Functional Application Areas

Application Note

Stability

Biological macromolecules like protein, lipids and nucleic acids are stabilized by non-covalent intramolecular interactions. Intermolecular non-covalent interactions stabilize any complexes formed between biomolecules. All biological processes depend on macromolecules 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 structure alone does not ensure accurate prediction of stability, function and biological activity. The complete characterization of any biopolymer requires stability determination and the forces which lead to stability.

Differential Scanning Calorimetry (DSC) is a powerful analytical tool which directly measures the change in heat associated with the unfolding of a protein, lipid, or nucleic acid.  In DSC, as the biomolecule is heated at a constant rate, a detectable heat change associated with thermal denaturation can be accurately measured. DSC experiments are:

• Label-free and use native materials
• True in-solution technique
• Easy to perform - require minimal assay development
• Conducted using a wide range of biological buffers, ionic strengths and pH’s
• Universal assay – measures the heat change associated with denaturation
• Non-optical – unaffected by colored or turbid samples
• Versatile - can be used with proteins, nucleic acids, lipids and other biomolecules
• Well documented – DSC has been cited in thousands of publications
• Widely used in academic, government and industrial laboratories worldwide

In a single DSC experiment, one can determine:

• Transition midpoint (T_m)
• Enthalpy (ΔH) and heat capacity change (∆Cp) associated with unfolding

A biomolecule 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 (∆G) of the system and the thermodynamic relationships between enthalpy (∆H) and entropy (∆S) changes.  A positive ∆G indicates the native state is more stable than the denatured state – the more positive the ∆G, 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.

DSC measures ∆H of unfolding due to heat denaturation.  The transition midpoint (T_m) is the temperature where 50% of the protein is in its native conformation and the other 50% is denatured. The higher the T_m, the more stable the molecule.  During the same experiment DSC also measures the change in heat capacity (∆Cp) 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 biopolymers, including hydrophobic interactions, hydrogen bonding, conformational entropy and the physical environment (pH, buffer, ionic strength, excipients, etc.)

DSC can be used to study the stability of a broad range of biomolecules, including protein, lipids and nucleic acids (see related links to the left).

Figure 1. Typical DSC thermogram. This DSC scan was carried out on a dilute protein solution. Protein unfolding is recognized as a sharp endothermic peak centered at a characteristic temperature called the transition midpoint (T_m). T_m, ∆H and ∆Cp of the transition are calculated by fitting the data to a two-state transition model using non-linear least squares regression analysis.

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