueous solution is in equilibrium between the native (folded) conformation and its denatured (unfolded) conformation.  The stability of the native state is based on the magnitude of the Gibbs free energy (ΔG) of the system and the thermodynamic relationships between enthalpy (ΔH) and entropy (ΔS) changes.  A positive ΔG indicates the native state is more stable than the denatured state – the more positive the ΔG, the greater the stability.  For a protein to unfold, stabilizing forces need to be broken.  Conformational entropy overcomes stabilizing forces allowing the protein to unfold at temperatures where entropy becomes dominant.

The transition midpoint Tm is the temperature where 50% of the protein is in its native confirmation, and the other 50% is denatured. In general, the higher the Tm, the more stable the protein.  Proteins which are more stable are less susceptible to unfolding and precipitation.

DSC measures ΔH of unfolding due to heat denaturation.  Protein unfolding is typically endothermic. During the same experiment, DSC also measures the change in heat capacity (ΔCp) for denaturation.  Heat capacity changes associated with protein unfolding are primarily due to changes in hydration of side chains that were buried in the native state, but become solvent exposed in the denatured state.

Many factors are responsible for the folding and stability of native proteins, including hydrophobic interactions, hydrogen bonding, conformational entropy, and the physical environment (pH, buffer, ionic strength, excipients, etc.).

DSC data, either used alone or in conjunction with sequence, stability and structural data, can provide information on:

  • Effects of protein mutagenesis and engineering
  • Effects of buffer, pH, salt, additives
  • Effects of post-translational modification
  • Reversibility of unfolding, by changes in ΔH
  • Presence of multiple unfolding domains
  • Stability contributions of individual domains
  • Formation of protein complexes
  • Effects of lipid, nucleic acid, or other biopolymer on protein stability

Protein stability characterization is a critical element of Biopharmaceutical and Vaccines Development.  Using Tm data from DSC, one can design and select the most stable engineered protein/variant, optimize process development, and screen for the most stable liquid formulations.

References

Microcalorimetry: a response to challenges in modern biotechnology.
Krell, T
Microbial Biotechnol 1, 126-136 (2008)

Differential scanning calorimetry in life science: thermodynamics, stability, molecular recognition and application in drug design.
Bruylants, G., Wouters, J., and Michaux, C.
Curr Med Chem 12, 2011-2020 (2005)

Advances in membrane receptor screening and analysis.
Cooper, M. A.
J Mol Recognit 17, 286-315 (2004)

The thermodynamic linkage between protein structure, stability, and function.
Freire, E.
Methods Mol Biol 168, 37-68 (2001)

Isothermal titration calorimetry and differential scanning calorimetry as complementary tools to investigate the energetics of biomolecular recognition.
Jelesarov, I. and Bosshard, H. R.  
J Mol Recognit 12, 3-18 (1999)

Physical methods for structure, dynamics and binding in immunological research.
Morikis, D. and Lambris, J. D.
Trends Immunol 25, 700-707 (2004)

Differential scanning calorimetry.
Spink, C. H.
Methods Cell Biol 84, 115-141 (2008)

Reference Lists

DSC – Protein Folding, Stability and Structural Studies Reference List

DSC – Protein Engineering and Mutagenesis Reference List

DSC – Liquid Protein Formulation Studies Reference List

DSC – Antibody Studies Reference List

DSC – Vaccine and Virus Studies Reference List

DSC – Protein-Small Molecule Interactions Reference List

DSC – Lipid-Protein Interactions Reference List

DSC – Protein-Nucleic Acid Interactions Reference List