Probing Site-Specific Energetics in Proteins and Nucleic Acids by Hydrogen Exchange and Nuclear Magnetic Resonance Spectroscopy

This chapter illustrates the use of hydrogen exchange and NMR spectroscopy to define how the free energy of stabilization of a biological molecule is being redistributed or lost as a result of a structural perturbation or of a functional change. The methodology relies on the potential of hydrogen exchange measurements to delineate and to quantify structural energetic changes throughout the molecule. At the same time, the methodology relies also on the resolving power of proton NMR spectroscopy. This resolving power generally decreases with increasing the size of the molecule observed. In large molecules, special environments of the proton can result in large shifts of its resonance. In these cases, observation of the proton by 1D NMR is possible, like in the case of hemoglobin discussed here. In general, however, at the present time, NMR spectroscopy can be used to monitor hydrogen exchange in molecules and molecular complexes of moderate size. Advances in isotopic labeling of proteins and nucleic acids, and in NMR techniques, promise to extend this limit to larger macromolecular systems.