Recent progress in protein and protein-related systems using DSC techniques

Abstract

As one family of the most important biomoleclues, proteins have gained more and more attention since the day they were discovered and the trend is still growing. Even so, many problems are emerging in this filed and thus new techiniques and ideas are required for solving these problems. In this regard, differential scanning calorimetry (DSC) is a conventional but useful method to obtain the relationship between heat capacity and temperature of peoteins. In the past decade, new ideas have been applied to DSC and new methods have been developed. This is reflected in this minireview. First, important progresses have been made on protein unfloding or folding mechanisms. A new method called “interruption-incubation protocol” was developd to judge wheather a protein unfolds in a two-state mechanism or not. Compared to the conventional van’t Hoff analysis method, the new approach breaks the limitations of old method on the requirement of protein properties, namely the reversibility of protein folding and the equilibrium assumption during the unfoding process. Also, this method avoids the inaccuracies of enthalpy change for protein unfolding caused by improper baseline determination. Thus, the “interruption-incubation protocol” has a lower possibility of misjudgement and turns out to be an effective way for analysing “two-state or non-two state” proteins. In addition, by combining DSC with phase transition models instead of chemical equilibrium assumption, several theories were proposed which can provide a rough landscape of protein folding or unfolding process. Among these theories, the “variable barrier model” is an interesting one, which can directly give the barrier heights in protein folding as long as the barrier heights are smaller than ~4 RT . Second, a new method was proposed to derive protein stability curve directly by combining DSC with isothermal chemical denaturation (ICD) method. This method targets those “problematic” proteins, when the denaturation process is not perfect reversible. Three cases were discussed, namely the unreliability of protein Δ C p determination, the irreversibility of protein thermal denaturation and the extreme stability of specific proteins. For the first case, a more reliable protein temperature-dependent Δ C p was obtained by a combantion of DSC and ICD method, which was used for further calculation of Δ G of the unfolding process. For the second case, the unfolding process of some proteins under low pH is reversible and protein stability result from DSC was highly consistent with that obtained by ICD method. Then, protein stability under other conditions such as high pH, where the denaturation would be irreversible, could be estimaed by ICD method. For the third case, a combination of several denaturants were used to obtain the protein stability curve. In addition, DSC can also be applied to optimize conditions for protein crystallization. Third, the application scope of DSC is expanded to complex protein-realted systems such as amyloid fibers or even biological samples like blood plasma. For amyloid fibers, DSC can provide useful information of their thermodynamic properties which can lead us to a better understanding of amyloid fiber formation; for biological samples, DSC thermograms can be used as “fingerprints” for clinical diagnosis since the features of DSC thermograms for healthy and sick individuals are different. To summarize, DSC is a powerful tool to obtain protein properties and will play a more important role in the future.