Backbone Amide Hydrogens as Thermodynamic Sensors

Formalisms to relate the observed rates of amide hydrogen exchange to thermodynamic stabilization of proteins have been developed [38]. Amide hydrogens of proteins in the native, folded state are proposed to exchange according to the following equation  

The closing equilibrium constant at each amide (Kd = kd/kop) is equal to the protection factor pf) and can be translated into the stabilization free energy of closed state (AGd) by Eq. (5). 

the measurement of exchange rates of backbone amide hydrogen serves as a precise thermodynamic sensor of the local environment. 

Dramatic advances in mass spectrometry and improvements in the various steps within the experimental hydrogen exchange procedures have resulted in the development of automated systems for high-throughput, high-resolution H/D exchange analysis [6, 8, 40-44]. 

The system (Fig. 12.1), described in this section, incorporates the latest of these enhancements, including solid phase proteolysis, automated liquid handling and streamlined data reduction software [8]. 

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