Temperature excursion infrared (TEIR) spectroscopy used to study hydrogen bonding between water and biomolecules.

Water is a highly polar molecule that is capable of making four H-bonding linkages. Stability and specificity of folding of water-soluble protein macromolecules are determined by the interplay between water and functional groups of the protein. Yet, under some conditions, water can be replaced with sugar or other polar protic molecules with retention of protein structure. Infrared (IR) spectroscopy allows one to probe groups on the protein that interact with solvent, whether the solvent is water, sugar or glycerol. The basis of the measurement is that IR spectral lines of functional groups involved in H-bonding show characteristic spectral shifts with temperature excursion, reflecting the dipolar nature of the group and its ability to H-bond. For groups involved in H-bonding to water, the stretching mode absorption bands shift to lower frequency, whereas bending mode absorption bands shift to higher frequency as temperature decreases. The results indicate increasing H-bonding and decreasing entropy occurring as a function of temperature, even at cryogenic temperatures. The frequencies of the amide group modes are temperature dependent, showing that as temperature decreases, the amide group H-bonds to water strengthen. These results are relevant to protein stability as a function of temperature. The influence of solvent relaxation is demonstrated for tryptophan fluorescence over the same temperature range where the solvent was examined by infrared spectroscopy.