High Pressure NMR Methods for Characterizing Functional Substates of Proteins.

Proteins usually exist in multiple conformational states in solution. High pressure NMR spectroscopy is a well-suited method to identify these states. In addition, these states can be characterized by their thermodynamic parameters, the free enthalpies at ambient pressure, the partial molar volumes, and the partial molar compressibility that can be obtained from the analysis of the high pressure NMR data. Two main types of states of proteins exist, functional states and folding states. There is a strong link between these two types, the functional states represent essential folding states (intermediates), other folding states may have no functional meaning (optional folding states). In this chapter, this concept is tested on the Ras protein, an important proto-oncogen in humans where all substates required by theory can be identified experimentally by high pressure NMR spectroscopy. Finally, we show how these data can be used to develop allosteric inhibitors of proteins.