Biophysical application of calorimetric methods to protein misfolding and aggregation examinations

The last two decades have seen important advances in our knowledge on protein folding and the stability of globular proteins, as a result of extensive studies using various biophysical approaches and theories. Calorimetry, particularly differential scanning calorimetry (DSC), has proven to be very useful for examining the thermodynamics of protein (un)folding and stability. A large number of DSC studies have revealed the general thermodynamic nature of thermal unfolding, i.e. cold and heat denaturation, by producing a series of thermodynamic parameters: ?G, ?H, ?S, and ?Cp, as well as the midpoint of unfolding temperature (TM). The signs of these parameters for thermal unfolding (i.e., positive values of ?S and ?Cp as well as negative values of ?G and ?H) are the same regardless of the type of protein; however, the value of each parameter differs from protein to protein. Stability curves for various proteins with increasing temperature, which are constructed using thermodynamic parameters and the Gibbs-Helmholtz equation, also explain how protein stability depends on solvent conditions, pressure, and mutations. Thus, DSC has also been successfully applied to the development of antibody drugs.

Advances in our understanding of the thermodynamics of protein folding have been achieved using DSC; however, knowledge on thermodynamic properties and molecular mechanisms leading to protein misfolding and aggregation is very limited due to the technical difficulties associated with the large size and heterogeneity of aggregates. Furthermore, the ability of calorimetric methods to investigate thermodynamics has not yet been applied to protein aggregation.

We herein concisely describe recent and unique applications of calorimetry (both DSC and ITC) to the examination of protein misfolding and aggregation. Observations of spontaneous and seeded amyloid formation, amyloid fibril associations, amorphous aggregation, and dissociation of aggregates using calorimetry are described. We also discuss calorimetry-based thermodynamic studies on protein aggregation and address structural features to characterize protein aggregates using the thermodynamic parameters of aggregation. These pioneering attempts are expected to promote calorimetry-based thermodynamic studies on protein misfolding and aggregation and may provide the basis for the development of treatments for and the prevention of aggregation-related diseases.

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